WO2009055760A1 - Promoter detection and analysis - Google Patents

Abstract

The present disclosure discloses an array -based method for promoter detection and analysis. Promoter sequence candidates are analyzed simultaneously in one reaction vial utilizing a vector comprising a TAG sequence wherein transcriptional products are tagged as they are synthesized, in such a way that one specific transcript is labeled with only one type of tag, and one tag labels only one type of transcript. The transcriptional output is analyzed on conventional arrays.

Description

PROMOTER DETECTION AND ANALYSIS

This application claims priority to U.S. Patent Application Ser. No. 11/925,837 filed 27 October 2007.

This invention was made with government support under Grant 1R43HG003559 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD The present disclosure relates to methods for detecting regulatory elements in a cell sample. More specifically, the disclosure relates to methods for detecting regulatory elements in multiple cell samples at the same time and uses arising there from. The present disclosure also provides a vector for detection and analysis of regulatory elements.

BACKGROUND The genes of all living organisms are encoded by the nucleic acids DNA and RNA. Each gene encodes a protein that may be produced by the organism through expression of the gene. The systems that regulate gene expression respond to a wide variety of developmental and environmental stimuli, thus allowing each cell type to express a unique and characteristic subset of its genes, and to adjust the dosage of particular gene products as needed. The importance of dosage control is underscored by the fact that targeted disruption of key regulatory molecules in mice often results in drastic phenotypic abnormalities (Johnson, R. S., et al., Cell, 71 :577-586 (1992)), just as inherited or acquired defects in the function of genetic regulatory mechanisms contribute broadly to human disease. Standard molecular biology techniques have been used to analyze the expression of genes in a cell by measuring nucleic acids. These techniques include PCR, northern blot analysis, or other types of DNA probe analysis such as in situ hybridization. Each of these methods allows one to analyze the transcription of only known genes and/or small numbers of genes at a time (Nucl. Acids Res. 19, 7097-7104 (1991); Nucl. Acids Res. 18, 4833-4842 (1990); Nucl. Acids Res. 18, 2789-2792 (1989); European J. Neuroscience 2, 1063-1073 (1990); Analytical Biochem. 187, 364-373 (1990); Genet. Annal Techn. Appl. 7, 64-70 (1990); GATA 8(4), 129-133 (1991); Pro. Natl. Acad. Sci. USA 85, 1696-1700 (1988); Nucl. Acids Res. 19, 1954 (1991); Proc. Natl. Acad. Sci. USA 88, 1943-1947 (1991); Nucl. Acids Res. 19, 6123-6127 (1991); Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988); Nucl. Acids Res. 16, 10937 (1988)). Measurement of the levels of mRNA has also been used to monitor gene expression. Since proteins are transcribed from mRNA, it is possible to detect transcription by measuring the amount of mRNA present. One common method, called "hybridization subtraction", allows one to look for changes in gene expression by detecting changes in mRNA expression (Nucl. Acids Res. 19, 7097-7104 (1991); Nucl. Acids Res. 18, 4833-4842 (1990); Nucl. Acids Res. 18, 2789-2792 (1989); European J. Neuroscience 2, 1063-1073 (1990); Analytical Biochem. 187, 364-373 (1990); Genet. Annal Techn. Appl. 7, 64-70 (1990); GATA 8(4), 129-133 (1991); Proc. Natl. Acad. Sci. USA 85, 1696-1700 (1988); Nucl. Acids Res. 19, 1954 (1991); Proc. Natl. Acad. Sci. USA 88, 1943-1947 (1991); Nucl. Acids Res. 19, 6123- 6127 (1991); Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988); Nucl. Acids Res. 16, 10937 (1988)). Gene expression has also been monitored by measuring levels of the gene product, (i.e., the expressed protein), in a cell, tissue, organ system, or even organism. Measurement of gene expression by measuring the protein gene product may be performed using antibodies known to bind to the particular protein to be detected. A difficulty arises in needing to generate antibodies to each protein to be detected. Measurement of gene expression via protein detection may also be performed using 2-dimensional gel electrophoresis, wherein proteins can be, in principle, identified and quantified as individual bands, and ultimately reduced to a discrete signal. In order to positively analyze each band, each band must be excised from the membrane and subjected to protein sequence analysis (e.g., Edman degradation). However, it tends to be difficult to isolate a sufficient amount of protein to obtain a reliable protein sequence. In addition, many of the bands often contain more multiple proteins. Another difficulty associated with quantifying gene expression by measuring an amount of protein gene product in a cell is that protein expression is an indirect measure of gene expression. It is impossible to know from a protein present in a cell when the expression of that protein occurred. Thus, it is difficult to determine whether the protein expression changes over time due to cells being exposed to different stimuli. The measurement of the amount of particular activated transcription factors has been used to monitor gene expression. Transcription in a cell is controlled by activated transcription factors which bind to DNA at sites outside the core promoter for the gene and activate transcription. Since activated transcription factors activate transcription, detection of their presence is useful for measuring gene expression Transcriptional activators are found in prokaryotes, viruses, and eukaryotes In molecular biology, a reporter gene (often simply reporter) is a gene that researchers often attach to another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are generally used to determine whether the gene of interest has been taken up by or expressed in the cell or organism population To introduce a reporter gene into an organism, researchers place the reporter gene and the gene of interest in the same DNA construct to be inserted into the cell or organism For bacteria or eukaryotic cells in culture, this is usually in the form of a circular DNA molecule called a plasmid It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent proteins; for example, green fluorescent protein (GFP) and the luciferase assay. Other reporters include, for example, beta-galactosidase, X-gal, and chloramphenicol acetyltransferase (CAT). Many methods of transfection and transformation - two ways of expressing a foreign or modified gene in an organism - are effective in only a small percentage of a population subjected to the techniques Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are normally expressed under their own promoter independent from that of the introduced gene of interest, the reporter gene can be expressed constitutively ("always on") or inducibly with an external intervention such as the introduction of IPTG in the beta-galactosidase system. As a result, the reporter gene's expression is independent of the gene of interest's expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions. 102 Reporter genes can also be used to assay for the expression of the gene of interest,

103 which may produce a protein that has little obvious or immediate effect on the cell culture or

104 organism. In these cases the reporter is directly attached to the gene of interest to create a

105 gene fusion. The two genes are under the same promoter and are transcribed into a single

106 polypeptide chain. In these cases it is important that both proteins be able to properly fold

107 into their active conformations and interact with their substrates despite being fused. In

108 building the DNA construct, a segment of DNA coding for a flexible polypeptide linker

109 region is usually included so that the reporter and the gene of interest will only minimally

110 interfere with one another.

111 Reporter genes can be used to assay for the activity of a particular promoter in a cell

112 or organism. In this case there is no separate "gene of interest"; the reporter gene is simply

113 placed under the control of the target promoter and the reporter gene product's activity is

114 quantitatively measured. The results are normally reported relative to the activity under a

115 "consensus" promoter known to induce strong gene expression.

116 In the past few years, the sequencing of numerous genomes, both eukaryotic and

117 prokaryotic, has generated an enormous amount of data. Although detection of coding

118 regions is common, the major challenge is to annotate the functional non-coding sequences,

119 in particular those involved in gene transcription. Because transcription plays a pivotal role

120 in regulating important processes such as morphogenesis, cell differentiation, tissue

121 specificity, hormonal communication, and cellular stress responses, a need for the

122 identification and functional characterization of transcriptional promoters exists. The

123 methods for detection and analysis of transcriptional promoters can be divided into two

124 categories: computational methods and experimental methods.

125 Computational methods for promoter studies incorporate the many public and private

126 databases containing information gathered from studies published by hundreds of laboratories

127 and conducted using conventional labor-intensive and time-consuming approaches. The

128 Eukaryotic Promoter Database (EPD) and the Transcription Regulatory Regions Database

129 (TRRD) contain 1,871 and 703 entries of human promoters, respectively. Other promoter

130 databases, such as TransFac and DBTSS, contain almost 9,000 promoter sequences.

131 However, most of these are derived from in silico primer extension assays (e.g., TransFac), or

132 contain only data about the putative transcriptional start site (e.g., DBTSS). The small

133 numbers of experimentally validated human promoters compared to the 35,000 expected

134 human genes indicate the magnitude of the work still to be done. 135 Numerous computer-based promoter prediction methods have been developed (Scherf

136 et al., J. MoI. Biol. 297(3):599-606, 2000; Werner, T. Brief Bioinform. l(4):372-80, 2000;

137 Loots et al , Gen. Res 12 832-839, 2002). These methods are limited by the lack of a

138 reliable, standard protocol to predict and identify promoter regions. Promoters are generally

139 only a few base pairs (bp) long, and are embedded within the massive genome. Thus,

140 promoters are much more difficult to find and are easier to confuse than long, patterned

141 coding sequences. Typical computer algorithms for promoter prediction are based on

142 comparisons of unknown sequences with known elements, a strategy which does not allow

143 for identification of new types of promoter elements. Thus, computer-based searches for

144 promoter elements are incomplete and always require experimental confirmation.

145 Computational methods based on microarray data have been used to investigate

146 genome-wide transcriptional regulation (Pilpel et al., Nat. Gen. 29(2):153-9, 2001) These

147 techniques allow for the identification of novel functional motif combinations in the

148 promoters of a given organism, and may provide a global view of transcription networks.

149 However, the data provided from these methods also need confirmation by experimental

150 means.

151 The experimental methods for investigation of a promoter region and subsequent

152 characterization usually follow a basic protocol. First, upon identification of a new coding

153 sequence, the transcription start site is defined with standard molecular biology tools such as

154 Sl mapping, primer extension, or 5'RACE. Second, the upstream genomic region (up to 10

155 kb) is cloned and demonstrated to have promoter activity by performing a reporter assay in a

156 transient transfection system. Third, deletion and point mutation analyses are performed to

157 define the important transcriptional c/s-acting elements; information about transcriptional

158 regulation may be obtained by applying different induction or repression agents in transient

159 transfection assays. Finally, the transcription factors involved in promoter regulation are

160 identified by Dnase I footprinting, electrophoresis mobility shift assay (EMSA) in the

161 presence or absence of mutant probes and competitors, and EMSA supershift assay.

162 Transient-transfection based experimental methods have several disadvantages.

163 These methods measure reporter protein level instead of mRNA level, which is the direct

164 product of the transcription; protein levels may not always correlate with mRNA levels.

165 There are a limited number of reporter assays available (e.g. chloramphenicol acetyl-

166 transferase, β-galactosidase, luciferase, green fluorescent protein (GFP), β-glucuronidase) and

167 the utilization of the same reporter to compare various promoters implies that these promoters

168 must be tested separately and thus these assays are labor-intensive and time-consuming. 169 Since each of the many steps involved (i.e. transfection, induction, harvest, reporter

170 detection) are performed separately for each promoter investigated, usually in duplicate or

171 triplicate, the handling of more than 20 constructs simultaneously is challenging. For each

172 step performed, the time difference between the first and last sample may be significant;

173 therefore incubation periods, cell and reagent quality, for example, may differ from one

174 sample to the other thus introducing more experimental variation. Large amounts of material

175 and reagents are required. Additionally, in order to compare a series of promoters to each

176 other, a second reporter cassette has to be included as an internal control. In some instances,

177 the detection of this control may be as time-consuming and labor-intensive as for the first

178 reporter, and subject to experimental errors. The expression of this internal control can also

179 compete with the gene expression driven by the promoter of interest, and affect the results of

180 the assay. Some assays, such as luciferase and GFP assays, require expensive

181 instrumentation.

182 Kim et al. reported an experimental method for isolation and identification of

183 promoters in the human genome (Kim et al. Genome Research 15:830-839, 2005). However,

184 the use of antibodies to identify regions that may be associated with active transcription and

185 the required binding of both RNAP and TFIID as criteria for promoters may lead to the

186 elimination of some promoters that only show partial binding.

187 Khambata-Ford et al. reported an experimental method for identification of promoter

188 regions in the human genome by using a retroviral plasmid library-based functional reporter

189 gene assay (Khambata-Ford et al., Gen. Res. 13 : 1765-1774, 2003). However, in addition to

190 allowing potentially lethal disruption of the target cell genome by random integration of the

191 retroviral vector, the assay relies on the fluorescent reporter GFP for detection and screens

192 the cells via fluorescence-activated cell sorting (FACS).

193 Trinklein et al, reported an experimental method for identification and functional

194 analysis of human transcriptional promoters (Trinklein et al, Gen. Res. 13:308-312, 2003) by

195 using a draft sequence of the human genome and cDNA libraries. However, for further

196 analysis and identification of promoter sequences they used a luciferase-based transfection

197 assay.

198 The sequencing of genomes has generated a huge amount of data that needs to be

199 annotated. Computational methods are available to detect putative transcriptional promoter

200 regions, but they are not 100% efficient and must be confirmed by experimentation.

201 Unfortunately, the experimental procedures that are currently available to study promoters are 202 time-consuming, laborious, and not easily adapted to large numbers of promoters. Therefore,

203 new techniques for transcriptional studies are needed. 204

205 SUMMARY

206 The foregoing disadvantages of the previously described methods are overcome by

207 providing a novel reporter system that incorporates unique, non-coding DNA sequences. The

208 object of the present disclosure is to provide a novel reporter system that is specific,

209 inexpensive, and provides an efficient means of promoter detection.

210 The present disclosure provides a method for the detection and analysis of DNA

211 promoter sequences. In a preferred embodiment, the present disclosure provides a method

212 for detecting DNA regulatory sequences comprising: a) inserting a promoter sequence

213 candidate into a vector wherein the vector comprises a TAG sequence and wherein the

214 promoter sequence candidate is inserted in a position to drive transcription of the TAG

215 sequence; b) the vector containing the inserted promoter sequence candidate is inserted into a

216 cloning host cell; c) cloning host cells containing different promoter sequence candidates are

217 grown to the same optical density, pooled and the vectors therein are extracted, purified and

218 inserted into a reporter cell line; d) mRNA is extracted from the reporter cell lines wherein

219 the mRNA is directly labeled or is used as template for cDNA or probe synthesis; and e) the

220 labeled mRNA, cDNA or probe is analyzed with an array wherein the array comprises

221 identical or complementary sequence to the TAG sequence. Preferably, the labeled mRNA,

222 cDNA or probe hybridizes to the array and the label of the mRNA, cDNA or probe has a

223 detectable response.

224 In another embodiment, the present disclosure provides a method for the detection

225 and analysis of DNA promoter sequence candidates wherein DNA promoter sequence

226 candidates are integrated into vectors that comprise a TAG sequence, one or more multiple-

227 cloning sites, one or more DNA recombination sequences, a negative selection marker,

228 nucleotide sequences useful for the detection of mRNA sequences such as a T7 promoter

229 sequence and a MA segment, a translation stop codon, a RNA stabilization fragment such as

230 the one from the alpha-globin gene, and a transcription termination signal, such as a poly A

231 signal, and wherein the DNA promoter sequence candidates are located such that they drive

232 the transcription of the TAG sequences. In another embodiment, the present disclosure

233 provides a method for the detection and analysis of DNA promoter sequences wherein DNA

234 promoter sequence candidates are integrated into a vector comprising a TAG sequence, one

235 or more multiple-cloning sites, both of attPl and attP2 sequences, a negative selection marker 236 wherein the negative selection marker is the ccdB gene, a T7 promoter sequence, a MA

237 segment, a translation stop codon, an alpha-globin RNA stabilization fragment, and a poly A- 238 signal, and wherein the DNA promoter sequence candidate drives the transcription of the

239 TAG sequence

240 In another embodiment, the present disclosure provides a method for the detection

241 and analysis of DNA promoter sequences wherein DNA promoter sequence candidates are

242 integrated into a vector wherein the vector comprises a TAG sequence, one or more multiple-

243 cloning sites, both of attPl and attP2 sequences, a negative selection marker, a T7 promoter

244 sequence, a MA sequence wherein the MA sequence is comprised of approximately 25% A,

245 25% T, 25% G, and 25% C, a translation stop codon, a RNA stabilization fragment, and a

246 transcription termination signal, and wherein the DNA promoter sequence candidate drives

247 the transcription of the TAG sequence Preferably, the vector is a plasmid Preferably, the

248 RNA stabilization fragment is from an alpha-globm gene. Preferably, the transcription

249 termination signal is a poly A signal.

250 In another embodiment, the present disclosure provides a method for the detection

251 and analysis of DNA promoter sequences wherein DNA promoter sequence candidates are

252 integrated into a vector wherein the vector comprises a TAG sequence, one or more multiple-

253 cloning sites, one or more DNA recombination sequences, a negative selection marker, a T7

254 promoter sequence, a MA sequence wherein the MA sequence is comprised of approximately

255 25% A, 25% T, 25% G, and 25% C, a translation stop codon wherein the translation stop is in

256 three frames, a RNA stabilization fragment, and a transcription termination signal, and

257 wherein the DNA promoter sequence candidate is located such that it drives the transcription

258 of the TAG sequence. Preferably, the vector is a plasmid. Preferably, the RNA stabilization

259 fragment is from an alpha-globm gene. Preferably, the transcription termination signal is a

260 poly A signal. Preferably, the DNA recombination sequences are attPl and att?2

261 In another embodiment, the present disclosure provides a method for the detection

262 and analysis of DNA promoter sequences comprising: (a) integrating DNA promoter

263 sequence candidates within TAG-vectors, wherein the DNA promoter sequence candidate is

264 located such that it drives the transcription of the TAG sequence, wherein the TAG-vector

265 comprises: multiple cloning sites (MCS) for inserting DNA promoter sequence candidate,

266 DNA recombination sequences, such as att? 1 and atiP2, between which DNA promoter

267 sequence candidates can be inserted; a negative selection marker to maximize the recovery of

268 clones containing promoter sequence inserts, such as ccdB; a nucleotide sequence useful to

269 enable RNA synthesis , preferably a T7 promoter sequence; a unique reporter TAG, a specific 270 MA segment useful to synthesize probes from RNA, wherein the MA segment is comprised

271 of approximately 25% A, 25% T, 25% G, and 25% C; a three frame translation stop codon;

272 RNA stabilization fragment, preferably from a hemoglobin or alpha-globin gene; and a

273 transcription termination signal, such as a poly A-signal; (b) the TAG-vectors with the

274 promoter sequence candidate inserts are cloned into a host, preferably Escherichia coli, and

275 the clones are arrayed into a 96-well plate and grown to about the same cell density; (c) the

276 resultant clones are pooled, and the vectors wherein are purified; (d) the purified vector

277 mixture is transfected into a cell line of interest; and (e) the RNA is extracted, labeled, and

278 quantified by hybridization to the DNA TAG sequences arrayed on a membrane or glass

279 support, or beads. Suitable bead compositions include those used in peptide, nucleic acid and

280 organic moeity synthesis, including but not limited to, plastics, ceramics, glass, polystyrene,

281 methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium

282 dioxide, latex or cross-linked dextrans such as sepharose, cellulose, nylon, cross-linked

283 micelles and teflon many all be used (see Microsphere Detection Guide, Bangs Laboratories,

284 Fishers Ind.). Preferably, the vector is a plasmid. Preferably, the label of the mRNA, cDNA

285 or probe has a detectable response.

286 In another embodiment of the present disclosure, a method is provided wherein each

287 DNA promoter sequence candidate under investigation (for example, computer-predicted

288 DNA promoter sequence candidates, DNA fragments from a collection of nucleotide

289 sequences, such as a genomic library, deletion or site-directed mutants of a specific DNA

290 promoter, tissue-specific promoters, artificial promoters, etc.) drives the transcription of a

291 unique mRNA that consists of a short oligonucleotide TAG embedded in the 5' end of a

292 luciferase coding sequence, wherein equimolar amounts of the various promoters under

293 investigation are pooled and transfected into a cell line, and wherein the mRNA levels are

294 quantified by hybridization to the TAG oligonucleotides in an array format. In another

295 embodiment, the reporters are short oligonucleotides TAGs. In another embodiment the

296 length the TAG sequence is between about 16 base pairs and about 200 base pairs, more

297 preferably between about 20 base pairs and about 175 base pairs, more preferably between

298 about 25 base pairs and about 150 base pairs, more preferably between about 30 base pairs

299 and about 125 base pairs, more preferably between about 45 base pairs and about 100 base

300 pairs, more preferably between about 50 base pairs and about 75 base pairs, more preferably

301 about 65 base pairs, and most preferably 60 bp. In another embodiment, all the TAG

302 sequences are designed to have approximately the same melting temperature; this feature

303 allows for the unbiased quantification of various mRNAs by hybridization under the same 304 temperature and ionic strength conditions. In another embodiment, the method enables the

305 detection and quantification of mRNA levels, instead of reporter protein levels, and is

306 unaffected by potentially interfering translation and posttranslational events as in the

307 conventional reporter assays. In another embodiment of the present disclosure, each of the

308 clones containing a TAG vector, preferably a plasmid, is grown to about the same cell

309 density, and the purified vectors, preferably plasmids, of these clonal cultures, containing

310 every DNA promoter sequence candidate, is mixed, and the resulting mixture transfected into

311 a single population of cells creating a competitive environment for the various promoters to

312 recruit transcription factors. In another embodiment, vectors, preferably plasmids, purified

313 from the clonal cell cultures of about equal cell density and containing about equimolar

314 amounts of all the DNA promoter sequences are mixed and used for transfection of a single

315 population of cells and the need for internal controls is eliminated. There are several ways to

316 obtain equimolar amounts of the vectors that carry the various candidate promoters-TAG

317 combinations that are used to transfect reporter cell lines. In another embodiment, equimolar

318 amounts of the vectors can be obtained by: 1) making the vector library; 2) array the vector

319 library (e.g., 96 well plate); 3) take an equal fraction from each clone and pool them all; 4)

320 grow all clones together assuming same growth rate and yield of the same amount of vector

321 per cell; 5) extract the transformation agent (e.g., a vector, plasmid or virus); and 6) transfect

322 the vector (or plasmid or infect virus) into a reporter cell line. Alternately, equimolar

323 amounts of the vector can be obtained by: 1) making the vector library; 2) array the vector

324 library (e.g., 96 well plate); 3) grow each clone individually (e.g., in a deep-well plate in case

325 of bacteria); 4) take an equal fraction from each clone and pool them all; 5) extract the

326 transformation agent (e.g., vector, plasmid or virus); and 6) transfect the vector (or plasmid or

327 infect virus) into the reporter cell line. Alternately, equimolar amounts of the vector can be

328 obtained by: 1) making the vector library; 2) array the vector library (e.g., 96 well plate); 3)

329 grow each clone individually (e.g., in a deep-well plate in case of bacteria); 4) extract the

330 transformation agent (e.g., vector, plasmid or virus) and quantify it; 5) take an equal fraction

331 from each clone (e.g., vector, plasmid or virus) and pool them all; and 6) transfect vector (or

332 plasmid or infect virus) into the reporter cell line. Alternately, equimolar amounts of the

333 vector can be obtained by: 1) making the vector library; 2) take a fraction from each clone,

334 and pool them all; 3) grow all the clones together and assume same growth rate and yield of

335 the same amount of vector per cell; 4) extract transformation agent (e.g., vector, or plasmid

336 or virus); 5) transfect vector (or plasmid or infect virus) into reporter cell line and determine 337 the TAG of interest (e.g., high level of expression); and 6) find the clone in the vector library

338 that contains TAG of interest (e.g., colony hybridization).

339 In another embodiment, the present disclosure provides a method for the detection

340 and analysis of DNA promoter sequences comprising: (a) integrating a DNA promoter

341 sequence candidate into a vector, preferably a plasmid, wherein the plasmid comprises a

342 TAG sequence, one or more multiple-cloning sites, at least one DNA recombination

343 sequence, preferably attP 1 or att?2, a negative selection marker, preferably ccdB, a

344 nucleotide sequence useful to enable RNA synthesis, such as a T7 promoter sequence, a MA

345 segment, a translation stop codon, a RNA stabilization fragment, preferably from the

346 hemoglobin or alpha-globin gene, and transcription termination signal, such as a poly A-

347 signal, and wherein the DNA promoter sequence candidate is located such that it drives the

348 transcription of the TAG sequence; (b) the vectors with the promoter sequence candidate

349 inserts are cloned into a host, preferably Escherichia coli, and the clones are arrayed into a

350 96-well plate and grown to the same cell density; (c) the resultant clones are pooled, and the

351 vectors wherein are purified; (d) the purified vector mixture is transfected into a cell line of

352 interest wherein the use of internal controls is eliminated and (e) the RNA is extracted,

353 labeled, and quantified by hybridization to the DNA TAG sequences arrayed on a membrane

354 or glass support. Preferably, the vector is a plasmid. Preferably, the label of the mRNA,

355 cDNA or probe has a detectable response.

356 In another embodiment, the disclosure provides a method for the detection and

357 analysis of DNA promoter sequences comprising integrating a DNA promoter sequence

358 candidate into a vector, preferably a plasmid, wherein the vector comprises a TAG sequence,

359 one or more multiple-cloning sites, at least one DNA recombination sequence, preferably

360 attΫ\ or att?2, a negative selection marker, such as ccdB, a nucleotide sequence useful to

361 enable RNA synthesis, preferably a T7 promoter sequence, a MA segment, a translation stop

362 codon, an RNA stabilization fragment, preferably a hemoglobin or alpha-globin gene, and

363 transcription termination signal, preferably a poly A-signal, and wherein the DNA promoter

364 sequence candidate is located such that it drives the transcription of the TAG sequence.

365 In another embodiment, the present disclosure provides a method for the detection

366 and analysis of DNA promoter sequences comprising: (a) integrating a DNA promoter

367 sequence candidate into a vector wherein the vector comprises a TAG sequence, one or more

368 multiple-cloning sites, at least one DNA recombination sequence, a negative selection

369 marker, a nucleotide sequence useful to enable RNA synthesis, a MA segment, a translation

370 stop codon, a RNA stabilization fragment, and a transcription termination signal, and wherein 371 the DNA promoter sequence candidate is located such that it drives the transcription of the

372 TAG sequence; (b) the vectors with the promoter sequence candidate inserts are cloned into a

373 host, preferably Escherichia coli, and the clones are arrayed into a 96-well plate and grown to

374 the same cell density; (c) the resultant clones are pooled, and the vectors wherein are purified;

375 (d) the purified vector mixture is transfected into a cell line of interest; and (e) the RNA is

376 extracted, labeled, and quantified by hybridization to the DNA TAG sequences arrayed on a

377 membrane or glass support. Preferably, the vector is a plasmid. Preferably, the DNA

378 recombination sequence is attVl or attV2. Preferably, the nucleotide sequence useful to

379 enable RNA synthesis is a T7 promoter sequence. Preferably, the transcription termination

380 signal is a poly A-signal. Preferably, the RNA stabilization fragment is from the hemoglobin

381 or alpha-globin gene. Preferably, the label of the mRNA, cDNA or probe has a detectable

382 response.

383 In another embodiment, the present disclosure provides a method for the detection

384 and analysis of DNA promoter sequences comprising: (a) integrating a DNA promoter

385 sequence candidate into a vector wherein the vector comprises a TAG sequence, one or more

386 multiple-cloning sites, at least one DNA recombination sequence, a negative selection

387 marker, a nucleotide sequence useful to enable RNA synthesis, a MA segment, a translation

388 stop codon, a RNA stabilization fragment, and a transcription termination signal, and wherein

389 the DNA promoter sequence candidate is located such that it drives the transcription of the

390 TAG sequence; (b) the vectors with the promoter sequence candidate inserts are cloned into a

391 host, preferably Escherichia coli, and the clones are arrayed into a 96-well plate and grown to

392 the same cell density; (c) the resultant clones are pooled, and the vectors wherein are purified;

393 (d) the purified vector mixture is transfected into a cell line of interest and wherein the use of

394 internal controls is eliminated upon transfecting the cells with vectors purified from the

395 clonal cell populations which are of the same cell density and (e) the RNA is extracted,

396 labeled, and quantified by hybridization to the DNA TAG sequences arrayed on a membrane

397 or glass support. Preferably, the vector is a plasmid. Preferably, the DNA recombination

398 sequence is αffPl or att?2. Preferably, the nucleotide sequence useful to enable RNA

399 synthesis is a T7 promoter sequence. Preferably, the RNA stabilization fragment is from the

400 hemoglobin or alpha-globin gene. Preferably, the transcription termination signal is a poly

401 A-signal. Preferably, the label of the mRNA, cDNA or probe has a detectable response.

402 In another embodiment of the present disclosure, the disclosure provides a method for

403 detection and analysis of DNA promoter nucleotide sequences in a collection of nucleotide

404 sequences, such as genomic library, comprising: (a) mixing promoter sequence candidates 405 with TAG-vectors, wherein the TAG-vector comprises: multiple cloning sites (MCS) for

406 inserting promoter sequence candidate, at least one DNA recombination sequence, such as

407 attP 1 or attP2, a negative selection marker to maximize the recovery of clones containing

408 promoter sequence inserts, such as, for example, a ccdB gene, a T7 promoter sequence to

409 enable RNA synthesis, a unique approximate 60 base pair reporter TAG, a specific MA

410 segment useful to synthesize probes from RNA, wherein the MA segment is comprised of

411 approximately 25% A, 25% T, 25% G, and 25% C, a three frame translation stop codon, a

412 RNA stabilization fragment, such as, for example, alpha-globin or hemoglobin, and

413 transcription termination signal, preferably a poly A-signal; (b) the TAG-vectors with the

414 promoter sequence candidate inserts are cloned into a host, preferably Escherichia coli, and

415 the clones are arrayed into a 96-well plate and grown to the same cell density; (c) the

416 resultant clones are pooled, and the vectors wherein are purified; (d) the purified vector

417 mixture is transfected into a cell line of interest; and (e) the RNA is extracted, labeled, and

418 quantified by hybridization to the DNA TAG sequences arrayed on a membrane or glass

419 support. Preferably, the TAG-vector is a TAG-plasmid. Preferably, the label of the mRNA,

420 cDNA or probe has a detectable response.

421 In another embodiment of the present disclosure, the disclosure provides a method for

422 the detection and analysis of DNA promoter nucleotide sequences in a collection of

423 nucleotide sequences, such as a genomic library, comprising: (a) mixing promoter sequence

424 candidates with TAG-vectors, wherein the TAG-vector comprises: multiple cloning sites

425 (MCS) for inserting promoter sequence candidate, at least one DNA recombination sequence,

426 a negative selection marker, a nucleotide sequence useful to enable RNA synthesis, a unique

427 approximate 60 base pair reporter TAG, a specific MA segment useful to synthesize probes

428 from RNA, wherein the MA segment is comprised of approximately 25% A, 25% T, 25% G,

429 and 25% C, a three frame translation stop codon, a RNA stabilization fragment, and

430 transcription termination signal; (b) the TAG-vectors with the promoter sequence candidate

431 inserts are cloned into a host, preferably Escherichia coli, and the clones are arrayed into a

432 96-well plate and grown to the same cell density; (c) the resultant clones are pooled, and the

433 vectors wherein are purified; (d) the purified vectors are transfected into a cell line of interest

434 and no internal controls are utilized and (e) the RNA is extracted, labeled, and quantified by

435 hybridization to the DNA TAG sequences arrayed on a membrane or glass support.

436 Preferably, the vectors are plasmids. Preferably, the DNA recombination sequence is attVl

437 or attV2. Preferably, the negative selection marker is ccdB. Preferably, the nucleotide 438 sequence to enable RNA synthesis is a T7 promoter sequence. Preferably, the RNA 439 stabilization fragment is from the alpha-globin gene. Preferably, the transcription termination

440 signal is a poly A-signal. Preferably, the label of the mRNA, cDNA or probe has a detectable

441 response

442 In another embodiment of the present disclosure, the disclosure provides a method for

443 detection and analysis of DNA promoter nucleotide sequences in a collection of nucleotide

444 sequences, such as a genomic library, comprising (a) mixing promoter sequence candidates

445 with TAG-vectors, wherein the TAG-vector comprises: multiple cloning sites (MCS) for

446 inserting promoter sequence candidate, at least one DNA recombination sequence, a negative

447 selection marker, a nucleotide sequence useful to enable RNA synthesis, a unique

448 approximate 60 base pair reporter TAG, a specific MA segment useful to synthesize probes

449 from RNA, wherein the MA segment is comprised of approximately 25% A, 25% T, 25% G,

450 and 25% C, a three frame translation stop codon, a RNA stabilization fragment, and a

451 transcription termination signal, (b) the TAG-vector with the promoter sequence candidate

452 inserts are cloned into a host, preferably Escherichia coli, and the clones are arrayed into a

453 96-well plate and grown to the same cell density, (c) the resultant clones, containing about

454 equal amounts of vectors are pooled, and the vectors wherein are purified; (d) the purified

455 vectors are transfected into a cell line of interest and wherein the use of internal controls is

456 not utilized, and (e) the RNA is extracted, labeled, and quantified by hybridization to the

457 DNA TAG sequences arrayed on a membrane or glass support. Preferably, the TAG-vectors

458 are TAG-plasmids. Preferably, the DNA recombination sequence is attP 1 or attP2.

459 Preferably, the negative selection marker is ccdB. Preferably, the nucleotide sequence to

460 enable RNA synthesis is a T7 promoter sequence. Preferably, the RNA stabilization

461 fragment is from the alpha-globm gene Preferably, the transcription termination signal is a

462 poly A-signal. Preferably, the label of the mRNA, cDNA or probe has a detectable response

463 In another embodiment, the disclosure provides a method for analysis and detection of

464 a plurality of DNA promoter nucleotide sequences in a plurality of samples, comprising: (a)

465 mixing DNA promoter sequence candidates, wherein the DNA promoter sequence

466 candidates are, for example, selected from computer-predicted promoter sequence candidates,

467 DNA fragments from a collection of nucleotide sequences, such as a genomic library,

468 deletion or site-directed mutants of a specific promoter, tissue-specific promoters, artificial

469 promoters, etc., with TAG vectors, wherein the TAG-vector comprises: multiple cloning sites

470 for inserting DNA promoter sequence candidate, DNA recombination sequences, a negative

471 selection marker, a nucleotide sequence useful to enable RNA synthesis, a unique

472 approximate 60 base pair reporter TAG, a specific MA segment useful to synthesize probes 473 from RNA, wherein the MA segment is comprised of about 25% A, 25% T, 25% G, and 25%

474 C, a three frame translation stop codon, a RNA stabilization fragment, and a transcription

475 termination signal; (b) the TAG-vectors with the promoter sequence candidate inserts are

476 cloned into a host, preferably Escherichia coli, and the clones are arrayed into a 96-well plate

477 and grown to the same cell density; (c) the resultant clones are pooled, and the vectors

478 wherein are purified; (d) the purified plasmid mixture is transfected into a cell line of interest;

479 and (e) the RNA is extracted, labeled, and quantified by hybridization to the DNA TAG

480 sequences arrayed on a membrane or glass support. Preferably, the TAG-vectors are TAG-

481 plasmids. Preferably, the DNA recombination sequence is attPl or attV2. Preferably, the

482 negative selection marker is ccdB. Preferably, the nucleotide sequence to enable RNA

483 synthesis is a T7 promoter sequence. Preferably, the RNA stabilization fragment is from the

484 alpha-globin gene. Preferably, the transcription termination signal is a poly A-signal.

485 Preferably, the label of the mRNA, cDNA or probe has a detectable response.

486 In another embodiment, the disclosure provides a method for detection and analysis of

487 a plurality of DNA promoter nucleotide sequences in a plurality of samples, comprising: (a)

488 mixing DNA promoter sequence candidates, wherein the promoter sequence candidates are,

489 for example, selected from computer-predicted promoter sequence candidates, DNA

490 fragments from a collection of nucleotide sequences, such as a genomic library, deletion or

491 site-directed mutants of a specific promoter, tissue-specific promoters, artificial promoters,

492 etc., with TAG vectors, wherein the TAG-vector comprises: multiple cloning sites for

493 inserting promoter sequence candidate, DNA recombination sequence, a negative selection

494 marker, a nucleotide sequence useful to enable RNA synthesis, a unique approximate 60 base

495 pair reporter TAG, a specific MA segment useful to synthesize probes from RNA, wherein

496 the MA segment is comprised of about 25% A, 25% T, 25% G, and 25% C, a three frame

497 translation stop codon, a RNA stabilization fragment, and a transcription termination signal;

498 (b) the TAG-vectors with the promoter sequence candidate inserts are cloned into a host,

499 preferably Escherichia coli, and the clones are arrayed into a 96-well plate and grown to the

500 same cell density; (c) the resultant clones contain about equal amounts of vector and are

501 pooled, and the vectors wherein are purified; (d) about equal amounts of the purified vectors

502 are transfected into a cell line of interest; and (e) the RNA is extracted, labeled, and

503 quantified by hybridization to the DNA TAG sequences arrayed on a membrane or glass

504 support. Preferably, the TAG-vectors are TAG-plasmids. Preferably, the DNA

505 recombination sequence is ati?\ or αftP2. Preferably, the negative selection marker is ccdB.

506 Preferably, the nucleotide sequence to enable RNA synthesis is a T7 promoter sequence. 507 Preferably, the RNA stabilization fragment is from the alpha-globin gene. Preferably, the

508 transcription termination signal is a poly A-signal. Preferably, the label of the mRNA, cDNA

509 or probe has a detectable response

510 In another embodiment, the disclosure provides a method for the detection and

511 analysis of a plurality of DNA promoter nucleotide sequences in a plurality of samples,

512 comprising (a) mixing DNA promoter sequence candidates, wherein the promoter sequence

513 candidates are, for example, selected from computer-predicted promoter sequence candidates,

514 DNA fragments from a collection of nucleotide sequences, such as a genomic library,

515 deletion or site-directed mutants of a specific promoter, tissue-specific promoters, artificial

516 promoters, etc., with TAG vectors, wherein the TAG-vector comprises: multiple cloning sites

517 for inserting promoter sequence candidate, DNA recombination sequence, a negative

518 selection marker, a nucleotide sequence useful to enable RNA synthesis, a unique

519 approximate 60 base pair reporter TAG, a specific MA segment useful to synthesize probes

520 from RNA, wherein the MA segment is comprised of about 25% A, 25% T, 25% G, and 25%

521 C, a three frame translation stop codon, a RNA stabilization fragment, and a transcription

522 termination signal, (b) the TAG-vectors with the DNA promoter sequence candidate inserts

523 are cloned into a host, preferably Escherichia coli, and the clones are arrayed into a 96-well

524 plate and grown to the same cell density, (c) the resultant clones are pooled, and the vectors

525 wherein are purified; (d) about equal amounts of the purified vectors are transfected into a

526 cell line of interest and wherein the use of internal controls is eliminated; and (e) the RNA is

527 extracted, labeled, and quantified by hybridization to the DNA TAG sequences arrayed on a

528 membrane or glass support Preferably, the TAG-vectors are TAG-plasmids. Preferably, the

529 DNA recombination sequence is α^Pl or attP2. Preferably, the negative selection marker is

530 ccclB Preferably, the nucleotide sequence to enable RNA synthesis is a T7 promoter

531 sequence. Preferably, the RNA stabilization fragment is from the alpha-globin gene.

532 Preferably, the transcription termination signal is a poly A-signal. Preferably, the label of the

533 mRNA, cDNA or probe has a detectable response.

534 The present disclosure provides a vector In a preferred embodiment, the present

535 disclosure provides a vector into which a DNA promoter sequence candidate is inserted into

536 comprising a TAG sequence, one or more multiple-cloning sites, at least one DNA

537 recombination sequence, a negative selection marker, a RNA polymerase promoter sequence,

538 a MA segment, a translation stop codon, a RNA stabilization fragment, and a transcription

539 termination signal, and wherein the DNA promoter sequence candidate is located such that it

540 can drive the transcription of the TAG sequence. Preferably, the vector is a plasmid. 541 In another embodiment, the present disclosure provides for a plasmid vector

542 comprising: a region for insertion of a putative promoter sequence wherein a MCS is located

543 both 5' and 3' to the putative promoter sequence, one or more DNA recombination

544 sequences; a T7 sequence; a TAG sequence; a luciferase gene sequence; a MA sequence; and

545 a translational stop sequence. Preferably, the MA sequence is either MA5 or MA4.

546 Preferably, the MA sequence is located 3' from the TAG sequence. Preferably, the luciferase

547 gene sequence is partial luciferase gene sequence or the full luciferase gene sequence.

548 Preferably, the translational stop sequence is a translational stop sequence in at least one

549 reading frame, more preferably at least two reading frames, and most preferably in three

550 reading frames. Preferably, the DNA recombination sequences are attP 1 and att?2.

551 In another embodiment, the present disclosure provides a plasmid vector into which a

552 DNA promoter sequence is inserted into comprising a TAG sequence, one or more multiple-

553 cloning sites, one or both of atiPl and attP2 sequences, a negative selection marker, a RNA

554 polymerase promoter sequence, a MA segment, a translation stop codon, a RNA stabilization

555 fragment, and a transcription termination signal, and wherein the DNA promoter sequence is

556 located such that it drives the transcription of the TAG sequence. Preferably, the vector is a

557 plasmid. Preferably, the TAG sequence is between about 16 base pairs to about 200 base

558 pairs, more preferably the vector of the TAG sequence is about 60 base pairs. Preferably, the

559 TAG sequence is located 3' to the inserted promoter sequence and 5' to a transcription

560 termination signal. Preferably, the DNA promoter sequence is an enhancer. Preferably, the

561 translation stop codon is a three frame translation stop codon. Preferably, the RNA

562 stabilization fragment is from an alpha-globin gene. Preferably, the transcription termination

563 signal is a poly-A signal. Preferably, the RNA polymerase promoter sequence is a T7

564 promoter sequence.

565 In another embodiment, the disclosure provides for a vector. The disclosure provides

566 a nucleotide sequence for use in the detection and analysis of a promoter nucleotide sequence

567 comprising: a T7 promoter, a TAG sequence, a MA sequence, and a poly A-signal. In

568 another embodiment of the disclosure, the promoter sequence candidate is selected from

569 promoter sequence candidates provided by a computer-predicted model, DNA fragments

570 from a collection of nucleotide sequences, such as a genomic library, deletion or site-directed

571 mutants of a specific promoter, tissue-specific promoters, artificial promoters, etc. In another

572 embodiment, the TAG sequence is a DNA sequence composed of random nucleotides. In

573 another embodiment, the length of the TAG sequence is short, preferably between about 16

574 base pairs to about 200 base pairs, more preferably between about 20 base pairs to about 150 575 base pairs, more preferably between about 30 base pairs to about 120 base pairs, more

576 preferably between about 40 base pairs to about 100 base pairs, more preferably between

577 about 50 base pairs to about 75 base pairs, and most preferably about 60 base pairs. Within a

578 plurality of TAG sequences, each TAG sequence will have approximately equivalent

579 amounts of the nucleotides A, T, G, and C such that each TAG sequence has approximately

580 the same melting temperature as the other the TAGs. A same melting temperature will allow

581 for the unbiased quantification of various mRNAs by hybridization under the same

582 temperature and ionic strength conditions. In another embodiment, the specific MA segment

583 is useful to synthesize probes from RNA, and the MA segment is comprised of about 25% A,

584 25% T, 25% G, and 25% C.

585 In another embodiment, the disclosure provides a method where a nucleotide

586 sequence is used for the detection and analysis of a promoter nucleotide sequence

587 comprising: a T7 promoter sequence, a TAG sequence, a MA sequence, and a poly A-signal.

588 A DNA promoter sequence candidate may be selected from promoter sequence candidates

589 provided by a computer-predicted model, DNA fragments from a collection of nucleotide

590 sequences, such as a genomic library, deletion or site-directed mutants of a specific promoter,

591 tissue-specific promoters, artificial promoters, etc. In preferred embodiments, the TAG

592 sequence is a DNA sequence comprised of short, random nucleotides preferably between

593 about 16 base pairs to about 200 base pairs, more preferably between about 20 base pairs to

594 about 150 base pairs, more preferably between about 30 base pairs to about 120 base pairs,

595 more preferably between about 40 base pairs to about 100 base pairs, more preferably

596 between about 50 base pairs to about 75 base pairs, and most preferably about 60 base pairs.

597 In another embodiment, the present disclosure provides a cloning vector comprising a

598 TAG sequence; a transcription termination signal, preferably a poly A-signal; a nucleotide

599 sequence useful to enable RNA synthesis, preferably a T7 promoter sequence; and a MA

600 sequence, wherein the nucleotide sequence useful to enable RNA synthesis, preferably a T7

601 promoter sequence, and the MA sequence are on the antisense DNA strand. In another

602 embodiment of the present disclosure, a cloning vector is provided wherein the cloning vector

603 is comprised of a DNA promoter sequence candidate, a TAG sequence, a transcription

604 termination signal, preferably a polyA signal; a nucleotide sequence useful to enable RNA

605 synthesis, preferably a T7 promoter sequence; and a MA sequence, wherein the DNA

606 promoter sequence candidate, the TAG sequence, and the transcription termination signal,

607 preferably a poly A-signal, are located on the sense DNA strand. 608 In another embodiment of the present disclosure, a cloning vector is provided wherein

609 the cloning vector is comprised of a TAG sequence, a transcription termination signal,

610 preferably a poly A-signal, a nucleotide sequence useful to enable RNA synthesis, preferably

611 a T7 promoter sequence, and a MA sequence, wherein the DNA promoter sequence candidate

612 is located 5' to the TAG sequence and wherein the TAG sequence is located 5' to the

613 transcription termination signal, preferably a poly A-signal In another embodiment of the

614 present disclosure, a cloning vector is provided wherein the cloning vector is comprised of a

615 TAG sequence, a transcription termination signal, preferably a poly A-signal; a nucleotide

616 sequence useful to enable RNA synthesis, preferably a T7 promoter sequence, and a MA

617 sequence, and the TAG sequence is located 3' to the DNA promoter sequence candidate and

618 the transcription termination signal, preferably a poly A-signal, is located 3 ' to the TAG

619 sequence

620 In another embodiment of the present disclosure, a cloning vector is provided wherein

621 the cloning vector is comprised of a TAG sequence, a transcription termination signal,

622 preferably a poly A-signal, a nucleotide sequence useful to enable RNA synthesis, preferably

623 a T7 promoter sequence, and a MA sequence, wherein the DNA promoter sequence is

624 operably linked to the TAG sequence In another embodiment of the present disclosure, a

625 cloning vector is provided wherein the cloning vector is comprised of a DNA promoter

626 sequence candidate, a TAG sequence, a transcription termination signal, preferably a poly A-

627 signal, a nucleotide sequence useful to enable RNA synthesis, preferably a T7 promoter

628 sequence, and a MA sequence, and the TAG sequence is operably linked to the transcription

629 termination signal, preferably a poly A-signal

630 In another embodiment of the present disclosure, a cloning vector is provided wherein

631 the cloning vector is comprised of a TAG sequence, a transcription termination signal,

632 preferably a poly A-signal, a nucleotide sequence useful to enable RNA synthesis, preferably

633 a T7 promoter sequence, and a MA sequence, wherein the DNA promoter sequence is located

634 5' to the TAG sequence, the TAG sequence is located 5' to the transcription termination

635 signal, preferably a poly A-signal, transcription termination signal is 3' to a DNA promoter

636 sequence candidate, and the DNA promoter sequence candidate is operably linked to the

637 TAG sequence and TAG sequence is operably linked to the transcription termination signal 638 In another embodiment of the present disclosure, a cloning vector is provided wherein

639 the cloning vector is comprised of a pair of MCS, a TAG sequence, a transcription

640 termination signal, preferably a poly A-signal, a nucleotide sequence useful to enable RNA

641 synthesis, preferably a T7 promoter sequence, and a MA sequence, and a MCS is located 5^' 642 of the DNA promoter sequence candidate and a MCS is located 3' of the DNA promoter

643 sequence candidate.

644 The present disclosure provides an array-based method for promoter detection and

645 analysis. The method provides for transcriptional products that are tagged as they are

646 synthesized, in such a way that one specific transcript is labeled with only one type of TAG,

647 and one TAG labels only one type of transcript. All promoter sequence candidates are

648 analyzed simultaneously in one reaction vial. The transcriptional output is analyzed on

649 conventional arrays and can be detected with procedures that do not require expensive

650 instrumentation. The method fulfills the need for reduction of labor, costs, and provides for

651 the detection of promoter regions from genomic libraries and other related advantages.

652 These and other embodiments of the present disclosure will become apparent upon

653 reference to the detailed description and illustrative examples which are intended to

654 exemplify non-limiting embodiments of the disclosure. All references disclosed herein are

655 hereby incorporated by reference in their entirety as if each was incorporated individually. 656

657 GLOSSARY

658 Unless defined otherwise, all technical and scientific terms used herein have the same

659 meaning as commonly understood by one of ordinary skill in the art to which this disclosure

660 belongs. Generally, the nomenclature used herein and the laboratory procedures in cell

661 culture, molecular genetics, and nucleic acid chemistry and hybridization described below are

662 those well known and commonly employed in the art. Standard techniques are used for

663 recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and

664 transformation (e.g., electroporation, lipofection). Generally, enzymatic reactions and

665 purification steps are performed according to the manufacturer's specifications. The

666 techniques and procedures are generally performed according to conventional methods in the

667 art and various general references (see generally, Sambrook et al. Molecular Cloning: A

668 Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

669 N. Y., which is incorporated herein by reference) which are provided throughout this

670 document. Units, prefixes, and symbols may be denoted in their SI accepted form. Unless

671 otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid

672 sequences are written left to right in amino to carboxyl orientation, respectively. Numeric

673 ranges are inclusive of the numbers defining the range and include each integer within the

674 defined range. Amino acids may be referred to herein by either their commonly known three

675 letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical 676 nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly

677 accepted single-letter codes. Unless otherwise provided for, software, electrical, and

678 electronics terms as used herein are as defined in The New IEEE Standard Dictionary of

679 Electrical and Electronics Terms (5.sup.th edition, 1993). As employed throughout the

680 disclosure, the following terms, unless otherwise indicated, shall be understood to have the

681 following meanings and are more fully defined by reference to the specification as a whole:

682 The term "amplified" refers to the construction of multiple copies of a nucleic acid

683 sequence or multiple copies complementary to the nucleic acid sequence using at least one of

684 the nucleic acid sequences as a template. Amplification systems include, for example, the

685 polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid

686 sequence based amplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicase

687 systems, transcription-based amplification system (TAS), and strand displacement

688 amplification (SDA) See, e.g., Diagnostic Molecular Microbiology: Principles and

689 Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington,

690 D.C. (1993). The product of amplification is termed an amplicon.

691 The term "array" refers to an array containing nucleic acid samples. An array may be

692 a "macroarray" or a "microarray." The term "microarray" refers to an array containing

693 nucleic acid samples, also referred to as microscopic DNA 'spots,' bound to solid substrates,

694 such as glass microscope slides, plastic, or silicon wafers. Because the physical area

695 occupied by each sample is usually 50-200 μm in diameter, nucleic acid samples representing

696 multiple samples, including, for example, entire genomes, genomic libraries, synthesized

697 DNA samples from computer predicted models, or in deletion mutants of promoters under

698 investigation etc., may be bound to the solid substrate. The solid substrate may include

699 membranes or beads. Macroarrays may be such as those available commercially (Clontech)

700 or synthesized manually. Beads may be of those used in peptide, nucleic acid and organic

701 moiety synthesis, including but not limited to, plastics, ceramics, glass, polystyrene,

702 methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium

703 dioxide, latex or cross-linked dextrans such as sepharose, cellulose, nylon, cross-linked

704 micelles and Teflon many all be used (see Microsphere Detection Guide, Bangs Laboratories,

705 Fishers Ind.). Microarrays allow the genes of a given sample to be simultaneously monitored

706 with respect to some experimental condition of interest. Microarrays may be fabricated by

707 the mechanical deposition of nucleic acid samples onto a solid substrate. Alternatively, the

708 nucleic acid samples may be manually deposited. The term "DNA microarray" may apply to 709 several different forms of the technology, each differing in the type of nucleic acid applied

710 and the method of application.

711 The term "assay marker" or a "reporter gene" refers to a gene that can be detected, or

712 'followed.' The expression of the reporter gene may be measured at either the RNA level, or

713 at the protein level. The gene product may be detected in experimental assay protocol, such

714 as marker enzymes, antigens, amino acid sequence markers, cellular phenotypic markers,

715 nucleic acid sequence markers, and the like. A "reporter gene" (or "reporter") is a gene that

716 researchers may attach to another gene of interest in cell culture, bacteria, animals, or plants.

717 Some reporters are selectable markers, or confer characteristics upon on organisms

718 expressing them allowing the organism to be easily identified and measured. To introduce a

719 reporter gene into an organism, researchers place the reporter gene and the gene of interest in

720 the same DNA construct to be inserted into the cell or organism. For bacteria or eukaryotic

721 cells in culture, this is usually in the form of a plasmid. Commonly used reporter genes may

722 include fluorescent proteins, luciferase, beta-galactosidase, and selectable markers, such as

723 chloramphenicol, and ccdB.

724 The term "cDNA" refers to DNA synthesized from a mature niRNA template. cDNA

725 is most often synthesized from mature mRNA using the enzyme reverse transcriptase. The

726 enzyme operates on a single strand of mRNA, generating its complementary DNA based on

727 the pairing of RNA base pairs (A, U, G, C) to their DNA complements (T, A, C, G). There

728 are several methods known for generating cDNA, for example, to obtain eukaryotic cDNA

729 whose introns have been spliced: a) an eukaryotic cell transcribes the DNA (from genes) into

730 RNA (pre-mRNA); b) the same cell processes the pre-mRNA strands by splicing out introns,

731 and adding a poly-A tail and 5' Methyl-Guanine cap; c) this mixture of mature mRNA

732 strands are extracted from the cell; d) a poly-T oligonucleotide primer is hybridized onto the

733 poly-A tail of the mature mRNA template. (Reverse transcriptase requires this double-

734 stranded segment as a primer to start its operation.); e) reverse transcriptase is added, along

735 with deoxynucleotide triphosphates (A, T, G, C); f) the reverse transcriptase scans the mature

736 mRNA and synthesizes a sequence of DNA that complements the mRNA template. This

737 strand of DNA is complementary DNA. (see also Current Protocols in Molecular Biology,

738 John Wiley & Sons).

739 The term "cloning host cell" refers to a host cell that contains a cloning vector.

740 The term "cloning vector" refers to a DNA molecule such as a plasmid, cosmid, or

741 bacterial phage, or virus, such as, for example retroviruses, adeno-associated adenoviruses,

742 lentivirus, baculoviruses and adenoviruses, that has the capability of replicating 743 autonomously in a host cell. Cloning vectors typically contain one or a small number of

744 restriction endomiclease recognition sites at which foreign DNA sequences can be inserted in

745 a determinable fashion without loss of essential biological function of the vector, as well as a

746 selectable marker gene that is suitable for use in the identification and selection of cells

747 transformed with the cloning vector. Selectable marker genes may include genes that provide

748 tetracycline resistance, ampicillin resistance, or other observable features, such as with the

749 ccdB gene.

750 The term "detectable marker" encompasses both the selectable markers and assay

751 markers. The term "selectable markers" refers to a variety of gene products to which cells

752 transformed with an expression construct can be selected or screened, including drug-

753 resistance markers, antigenic markers useful in fluorescence-activated cell sorting, adherence

754 markers such as receptors for adherence ligands allowing selective adherence, and the like.

755 When the nucleic acid is prepared or altered synthetically, advantage can be taken of known

756 codon preferences of the intended host where the nucleic acid is to be expressed.

757 The term "detectable response" refers to any signal or response that may be detected

758 in an assay, which may be performed with or without a detection reagent. Detectable

759 responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent,

760 ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence,

761 phosphorescence, transmission, reflection or resonance transfer. Detectable responses also

762 include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum,

763 ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray

764 diffraction. Alternatively, a detectable response may be the result of an assay to measure one

765 or more properties of a biologic material, such as melting point, density, conductivity, surface

766 acoustic waves, catalytic activity or elemental composition. A "detection reagent" is any

767 molecule that generates a detectable response indicative of the presence or absence of a

768 substance of interest. Detection reagents include any of a variety of molecules, such as

769 antibodies, nucleic acid sequences and enzymes. To facilitate detection, a detection reagent

770 may comprise a marker.

771 The term "DNA recombination sequences" refers to nucleic acid sequence that

772 provides for efficient transfer of DNA fragments across multiple systems and into multiple

773 vectors. Any DNA fragment flanked by a recombination site can be transferred into any

774 vector that has a corresponding site. Orientation and reading frame are maintained with

775 efficiencies (typically 99%), effectively eliminating the need for secondary sequencing or

776 subcloning after the initial entry clone is made. The transfer of DNA fragments makes use of 777 lambda phage-based site-specific recombination instead of restriction endonuclease and

778 ligase to insert a gene of interest into an expression vector. The DNA recombination

779 sequences, for example, attL, attR, attB, and attV, and enzyme mixtures, for example, LR and

780 BP Clonase, may be used to mediate the lambda recombination reactions. Transferring a gene

781 into a destination vector is accomplished in two steps: 1) clone the gene of interest into an

782 entry vector and 2) mix the entry clone containing the gene of interest in vitro with the

783 appropriate expression vector (destination vector) and enzyme mix. Site-specific

784 recombination between the att sites (attR x attL attB x att?) generates an expression clone

785 and a by-product. The expression clone contains the gene of interest recombined into the

786 destination vector backbone. Following transformation and selection in E. coli, the expression

787 clone is ready to be used for expression in the appropriate host. This lambda-based system is

788 also known as the Gateway® cloning system (Invitrogen Inc., Carlsbad, CA).

789 The term "electroporation" refers to a significant increase in the electrical

790 conductivity and permeability of the cell plasma membrane caused by an externally applied

791 electrical field. It is used as a way of introducing some substance into a cell, such as loading

792 it with a piece of coding DNA, a molecular probe, or a drug. Pores are formed when the

793 voltage across a plasma membrane exceeds its dielectric strength. If the strength of the

794 applied electrical field and/or duration of exposure to it are properly chosen, the pores formed

795 by the electrical pulse reseal after a short period of time, during which extracellular

796 compounds have a chance to enter into the cell. However, excessive exposure of live cells to

797 electrical fields can result in cell death. Electroporation is done with electroporators,

798 instruments which create the electric current and send it through the cell solution, typically

799 bacteria. The solution is pipetted into a glass or plastic cuvette which has two Al electrodes

800 on its sides. For example, for bacterial electroporation, a suspension of around 50 μl is

801 usually used. Prior to electroporation it is mixed with the plasmid to be transformed. The

802 mixture is pipetted into the cuvette, the voltage is set on the electroporator (2,400 volts is

803 often used) and the cuvette is inserted into the electroporator and an electric current is

804 applied. Immediately after electroporation 1 ml of liquid medium is added to the bacteria (in

805 the cuvette or in a microcentrifuge tube), and the tube is incubated at the bacteria's optimal

806 temperature for an hour or more and then it is spread on an agar plate (see Ausubel, Current

807 Protocols in Molecular Biology, Wiley).

808 The term "equimolar" refers to having an equal concentration of moles in one liter of

809 solution. 810 The term "expression system" refers to a genetic sequence which includes a protein

811 encoding region which is operably linked to all of the genetic signals necessary to achieve

812 expression of the protein encoding region. Traditionally, the expression system will include a

813 regulatory element such as a promoter or enhancer, to increase transcription and/or

814 translation of the protein encoding region, or to provide control over expression. The

815 regulatory element may be located upstream or downstream of the protein encoding region,

816 or may be located at an intron (non coding portion) interrupting the protein encoding region.

817 Alternatively it is also possible for the sequence of the protein encoding region itself to

818 comprise regulatory ability.

819 The term "expression vector" refers a DNA molecule comprising a gene that is

820 expressed in a host cell. Typically, gene expression is placed under the control of certain

821 regulatory elements including promoters, tissue specific regulatory elements, and enhancers.

822 Such a gene is said to be "operably linked to" the regulatory elements.

823 The term "functional splice acceptor" refers to any individual functional splice

824 acceptor or functional splice acceptor consensus sequence that permits the construct of the

825 disclosure to be processed such that it is included in any mature, biologically active mRNA,

826 provided that it is integrated in an active chromosomal locus and transcribed as a contiguous

827 part of the pre-messenger RNA of the chromosomal locus.

828 The term "homing endonucleases" refers to double stranded DNases that have large,

829 asymmetric recognition sites (12-40 base pairs) and coding sequences that are usually

830 embedded in either introns or inteins. Introns are spliced out of precursor RNAs, while

831 inteins are spliced out of precursor proteins. Homing endonucleases are named using

832 conventions similar to those of restriction endonucleases with intron-encoded endonucleases

833 containing the prefix, "I-" and intein endonucleases containing the prefix, "PI-". Homing

834 endonuclease recognition sites are extremely rare. For example, an 18 base pair recognition

835 sequence will occur only once in every 7 x 10¹⁰ base pairs of random sequence. This is

836 equivalent to only one site in 20 mammalian-sized genomes. However, unlike standard

837 restriction endonucleases, homing endonucleases tolerate some sequence degeneracy within

838 their recognition sequence. As a result, their observed sequence specificity is typically in the

839 range of 10-12 base pairs. Homing endonucleases do not have stringently-defined

840 recognition sequences in the way that restriction enzymes do. That is, single base changes do

841 not abolish cleavage but reduce its efficiency to variable extents. The precise boundary of

842 required bases is generally not known. 843 The term "host cell" encompasses any cell which contains a vector and preferably

844 supports the replication and/or expression of the vector. Host cells may be prokaryotic cells

845 such as Escherichia coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian

846 cells. The term as used herein means any cell which may be in culture or in vivo as part of a

847 unicellular organism, part of a multicellular organism, or a fused or engineered cell culture.

848 The term "hybridization" refers to the process of combining complementary, single-

849 stranded nucleic acids into a single molecule. Nucleotides will bind to their complement

850 under normal conditions, so two perfectly complementary strands will bind (or 'anneal') to

851 each other readily. However, due to the different molecular geometries of the nucleotides, a

852 single inconsistency between the two strands will make binding between them more

853 energetically unfavorable. Measuring the effects of base incompatibility by quantifying the

854 rate at which two strands anneal can provide information as to the similarity in base sequence

855 between the two strands being annealed.

856 The term "internal ribosome entry site" (IRES) refers to an element which permits

857 attachment of a downstream coding region or open reading frame with a cytoplasmic

858 polysomal ribosome for purposes of initiating translation thereof in the absence of any

859 internal promoters. An IRES is included to initiate translation of selectable marker protein

860 coding sequences. Examples of suitable IRESes that can be used include the mammalian

861 IRES of the immunoglobulin heavy-chain-binding protein (BiP). Other suitable IRESes are

862 those from the picomaviruses. For example, such IRESes include those from

863 encephalomyocarditis virus (preferably nucleotide numbers 163-746), poliovirus (preferably

864 nucleotide numbers 28-640) and foot and mouth disease virus (preferably nucleotide numbers

865 369-804). Thus, the IRES are located in the long 5' untranslated regions of the picomaviruses

866 which can be removed from their viral setting in length to unrelated genes to produce

867 polycistronic mRNAs.

868 The term "isolated" refers to material, such as a nucleic acid or a protein, which is: (1)

869 substantially or essentially free from components that normally accompany or interact with it

870 as found in its naturally occurring environment. The isolated material optionally comprises

871 material not found with the material in its natural environment; or (2) if the material is in its

872 natural environment, the material has been synthetically (non-naturally) altered by deliberate

873 human intervention to a composition and/or placed at a location in the cell (e.g., genome or

874 subcellular organelle) not native to a material found in that environment. The alteration to

875 yield the synthetic material can be performed on the material within or removed from its

876 natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic 877 acid if it is altered, or if it is transcribed from DNA which has been altered, by means of

878 human intervention performed within the cell from which it originates. See, e.g., Compounds

879 and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No.

880 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,

881 PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes

882 isolated if it is introduced by non-naturally occurring means to a locus of the genome not

883 native to that nucleic acid. Nucleic acids which are "isolated" as defined herein are also

884 referred to as "heterologous" nucleic acids.

885 The term "inserted" or "introduced" in the context of inserting a nucleic acid into a

886 cell, refers to "transfection" or "transformation" or "transduction" and includes reference to

887 the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid

888 may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or

889 mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g.,

890 transfected mRNA).

891 The terms "label" or "labeled" refers to incorporation of a detectable marker or

892 molecule, e.g., by incorporation of a radiolabeled nucleoside triphosphates or radioisotopes to

893 a nucleic acid that can be detected or measured. Various methods of labeling nucleic acids are

894 known in the art (see Short Protocols in Molecular Biology, 5^th Ed., John Wiley & Sons,

895 2002) and may be used. Examples of labels for nucleic acids include, but are not limited to,

896 the following: radioisotopes (e.g., ³²P-labeled NTPs and dNTPs; ³⁵S-labeled NTPs and

897 dNTPs; ³H' ¹⁴C; ¹²⁵I), fluorophores and fluorescent labels (e.g., FITC; rhodamine; lanthanide

898 phosphors; cyanine (Cy3, Cy5); fluorescein; coumarin, SYBR Green); and digoxygenin-1 1-

899 dUTP.

900 The term "MA segment", also referred to as a "MA sequence," refers to a nucleotide

901 sequence located downstream from the TAG and upstream of the transcription termination

902 signal in the TAG plasmids and their derivatives. All mRNAs synthesized from the various

903 promoters studied in a single experiment will contain the same MA sequence, to which a

904 complementary primer can anneal and initiate the synthesis of the first strand cDNA in order

905 to make hybridization probes. The MA sequence is usually 20 to 30 nucleotides in length,

906 but may be longer provided the MA sequence does not contain any secondary structure, such

907 as hairpin loops, which would prevent an efficient cDNA synthesis. The MA sequence is

908 composed of approximately 50% GC, such that the melting temperature ranges from about

909 7O⁰C to about 75⁰C. MA sequences are unique among all published nucleotide databases, so

910 that only the TAG-transcripts will serve as template for cDNA synthesis. MA sequences do 911 not contain any of the restriction sites that are used elsewhere in the TAG plasmids for

912 cloning purposes. It cannot function as (or does not contain) a transcriptional promoter or

913 transcription termination signal

914 The term "mixing" refers to combining, joining, uniting, associating, fusing, or

915 hgating at least two distinct nucleotide sequences such that they become one fragment.

916 The term "multiple cloning site," also referred to as an "MCS" or a "polylinker"

917 refers to a short segment of DNA which contains many (usually 20+) sites recognized by

918 restriction enzymes or other endonucleases such as homing endonucleases.

919 The term "nucleic acid" refers to a deoxyribomicleotide or ribonucleotide polymer in

920 either single- or double-stranded form, and unless otherwise limited, encompasses known

921 analogues having the essential nature of natural nucleotides in that they hybridize to single-

922 stranded nucleic acids in a manner similar to naturally occurring nucleotides (e g , peptide

923 nucleic acids)

924 The term "nucleotide" refers to a chemical compound that consists of a heterocyclic

925 base, a sugar, and one or more phosphate groups In the most common nucleotides the base

926 is a derivative of purine or pyrimidme, and the sugar is the pentose deoxyribose or πbose

927 Nucleotides are the monomers of nucleic acids, with three or more bonding together in order

928 to form a nucleic acid. Nucleotides are the structural units of RNA, DNA, and several

929 cofactors: CoA, FAD, DMN, NAD, and NADP. The purines include adenine (A), and

930 guanine (G); the pyπmidines include cytosine (C), thymine (T), and uracil (U).

931 The terms "oligoclonal", "polyclonal" applied to cell populations indicates a

932 population of cells where some cells within that population are not genetically identical to the

933 rest of the cells of that population Conversely, the term "monoclonal" or "monoclonal cell

934 population" indicates that all cells within that population are genetically identical.

935 Differences in the "genetic identity" of a population of cells in the context of this disclosure

936 arise by random retroviral integration into different genomic insertion sites.

937 The term "operably linked" refers to a functional linkage between a promoter and a

938 second sequence, wherein the promoter sequence initiates and mediates transcription of the

939 DNA sequence corresponding to the second sequence. Generally, operably linked means that

940 the nucleic acid sequences being linked are contiguous and, where necessary to join two

941 protein coding regions, contiguous and in the same reading frame.

942 The term "optical density^"' refers to the absorbance of an optical element for a given

943 wavelength per unit distance. Typically, bacterial cultures are measured at a wavelength of

944 600 nm. 945 The term "polymerase chain reaction" or "PCR" refers to a procedure described in

946 U.S. Pat. No. 4,683,195, the disclosure of which is incorporated herein by reference.

947 The term "polynucleotide" refers to a deoxyribopolynucleotide, ribopolymicleotide, or

948 analogs thereof that have the essential nature of a natural ribonucleotide in that they

949 hybridize, under stringent hybridization conditions, to substantially the same nucleotide

950 sequence as naturally occurring nucleotides and/or allow translation into the same amino

951 acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a

952 subsequence of a native or heterologous structural or regulatory gene. Unless otherwise

953 indicated, the term includes reference to the specified sequence as well as the complementary

954 sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other

955 reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs

956 comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to

957 name just two examples, are polynucleotides as the term is used herein. It will be appreciated

958 that a great variety of modifications have been made to DNA and RNA that serve many

959 useful purposes known to those of skill in the art. The term polynucleotide as it is employed

960 herein embraces such chemically, enzymatically or metabolically modified forms of

961 polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses

962 and cells, including among other things, simple and complex cells.

963 The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to

964 refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which

965 one or more amino acid residue is an artificial chemical analogue of a corresponding

966 naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The

967 essential nature of such analogues of naturally occurring amino acids is that, when

968 incorporated into a protein that protein is specifically reactive to antibodies elicited to the

969 same protein but consisting entirely of naturally occurring amino acids. The terms

970 "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not

971 limited to, glycosylation, lipid attachment, sulfation, gamma. -carboxylation of glutamic acid

972 residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as

973 noted above, that polypeptides are not entirely linear. For instance, polypeptides may be

974 branched as a result of ubiquitination, and they may be circular, with or without branching,

975 generally as a result of posttranslational events, including natural processing event and events

976 brought about by human manipulation which do not occur naturally. Circular, branched and

977 branched circular polypeptides may be synthesized by non-translation natural process and by

978 entirely synthetic methods, as well. 979 The term "primer" refers to a nucleic acid which, when hybridized to a strand of

980 DNA, is capable of initiating the synthesis of an extension product in the presence of a

981 suitable polymerization agent. The primer preferably is sufficiently long to hybridize

982 uniquely to a specific region of the DNA strand. A primer may also be used on RNA, for

983 example, to synthesize the first strand of cDNA.

984 The term "promoter" refers to a region of DNA upstream, downstream, or distal, from

985 the start of transcription and involved in recognition and binding of RNA polymerase and

986 other proteins to initiate transcription. For example, T7, T3 and Sp6 are RNA polymerase

987 promoter sequences. In RNA synthesis, promoters are a means to demarcate which genes

988 should be used for messenger RNA creation and by extension, control which proteins the cell

989 manufactures. Promoters represent critical elements that can work in concert with other

990 regulatory regions (enhancers, silencers, boundary elements/insulators) to direct the level of

991 transcription of a given gene.

992 The term "promoter sequence candidate" refers to a nucleotide sequence that contains

993 a putative promoter sequence. A promoter sequence candidate may be provided by a

994 computer-predicted model, DNA fragments from a collection of nucleotide sequences, such

995 as a genomic library, deletion or site-directed mutants of a specific promoter, tissue-specific

996 promoters, artificial promoters, etc.

997 The term "promoterless" refers to a protein coding sequence contained in a vector,

998 retrovirus, adenovirus, adeno-associated virus or retroviral provirus that is not directly or

999 significantly under the control of a promoter within the vector, whether it be in RNA or DNA

1000 form. The vector, plasmid, viral or otherwise, may contain a promoter, but that promoter

1001 cannot be positioned or configured such that it directly or significantly regulates the

1002 expression of the promoterless protein coding sequence.

1003 The term "protein coding sequence" refers a nucleotide sequence encoding a

1004 polypeptide gene which can be used to distinguish cells expressing the polypeptide gene from

1005 those not expressing the polypeptide gene. Protein coding sequences include those commonly

1006 referred to as selectable markers. Examples of protein coding sequences include those coding

1007 a cell surface antigen and those encoding enzymes. A representative list of protein coding

1008 sequences include thymidine kinase, beta.-galactosidase, tryptophan synthetase, neomycin

1009 phosphotransferase, histidinol dehydrogenase, luciferase, chloramphenicol acetyltransferase,

1010 dihydrofolate reductase (DHFR); hypoxanthine guanine phosphoribosyl transferase

1011 (HGPRT), CD4, CD8 and hygromycin phosphotransferase (HYGRO). 1012 The term "recombinant" refers to a cell or vector that has been modified by the

1013 introduction of a heterologous nucleic acid or the cell that is derived from a cell so modified.

1014 Thus, for example, recombinant cells express genes that are not found in identical form

1015 within the native (non-recombinant) form of the cell or express native genes that are

1016 otherwise abnormally expressed, under-expressed or not expressed at all as a result of

1017 deliberate human intervention. The term "recombinant" as used herein does not encompass

1018 the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation,

1019 natural transformation transduction/transposition) such as those occurring without deliberate

1020 human intervention.

1021 The term "recombinant expression cassette" refers to a nucleic acid construct,

1022 generated recombinantly or synthetically, with a series of specified nucleic acid elements

1023 which permit transcription of a particular nucleic acid in a host cell. The recombinant

1024 expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA,

1025 virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an

1026 expression vector includes, among other sequences, a nucleic acid to be transcribed, a

1027 promoter, and a transcription termination signal such as a poly-A signal.

1028 The term "recombinant host" refers to any prokaryotic or eukaryotic cell that contains

1029 either a cloning vector or an expression vector. This term also includes those prokaryotic or

1030 eukaryotic cells that have been genetically engineered to contain the cloned genes, or gene of

1031 interest, in the chromosome or genome of the host cell.

1032 The term "regulatory sequence" (also called regulatory region or regulatory element)

1033 refers to a promoter, enhancer or other segment of DNA where regulatory proteins such as

1034 transcription factors bind preferentially. They control gene expression and thus protein

1035 expression.

1036 The term "reporter cell line" refers to prokaryotic or eukaryotic cells that contain a

1037 reporter or as say marker.

1038 The term "restriction digestion'^" refers to a procedure used to prepare DNA for

1039 analysis or other processing. Also known as DNA fragmentation, it uses a restriction enzyme

1040 to selectively cleave strands of DNA into shorter segments.

1041 The term "restriction enzyme^"' (or restriction endonuclease) refers to an enzyme that

1042 cuts double-stranded DNA. The enzyme makes two incisions, one through each of the

1043 phosphate backbones of the double helix without damaging the bases. Restriction enzymes

1044 are classified biochemically into four types, designated Type 1, Type II, Type III, and Type

1045 IV. In Type I and Type III systems, both the methylase and restriction activities are carried 1046 out by a single large enzyme complex Although these enzymes recognize specific DNA

1047 sequences, the sites of actual cleavage are at variable distances from these recognition sites,

1048 and can be hundreds of bases away Both require ATP for their proper function In Type II

1049 systems, the restriction enzyme is independent of its methylase, and cleavage occurs at very

1050 specific sites that are within or close to the recognition sequence Type II enzymes are

1051 further classified according to their recognition site Most Type II enzymes cut palindromic

1052 DNA sequences, while Type Ha enzymes recognize non-palmdromic sequences and cleavage

1053 outside of the recognition site. Type lib enzymes cut sequences twice at both sites outside of

1054 the recognition sequence In Type IV systems, the restriction enzymes target only methylated

1055 DNA

1056 The term "restriction sites" or "restriction recognition sites" refer to particular

1057 sequences of nucleotides that are recognized by restriction enzymes as sites to cut the DNA

1058 molecule The sites are generally, but not necessarily, palindromic, (because restriction

1059 enzymes usually bind as homodimers) and a particular enzyme may cut between two

1060 nucleotides withm its recognition site, or somewhere nearby

1061 The term "reverse transcription^"' or "reverse transcription polymerase chain reaction"

1062 (RT-PCR) refers to amplifying a defined piece of a ribonucleic acid (RNA) molecule The

1063 RNA strand is first reverse transcribed into its DNA complement or complementary DNA,

1064 followed by amplification of the resulting DNA using polymerase chain reaction

1065 The term "selectable marker" refers to a gene introduced into a cell, especially a

1066 bacterium or to cells in culture that confers a trait suitable for artificial selection. They are a

1067 type of reporter gene used in laboratory microbiology, molecular biology, and genetic

1068 engineering to indicate the success of a transfection or other procedure meant to introduce

1069 foreign DNA into a cell For example, analysis of gene function frequently requires the

1070 formation of cells that contain the studied gene m a stably integrated form In some

1071 situations, few cells may stably integrate DNA thus a dominant selectable marker is used to

1072 permit isolation of stable transfectants Selectable markers may include antibiotics

1073 (ampicillin) and 'suicide' genes (for example ccdB) Positive selective markers may utilize

1074 adenosine deaminase (thymidine, hypoxanthme, 9-β-D-xylofuranosyl adenine, T-

1075 deoxycoformycin), aminoglycoside phosphotransferase (neomycin, G418, gentamycm,

1076 kanamycin), Bleomycin (bleomycin, phleomycm, zeocin), cytosine deaminase (N-

1077 (phosphonacetyl) L aspartate, inosine, cytosine); dehydrofolate reductase (methotrexate,

1078 aminoptenn); histidmol dehydrogenase (histmdol); hygromycm-B-phosphotransferase

1079 (hygromycm-B), puromycin-Ν-acetyl transferase (puromycm), thymidine kinase 1080 (hypoxanthine, aminopterin, thymidine, glycine); and xanthine-guanine

1081 phosphorriobsyltransferase (xanthine, hypoxanthine, thymidine, aminopterin, mycophenolic

1082 acid, L-glutamine). Negative selectable markers may utilize: cytosine deaminase (5-

1083 fluorocytosine); diptheria toxin; ccdB, and HSV-TK.

1084 The term "selectively hybridizes" refers to hybridization, under stringent

1085 hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target

1086 sequence to a detectably greater degree (e.g., at least 2-fold over background) than its

1087 hybridization to non-target nucleic acid sequences and to the substantial exclusion of non- 1088 target nucleic acids. Selectively hybridizing sequences typically have about at least 80%

1089 sequence identity, preferably 90% sequence identity, and most preferably 100% sequence

1090 identity (i.e., complementary) with each other.

1091 The term "sense" refers to the general concept used to compare the polarity of nucleic

1092 acid molecules to other nucleic acid molecules. Generally, a DNA sequence is called "sense"

1093 if its sequence is the same as that of a messenger R]SfA copy that is translated into protein.

1094 The sequence on the opposite strand is complementary to the sense sequence and is therefore

1095 called the "antisense" sequence.

1096 The term "TAG" refers to a DNA sequence composed of random nucleotides, in

1097 which each position has an equal probability of having any of the four deoxynucleotides (A,

1098 C, T, and G). Other bases, such as inosine, uracil, 5-methylcytosine, 8-azaguanine, 2,6-

1099 diaminopurine, 5 bromouracil, and other derivatives may be incorporated in their nucleotide

1100 form into the sequences. The length of the TAG sequence is short, preferably between about

1101 16 bp to about 200 bp, more preferably between about 20 to about 150 bp, more preferably

1102 between about 30 to about 120 bp, more preferably between about 40 to about 100 bp, more

1103 preferably between about 50 to about 75 bp, and most preferably about 60 bp. The sequences

1104 are preferably different or distinct enough to avoid annealing to each other at times when the

1105 oligonucleotide is present as a single strand. In addition, the sequence should not be self-

1106 complementary, so as to avoid the formation of primer-dimers during amplification. Within a

1107 plurality of TAG sequences, each TAG sequence will have approximately equivalent

1108 amounts of the nucleotides A, T, G, and C such that each TAG sequence has approximately

1109 the same melting temperature as the other TAGs. A same melting temperature will allow for

1110 the unbiased quantification of various mRNAs containing each a different TAG sequence by

1111 hybridization under the same temperature and ionic strength conditions. Within a plurality of

1112 TAG sequences, the nucleotide sequence of each individual TAG sequence is unique to the

1113 individual TAG of the plurality. 1114 The term "transcription termination signal" refers to a section of genetic sequence that

1115 marks the end of gene or operon on genomic DNA for transcription. In prokaryotes, two

1116 classes of transcription termination signals are known 1) intrinsic transcription termination

1117 signals where a hairpin structure forms within the nascent transcript that disrupts the mRNA-

1118 DNA-RNA polymerase ternary complex; and 2) Rho-dependent transcription termination

1119 signal that require Rho factor, an RNA helicase protein complex to disrupt the nascent

1120 mRNA-DNA-RNA polymerase ternary complex. In eukaryotes, transcription termination

1121 signals are recognized by protein factors that co-transcπptionally cleave the nascent RNA at a

1122 polyadenlyation signal (i e, "poly-A signal" or "poly-A tail") halting further elongation of the

1123 transcript by RNA polymerase. The subsequent addition of the poly-A tail at this site

1124 stabilizes the mRNA and allows it to be exported outside the nucleus. Termination sequences

1125 are distinct from termination codons that occur in the mRNA and are the stopping signal for

1126 translation, which may also be called nonsense codons.

1127 The term "translational stop sequence" refers to a sequence which codes for the

1128 translational stop codons In some embodiments, the translational stop sequence may be in

1129 one, two, or three reading frames

1130 The term "transfection" refers to the introduction of foreign DNA into eukaryotic or

1131 prokaryotic cells. Transfection typically involves opening transient holes in cells to allow the

1132 entry of extracellular molecules, typically supercoiled plasmid DNA, but also siRNA, among

1133 others. There are various methods of transfectmg cells. One method is by calcium

1134 phosphate. HEPES-buffered saline solution containing phosphate ions is combined with a

1135 calcium chloride solution containing the DNA to be transfected. When the two are

1136 combined, a fine precipitate of calcium phosphate will form, binding the DNA to be

1137 transfected on its surface The suspension of the precipitate is then added to the cells to be

1138 transfected. The cells take up precipitate and the DNA. Alternatively, MgCi₂ or RbCl can be

1139 used. Other methods of transfection include electroporation, heat shock, proprietary

1140 transfection agents, dendrimers, and the use of liposomes. Liposomes are small, membrane-

1141 bounded bodies that fuse to the cell membrane releasing DNA into the cell. For eukaryotic

1142 cells, lipid-cation based transfection is typically used. Other methods of transfection include

1143 use of the gene gun and viruses. For stable transfection another gene is co-trans fected, which

1144 gives the cell some selection advantage, such as resistance towards a certain toxin. If the

1145 toxin, towards which the co-transfected gene offers resistance, is then added to the cell

1146 culture, only those cells with the foreign genes inserted into their genome will be able to

1147 proliferate, while other cells will die. After applying this selection pressure for some time, 1148 only the cells with a stable transfection remain and can be cultivated further. A common

1149 agent for stable transfection is Geneticin, also known as G418, which is a toxin that can be

1150 neutralized by the product of the neomycin resistant gene (see Bacchetti and Graham.

1151 Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by

1152 purified herpes simplex viral DNA. 1977. Proc. Natl. Acad. Sci. USA 74(4): 1590-94).

1153 Conventional transient transfection assays may incorporate internal controls, such as pRL-

1154 SV40 (Promega, Inc.) and may be used in combination with any experimental reporter vector

1155 to co-transfect mammalian cells.

1156 The term "transformation" refers to the genetic alteration of a cell resulting from the

1157 introduction, uptake, and expression of foreign genetic material (DNA or RNA). In bacteria,

1158 transformation refers to a genetic change brought about by taking up and expressing DNA,

1159 and "competence" refers to a state of being able to take up DNA. Competent cells may be

1160 generated by a laboratory procedure in which cells are passively made permeable to DNA,

1161 using conditions that do not normally occur in nature, thus cells that have been manipulated

1162 to accept foreign DNA are called "competent cells". These procedures are comparatively

1163 easy and simple, and can be used to genetically engineer bacteria. These procedures may

1164 include chilling cells in the presence of divalent cations, such as CaCi₂, which prepares the

1165 cell walls to become permeable to plasmid DNA. Cells are incubated with the DNA and then

1166 briefly heat shocked (e.g., 42⁰C for 30-120 seconds), which causes the DNA to enter the cell.

1167 This method works well for circular plasmid DNAs. Electroporation is another way to allow

1168 DNA to enter cells and involves briefly shocking cells with an electric field of 100-200 V.

1169 Plasmid DNA enters cells via the holes created in the cell membrane by the electric shock;

1170 natural membrane-repair mechanisms close these holes afterwards. Yeasts may be

1171 transformed, for example, by High Efficiency Transformation (see Gietz, R. D., and R. A.

1172 Woods . 2002 Transformation of Yeast by the Liac/SS Carrier DN A/PEG Method. Methods in

1173 Enzymology 350:87-96); the Two-hybrid System Protocol (see Gietz, R.D., B. Triggs-Raine,

1174 A. Robbins, K.C. Graham, and R.A. Woods. 1997 Identification of proteins that interact with

1175 a protein of interest: Applications of the yeast two-hybrid system. MoI Cell Biochem 172:67-

1176 79); and the Rapid Transformation Protocol (see Gietz, R. D₁, and R. A. Woods. 2002

1177 Transformation of Yeast by the Liac/SS Carrier DNA/PEG Method. Methods in Enzymology

1178 350:87-96).

1179 The term "vector" refers to a nucleic acid used in transfection of a host cell and into

1180 which can be inserted a polynucleotide. Vectors are frequently replicons. Expression vectors

1181 permit transcription of a nucleic acid inserted therein. Some common vectors include 1182 plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. The

1183 term "vector" may also refer to an element which aids in the transfer of a gene from one

1184 location to another. Vectors may include expression vectors and cloning vectors.

1185 The following terms are used to describe the sequence relationships between two or

1186 more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window",

1187 (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".

1188 The term "reference sequence" refers to a sequence used as a basis for sequence comparison.

1189 A reference sequence may be a subset or the entirety of a specified sequence; for example, as

1190 a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

1191 The term "comparison window" refers to a contiguous and specified segment of a

1192 polynucleotide sequence, wherein the polynucleotide sequence may be compared to a

1193 reference sequence and wherein the portion of the polynucleotide sequence in the comparison

1194 window may comprise additions or deletions (i.e., gaps) compared to the reference sequence

1195 (which does not comprise additions or deletions) for optimal alignment of the two sequences.

1196 Generally, the comparison window is at least 20 contiguous nucleotides in length, and

1197 optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid

1198 a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide

1199 sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

1200 All TAGs should lack homology to other TAGs used within the same assay.

1201 Dependent upon the method the probe is made, the homology of the TAG with known

1202 nucleic acid sequences may be acceptable. For example, if the probe is made by labeling

1203 mRNA directly, for example with polyA polymerase (see, for example, Aviv and Leder, Proc

1204 Natl Acad Sci U S A. 1972 Jun;69(6): 1408-12), the TAG-containing mRNAs, the

1205 endogenous mRNAs and possibly the tRNA, and rRNA may be labeled as well.

1206 Hybridization by these latter RNAs may interfere with detection by the probe. The TAGs

1207 should not have homology with any known sequence that is transcribed into RNA, including

1208 mRNA, tRNA, rRNA, etc. If the probe is made by labeling the first-strand cDNA, there are

1209 two possibilities: 1) if oligo(dT) is used as a primer, all first strand cDNA synthesized from

1210 mRNAs will be labeled, including the TAG-containing mRNAs and the endogenous mRNAs.

1211 These latter cDNAs may interfere with detection by the probe, thus the TAGs should not

1212 have homology with any known sequence that is transcribed into RNA; and 2) if

1213 oligo(dT)+anchor is used as a primer "B" (where the anchor would be a short stretch of

1214 nucleotides corresponding to the 3' end of the mRNA, immediately preceding the poly A)

1215 only cDNAs synthesized from mRNAs terminated by the same or similar transcription 1216 termination signal as the one used for the TAG constructs will be labeled. Thus if a particular

1217 kind of endogenous mRNA is recognized by the oligo(dT)-anchor primer, that specific

1218 mRNA would interfere with detection by the probe, therefore the TAG should not share

1219 homology with that specific mRNA. If the probe is made by PCR, in addition to the

1220 homology considerations discussed above with regard to the synthesis of the first strand

1221 cDNA, there are two additional considerations. First, linear amplification of the first strand

1222 cDNA is made using a primer (A) corresponding to a region common to all the TAG-mRNAs

1223 that is located 5 ' to the TAG. This situation may arise when the vector (plasmid or viral

1224 DNA), from which the probe may be made from, is removed and the primer B used for the

1225 first strand cDNA synthesis is removed as well. Accordingly, if the first strand cDNA was

1226 synthesized using oligo(dT) as the primer, then the TAGs may not have homology with any

1227 known sequence that is transcribed into mRNA, and that shares sequence identity with primer

1228 A, and if the first strand cDNA was synthesized using oligo(dT)-anchor as the primer, then

1229 the TAGs may not have homology with any known sequence that is transcribed into mRNA

1230 that shares sequence identity with both the 3 ' end as the TAG-mRNA and primer A Second,

1231 exponential amplification of the first strand cDNA using primer (A) and the oligo(dT)-based

1232 primer occurs In this situation, the antisense strand may be used as a probe and the printing

1233 of the assay membrane with the sense-strand oligonucleotides so that the vector does not have

1234 to be removed, as discussed above. Thus, at times, one can use TAGs with sequences that are

1235 found elsewhere in databases A specific TAG should not share sequence homology with any

1236 other TAG used simultaneously in the same assay and with any DNA or RNA molecule that

1237 will be labeled during the synthesis of the probe, regardless of the method used to synthesize

1238 the probe.

1239 Methods of alignment of sequences for comparison are well-known in the art.

1240 Optimal alignment of sequences for comparison may be conducted by the local homology

1241 algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology

1242 alignment algorithm of Needleman and Wunsch, J. MoI. Biol. 48:443 (1970); by the search

1243 for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); by

1244 computerized implementations of these algorithms, including, but not limited to: CLUSTAL

1245 in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST,

1246 FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer

1247 Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well

1248 described by Higgins and Sharp, Gene 73 :237-244 (1988); Higgins and Sharp, CABIOS

1249 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., 1250 Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in

1251 Molecular Biology 24:307-331 (1994). The BLAST family of programs which can be used

1252 for database similarity searches includes BLASTN for nucleotide query sequences against

1253 nucleotide database sequences; BLASTX for nucleotide query sequences against protein

1254 database sequences, BLASTP for protein query sequences against protein database

1255 sequences, TBLASTN for protein query sequences against nucleotide database sequences,

1256 and TBLASTX for nucleotide query sequences against nucleotide database sequences. See,

1257 Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing

1258 and Wiley-Interscience, New York (1995)

1259 Unless otherwise stated, sequence identity/similarity values provided herein refer to

1260 the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul

1261 et al , Nucleic Acids Res 25 3389-3402 (1997) Software for performing BLAST analyses is

1262 publicly available, e.g., through the National Center for Biotechnology-Information

1263 (http:/'www.hcbi nlm nih gov/) This algorithm involves first identifying high scoring

1264 sequence pairs (HSPs) by identifying short words of length W in the query sequence, which

1265 either match or satisfy some positive-valued threshold score T when aligned with a word of

1266 the same length in a database sequence T is referred to as the neighborhood word score

1267 threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for

1268 initiating searches to find longer HSPs containing them. The word hits are then extended in

1269 both directions along each sequence for as far as the cumulative alignment score can be

1270 increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters

1271 M (reward score for a pair of matching residues; always>0) and N (penalty score for

1272 mismatching residues; alwaysO) For amino acid sequences, a scoring matrix is used to

1273 calculate the cumulative score Extension of the word hits in each direction are halted when.

1274 the cumulative alignment score falls off by the quantity X from its maximum achieved value,

1275 the cumulative score goes to zero or below, due to the accumulation of one or more negative-

1276 scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm

1277 parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN

1278 program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation

1279 (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid

1280 sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E)

1281 of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henrkoff (1989) Proc Natl.

1282 Acad ScL USA 89:10915). 1283 In addition to calculating percent sequence identity, the BLAST algorithm also

1284 performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &

1285 Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity

1286 provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an

1287 indication of the probability by which a match between two nucleotide or amino acid

1288 sequences would occur by chance. BLAST searches assume that proteins can be modeled as

1289 random sequences. However, many real proteins comprise regions of nonrandom sequences

1290 which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more

1291 amino acids. Such low-complexity regions may be aligned between unrelated proteins even

1292 though other regions of the protein are entirely dissimilar. A number of low-complexity filter

1293 programs can be employed to reduce such low-complexity alignments. For example, the SEG

1294 (Wooten and Federhen, Comput. Chem, 17: 149-163 (1993)) and XNU (Claverie and States,

1295 Comput. Chem., 17: 191-201 (1993)) low-complexity filters can be employed alone or in

1296 combination. As used herein, "sequence identity" or "identity" in the context of two nucleic

1297 acid or polypeptide sequences refers to the residues in the two sequences which are the same

1298 when aligned for maximum correspondence over a specified comparison window. When

1299 percentage of sequence identity is used in reference to proteins it is recognized that residue

1300 positions which are not identical often differ by conservative amino acid substitutions, where

1301 amino acid residues are substituted for other amino acid residues with similar chemical

1302 properties (e.g. charge or hydrophobicity) and therefore do not change the functional

1303 properties of the molecule. Where sequences differ in conservative substitutions, the percent

1304 sequence identity may be adjusted upwards to correct for the conservative nature of the

1305 substitution. Sequences which differ by such conservative substitutions are said to have

1306 "sequence similarity" or "similarity". Means for making this adjustment are well-known to

1307 those of skill in the art. Typically this involves scoring a conservative substitution as a partial

1308 rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for

1309 example, where an identical amino acid is given a score of 1 and a non-conservative

1310 substitution is given a score of zero, a conservative substitution is given a score between zero

1311 and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm

1312 of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in

1313 the program PC/GENE (Intelligenetics, Mountain View, Calif, USA).

1314 As used herein, "percentage of sequence identity" means the value determined by

1315 comparing two optimally aligned sequences over a comparison window, wherein the portion

1316 of the polynucleotide sequence in the comparison window may comprise additions or 1317 deletions (i.e., gaps) as compared to the reference sequence (which does not comprise

1318 additions or deletions) for optimal alignment of the two sequences. The percentage is

1319 calculated by determining the number of positions at which the identical nucleic acid base or

1320 amino acid residue occurs in both sequences to yield the number of matched positions,

1321 dividing the number of matched positions by the total number of positions in the window of

1322 comparison and multiplying the result by 100 to yield the percentage of sequence identity.

1323 The term "substantial identity" of polynucleotide sequences means that a polynucleotide

1324 comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more

1325 preferably at least 90% and most preferably at least 95%, compared to a reference sequence

1326 using one of the alignment programs described using standard parameters. One of skill will

1327 recognize that these values can be appropriately adjusted to determine corresponding identity

1328 of proteins encoded by two nucleotide sequences by taking into account codon degeneracy,

1329 amino acid similarity, reading frame positioning and the like. Substantial identity of amino

1330 acid sequences for these purposes normally means sequence identity of at least 60%, or

1331 preferably at least 70%, 80%, 90%, and most preferably at least 95%. Another indication that

1332 nucleotide sequences are substantially identical is if two molecules hybridize to each other

1333 under stringent conditions. However, nucleic acids which do not hybridize to each other

1334 under stringent conditions are still substantially identical if the polypeptides which they

1335 encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is

1336 created using the maximum codon degeneracy permitted by the genetic code. One indication

1337 that two nucleic acid sequences are substantially identical is that the polypeptide which the

1338 first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by

1339 the second nucleic acid. The terms "substantial Identity" in the context of a peptide indicates

1340 that a peptide comprises a sequence with at least 70% sequence identity to a reference

1341 sequence, preferably 80%, ore preferably 85%, most preferably at least 90% or 95% sequence

1342 identity to the reference sequence over a specified comparison window. Optionally, optimal

1343 alignment is conducted using the homology alignment algorithm of Needleman and Wunsch,

1344 J. MoI. Biol. 48:443 (1970). An indication that two peptide sequences are substantially

1345 identical is that one peptide is immunologically reactive with antibodies raised against the

1346 second peptide. Thus, a peptide is substantially identical to a second peptide, for example,

1347 where the two peptides differ only by a conservative substitution. Peptides which are

1348 "substantially similar" share sequences as noted above except that residue positions which are

1349 not identical may differ by conservative amino acid changes. 1350 Methods of extraction of RNA are well-known in the art and are described, for

1351 example, in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" (Cold Spring

1352 Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989), vol. 1, ch. 7, "Extraction,

1353 Purification, and Analysis of Messenger RNA from Eukaryotic Cells," incorporated herein by

1354 this reference. Other isolation and extraction methods are also well-known, for example in F.

1355 Ausubel et al., "Current Protocols in Molecular Biology, John Wiley & Sons). Typically,

1356 isolation is performed in the presence of chaotropic agents such as guanidinium chloride or

1357 guanidinium thiocyanate, although other detergents and extraction agents can alternatively be

1358 used. Typically, the mRNA is isolated from the total extracted RNA by chromatography over

1359 oligo(dT)-cellulose or other chromatographic media that have the capacity to bind the

1360 polyadenylated 3'-portion of mRNA molecules. Alternatively, but less preferably, total RNA

1361 can be used. However, it is generally preferred to isolate poly(A)+RNA.

1362 The method employs several basic steps to achieve its objective. First, a library of

1363 DNA TAGs is designed. The DNA TAG sequences are composed of random nucleotides.

1364 Each DNA TAG sequence, in one embodiment of approximately 60 bp in length, is unique

1365 among a plurality of TAG sequences, i.e. a specific TAG does not share sequence homology

1366 with any other TAG used simultaneously in the same assay and with any DNA or RNA

1367 molecule that will be labeled during the synthesis of the probe, regardless of the method used

1368 to synthesize the probe. The TAG sequences have similar physical properties so that a

1369 plurality of the TAG sequences can be used for hybridization under similar conditions.

1370 Second, pTAG-basic plasmids are constructed. Third, the TAG sequences are inserted into

1371 the pTAG-basic plasmids. Fourth, promoter array membranes are prepared. Fifth, promoter

1372 sequence candidates are inserted into the pTAG plasmids. Sixth, the pTAG plasmids with the

1373 promoter sequence candidate inserts are transfected into host cells, and the RNA extracted.

1374 The RNA or the resultant cDNA derived from the extracted RNA is then labeled, hybridized

1375 to the promoter array membrane, and analysis performed. Thus, the present disclosure

1376 discloses an array-based method for promoter detection and analysis. The method provides

1377 for transcriptional products that are tagged as they are synthesized, in such a way that one

1378 specific transcript is labeled with only one type of TAG, and one TAG labels only one type of

1379 transcript. All promoter sequence candidates are analyzed simultaneously in one reaction

1380 vial. The transcriptional output is analyzed on conventional arrays.

1381

1382 BRIEF DESCRIPTION OF THE DRAWINGS

1383 1384 FIGURE 1. Flow diagram of array-based promoter detection and analysis.

1385 FIGURE 2. BrightStar-Plus membranes spotted manually (left) or using a robot (right) with a

1386 collection of reverse-strand TAG oligonucleotides.

1387 FIGURES 3A and 3B. Comparative analysis of the activity of 42 promoters in a single

1388 population of HEK 293 cells. The 42 promoter-TAG plasmids and 8 promoter-less TAG-

1389 reporter plasmids were mixed in equimolar amounts and transfected into the same cell

1390 population. Total RNA was extracted 14 hours after transfection. RNA was labeled using

1391 the linear amplification method, and biotin-labeled probes were hybridized on the TAG-

1392 spotted membranes (Fig 3A). Hybridization was revealed by chemiluminescence, and

1393 quantified by densitometry (Fig 3B). The macro array membrane was made by spotting

1394 manually each oligonucleotide as a diagonal doublet.

1395 FIGURES 4A and 4B. Comparison of the transcriptional activities of 92 promoters in a single

1396 cell population. The 92 promoter-TAG plasmids and 8 promoter-less TAG-reporter plasmids

1397 were mixed in equimolar amounts and transfected into the same cell population. Total RNA

1398 was extracted 14 hours after transfection. RNA was labeled using the linear amplification

1399 method, and biotin-labeled probes were hybridized on the TAG-spotted membranes (Fig.

1400 4A). Hybridization was revealed by chemiluminescence, and quantified by densitometry

1401 (plain bars) (Fig. 4B). The relative luciferase activities obtained with each plasmid construct

1402 were obtained from previously published work and are shown at the bottom (empty bars)

1403 (Fig. 4B). The numbers at the bottom of the figure refer to the list of promoters described in

1404 Table 1. The luciferase data obtained with the various OM promoters (# 59-73), defensin

1405 promoters (# 74-85), and other promoters studied by Coleman (Coleman, S., et al.

1406 Experimental analysis of the annotation of promoters in the public database. Hum. MoL

1407 Genet., 2002. 11(16): 1817-1821) were generated in different experimental conditions and

1408 should not be compared between each other. The macroarray membrane was made by

1409 spotting each oligonucleotide as a quadruplet, using a Biorobotics MicroGrid array spotting

1410 robot (Genomic Solutions, Ann Arbor, MI) at the microarray facility of the University of

1411 Idaho Environmental Biotechnology Institute (Moscow, ID).

1412 FIGURES 5A and 5B. Validation of the Promoter Detective method with a set of 35

1413 promoter-TAG plasmids. The autoradiogram (Fig. 5A) was obtained by hybridizing

1414 radioactive TAG-cDNA probes to a membrane spotted with the complementary TAG strands.

1415 The identity of the spots is indicated by numbers on the left side of the autoradiogram, and on 1416 the bottom of the bar chart (Fig. 5B). The bar chart summarizes the intensities of the various

1417 spots, relative to the signal obtained with the CMV promoter (= 100).

1418 FIGURE 6. Flow diagram for the construction of the pTAG reporter plasmid.

1419 FIGURE 7. Plasmid map of the pT AG basic vector.

1420 TABLE 1. List of 100 promoter sequences used within the examples. Each promoter is

1421 described with its symbol, length, and Refseq or GenBank accession number. The TAG

1422 identification number to which it is associated is also indicated. 1423

1424 DETAILED DESCRIPTION

1425 The present disclosure provides a method for the detection and analysis of DNA

1426 promoter sequences. Figure 1 provides a general flow chart. The disclosure provides for the

1427 construction of a vector library containing potential DNA promoter sequence candidates that

1428 may be present, for example, in a collection of nucleotide sequences, such as a genomic

1429 library, in computer-predicted promoter regions, or in deletion mutants of promoters under

1430 investigation, etc. Each clone generated potentially drives the transcription of a unique

1431 reporter gene composed of a well-defined, approximately 60-bp long DNA TAG composed

1432 of random nucleotides. The transcriptional properties of the various constructs are analyzed

1433 by pooling equimolar amounts of vectors and transfecting them into a cell line of interest.

1434 RNA is extracted, cDNA synthesized and labeled, directly or indirectly, and quantified by

1435 hybridization to the DNA TAGs arrayed on a membrane, glass, or bead support (see Figure 1

1436 for a general schematic diagram). Suitable bead compositions may include those used in

1437 peptide, nucleic acid and organic moeity synthesis, including but not limited to, plastics,

1438 ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria

1439 sol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as sepharose,

1440 cellulose, nylon, cross-linked micelles and teflon many all be used (see Microsphere

1441 Detection Guide, Bangs Laboratories, Fishers Ind.).

1442 The design, operation and applications for the present disclosure will now be

1443 described in greater detail.

1444 1. Design of a library of DNA TAGs that will be transcribed by the putative DNA promoter

1445 sequences.

1446 The TAG DNA sequences were DNA sequences composed of random nucleotides,

1447 that is each position had an equal probability of having any of the four deoxynucleotides (A,

1448 C, T, and G). Other bases, such as inosine, uracil, 5-methylcytosine, 8-azaguanine, 2,6-

1449 diaminopurine, 5 bromouracil, and other derivatives may be incorporated in their nucleotide 1450 form into the oligonucleotides. The length of the TAG sequence was short, preferably

1451 between about 16 bp to about 200 bp, although a shorter or longer length may be used, but

1452 typically about 60 bp. Within a plurality of TAG sequences, each TAG sequence had

1453 approximately equivalent amounts of the nucleotides A, T, G, and C such that each TAG

1454 sequence had approximately the same melting temperature as the other TAGs. A same

1455 melting temperature allowed for the unbiased quantification of various mRNAs by

1456 hybridization under the same temperature and ionic strength conditions. Within a plurality of

1457 TAG sequences, the nucleotide sequence of each individual TAG sequence was unique

1458 amongst the plurality of TAGs. Each TAG did not share sequence homology with any other

1459 TAG used simultaneously in the same assay and with any DNA or RNA molecule that was

1460 labeled during the synthesis of the probe, regardless of the method used to synthesize the

1461 probe. A 60 bp length of random nucleotides of the TAG sequence allowed for generation of

1462 a large number of unique TAGs that were highly unlikely to be found in nature.

1463 Additionally, the longer length of the TAG (e.g., about 60 bp) allowed for use of

1464 hybridization temperatures (e.g., 70° C) that were high enough to prevent unspecific

1465 hybridization with partially homologous sequences. The GC content and thus melting

1466 temperature was normalized across the plurality of TAGs to ensure identical hybridization

1467 conditions for all of the TAG probes. To minimize cross-hybridization and for the highest

1468 specificity, all oligonucleotides were selected with a minimal length of sequence identity of

1469 no longer than six (6) bases. Low-complexity sequences with stretches of more than four (4)

1470 identical nucleotides were not allowed, thus avoiding difficulties in sequence similarity

1471 searching. Upon generation of the TAG sequences, the sequences were verified for the

1472 absence of homology amongst themselves. In some embodiments, the TAG sequences may

1473 be examined against sequences deposited in public databases such as GenBank, EMBL,

1474 DDBJ, and PDB using NCBI BLASTN to aid in determining if non-intended binding may

1475 occur. Oligonucleotides are generally synthesized as single strands by standard chemistry

1476 techniques, including automated synthesis. Many methods have been described for

1477 synthesizing oligonucleotides containing a randomized base. For example, a randomized

1478 position can be achieved by in-line mixing or using pre-mixed phosphoramidite precursors

1479 during an automated procedure (see, Ausbel et al., Current Protocols in Molecular Biology,

1480 Green Publishing, N.Y., 1995). Oligonucleotides are subsequently deprotected and may be

1481 purified by precipitation with ethanol, chromatographed using a size-exclusion or reversed-

1482 phase column, denaturing polyacrylamide gel electrophoresis, high-pressure liquid

1483 chromatography (HPLC), or other suitable method. 1484

1485 2. Construction of TAG-plasmids

1486 The TAG plasmids were derived from pTAG-basic (Figure 7) This plasmid

1487 incorporates a pair of Sfil sites which generate two distinct 3 nucleotide-long nonsymmetrical

1488 sticky ends suitable for the directional insertion of the TAG oligonucleotides. The plasmid

1489 also incorporates a modified cDNA encoding firefly luciferase (luc+) This 1650 bp cDNA

1490 was excised from the commercially available pGL3 using the restriction enzymes Ncol and

1491 Xbal. The wild-type coding region had been modified, in order to eliminate consensus

1492 sequences recognized by genetic regulatory proteins, thus helping to ensure that this reporter

1493 gene is unaffected by spurious host transcriptional signals. The plasmid also incorporates a

1494 97 bp long α-globm 3'UTR. The high level stability of α-globin mRNA, with a half-life from

1495 24 to 60 hours, is attributed to a C-πch as element in its 3'UTR, to which a protein complex

1496 binds to stabilize the mRNA. This protein complex is highly conserved from mouse to

1497 human and is found in a wide spectrum of tissues and cell lines This sequence is sufficient

1498 to increase luciferase mRNA stability, with a half-life of 7 hours The plasmid also

1499 incorporates the SV40 polyA signal to efficiently polyadenylate the luciferase transcript, thus

1500 resulting in up to a five-fold increase of steady-state mRNA levels The plasmid also

1501 incorporates a high copy number origin of replication from pUC19, but may alternatively

1502 contain a low copy number origin of replication, such as pBR322 CoIE 1 oπ'rop (15-20

1503 copies per chromosome), pACYC177 pl5A on (10-12 copies per chromosome) or the

1504 CopyControl system (1, 10-50 copies per chromosome). Additionally, the plasmid

1505 incorporates the ampicillin and kanamycin resistance genes for selection of the pTAG

1506 derivatives in E coli, the λ attVX and α#P2 sites for inserting promoter sequences by

1507 recombination using the Gateway system, and a MCS for inserting promoter sequence

1508 candidates by DNA ligation. The MCS was present in two structurally different but

1509 functionally equivalent copies flanking the ccdB gene, a configuration that allows for using

1510 the ccdB gene as a selection marker for plasmids that incorporates promoter sequences, by

1511 recombination or by ligation. The CcdB protein targets DNA gyrase and inhibits its catalytic

1512 reactions. Cells taking up unreacted vectors with the ccdB gene will not grow. The plasmid

1513 also incorporates a short, synthetic polyA signal based on the highly efficient polyA signal of

1514 the rabbit β-globin gene. Placed upstream of the MCS, it will terminate spurious

1515 transcription, which may initiate within the vector backbone 1516

1517 3. Insertion of DNA TAGs into pTAG-basic 1518 Typically, TAGs were obtained by annealing complementary 63 bp oligonucleotides

1519 [(+)strand: (N)₆₀: ATA; (-)strand: (N)₅o:GTG] that are then ligated into Sfil digested pTAG-

1520 basic, although oligonucleotides of differing lengths can be used, preferably between about

1521 16 bp to about 200 bp, more preferably between about 20 to about 150 bp, more preferably

1522 between about 30 to about 120 bp, more preferably between about 40 to about 100 bp, more

1523 preferably between about 50 to about 75 bp, and most preferably about 60 bp. The ligation

1524 reaction was electroporated into a host strain, for example E. coli DB3.1, which contains a

1525 gyrase mutation (gyrA462) that renders it resistant to the ccdB. Because the sticky ends

1526 generated by both Sfil sites are incompatible, a very low background of self-circularized

1527 pTAG-basic vectors, or vectors with multiple TAGs in tandem, was generated. The presence

1528 of the TAGs in the various plasmids was verified by DNA sequencing. High-throughput

1529 production of TAGs followed a similar methodology. Synthesis of 63 bp oligonucleotides

1530 was performed in two 96-well plates ((+) and (-) strands, respectively). The (+) and (-)

1531 strands were annealed in a 96-well plate, and ligated with Sfil digested, gel-purified pTAG

1532 basic. The ligation mixture was electroporated into electro-competent the E. coli DB3.1 host

1533 cells, using a 96-well electroporation plate. The bacterial clones were seeded into a 96-Deep-

1534 Well plate and the cultures were incubated for 18-24 hours at 37⁰C at 250 rpm using a

1535 microtiter plate incubator shaker. Plasmid DNA purification was performed, either manually

1536 or via automation, for example using a BioRobot 3000 (Qiagen, Valencia, CA), and the

1537 presence of the TAGs verified via DNA sequencing (96-well format). 1538

1539 4. Preparation of Promoter Array Membranes

1540 Oligonucleotide arrays were manufactured using nylon membranes. The (-) strand

1541 TAG oligonucleotides were synthesized in a 96-well plate format and resuspended in buffer,

1542 for example TE, pH 7.5, at a concentration of 100 μg/ml. Nylon membranes, for example

1543 Nytran SuPerCharge (Whatman PLC, Middlesex, UK), were cut (2 cm x 4 cm) to fit 5.0 ml

1544 glass hybridization tubes. Oligonucleotides were either spotted manually in duplicate on the

1545 membranes (0.2 μl/spot) or oligonucleotide arrays printed using an array spotting robot, for

1546 example a Biorobotics MicroGrid (Genomic Solutions, Ann Arbor, MI), After spotting, the

1547 membranes were UV cross-linked twice using a Stratalinker 1800 at 120 mJ/sec, then baked

1548 at 7O⁰C for 1-2 hours. The printed membranes were sealed in parafilm and stored at -20⁰C.

1549 The quality of the membranes was validated by hybridizing 10% of the membranes with

1550 biotin-labeled (+) strand oligonucleotide TAGs. The 3' end of the TAG oligonucleotides was

1551 labeled using terminal transferase and biotin-16-ddUTP. All TAGs were mixed together in 1552 equimolar amounts. The TAG mixture (100 pmol) was incubated in the presence of 1.0 nmol

1553 biotin-16-ddUTP and 50 U terminal transferase, following the manufacturer's

1554 recommendations. After a 15 minute incubation at 37⁰C, the end-labeled TAG probes were

1555 precipitated with LiCl, centrifuged and resuspended in ddH₂O. The labeling efficiency was

1556 checked by spotting a serial dilution of the labeling reaction and a standard on the nylon

1557 membrane. Detection was performed by chemiluminescence, for example with alkaline

1558 phosphatase-conjugated streptavidin, following the manufacturer's recommendations.

1559 Quantification was performed by densitometry. Upon validation of the quality of the biotin-

1560 labeled probes, the quality of the arrays was assessed by hybridizing the probes to the

1561 membranes using standard procedures, detecting them by chemiluminescence, and measuring

1562 the intensity of each spot by densitometry. The membranes were accepted upon observation

1563 of less than a variation of 5% of intensity and spot size. 1564

1565 5. Construction of promoter-TAG plasmids

1566 Promoter sequence candidates were inserted into TAG plasmids using two methods.

1567 First, promoter sequence candidates were extracted from existing plasmids using

1568 endonucleases such as restriction enzymes and inserted into the pTAG plasmids, between

1569 sites located in the multiple cloning sites. Promoter sequence and pTAG plasmids were

1570 assembled by DNA ligation using standard protocols (see Crowe et al., Improved cloning

1571 efficiency of polymerase chain reaction (PCR) products after proteinase K digestion.

1572 Nucleic Acids Res. 1991 Jan 11; 19(1):184); Ausubel, F. M., et al., Short Protocols in

1573 Molecular Biology). Alternatively, promoter sequences were amplified by PCR, using

1574 primers carrying attBl and attB2 extensions, and using mammalian genomic DNA or other

1575 plasmids as templates. The PCR products were inserted into the pTAG plasmids using the

1576 Gateway® recombination system. A promoter sequence candidate may be provided by a

1577 computer-predicted model, DNA fragments from a collection of nucleotide sequences, such

1578 as a genomic library, deletion or site-directed mutants of a specific promoter, tissue-specific

1579 promoters, artificial promoters, etc. Clones containing the pTAG plasmids with the promoter

1580 inserts were cultured in LB medium in the presence of 50 μg/ml ampicillin or 25 μg/ml

1581 kanamycin. At various time points during cell growth, aliquots of each culture were taken,

1582 the cell density measured spectrophotometrically at 600 nm, and equal volumes of culture

1583 pooled. Plasmid DNA was extracted using an alkaline lysis method and purified using anion-

1584 exchange resin. In order to verify that all plasmids were present in the mixture in equimolar

1585 concentrations, the following manipulation was performed. All plasmids in the DNA mixture 1586 were linearized by restriction digestion, and separated on an agarose gel (0.7%). The

1587 resultant DNA fragments, with sizes ranging from 5 to 15 kb, were stained with ethidium

1588 bromide and quantitated by densitometry using a gel documentation system. The linearity of

1589 the assay was verified by quantifying serial dilutions of the plasmid restriction digestion. 1590

1591 6. Transfection and RNA extraction

1592 The purified plasmid DNA mixture containing equimolar amounts of the promoter

1593 plasmids was transfected into HL60, U937, and 293 cell lines. Per transfection, 1 x 10⁷

1594 viable U937 cells were washed and resuspended in 0.4 ml RPMI medium. Plasmid DNA (20

1595 μg) was added and the cell/DNA suspension was mixed gently by inversion. After a 5

1596 minute incubation at 25⁰C, the cells were electroporated using a BTX ECM-600

1597 electroporator with the following settings: 500 V capacitance and resistance, 950 μF

1598 capacitance, 186 ohms resistance, 200 V charging voltage. After the electrochoc, the cells

1599 were transferred into a 10 cm diameter tissue culture dish containing 10 ml RPMI medium

1600 supplemented with 10% FBS. After 2 to 5 hours incubation at 37⁰C, cells were harvested by

1601 centrifugation at 10 krpm for 30 seconds. Cell pellets were lysed by addition of 300 μl Trizol

1602 reagent and total RNA was extracted according to the manufacturers protocol (Invitrogen,

1603 Carlsbad, CA) (see also Current Protocols in Molecular Biology, John Wiley & Sons). RNA

1604 was precipitated with isopropyl alcohol, resuspended in RNase-free TE, pH 7.5, and

1605 quantified by measuring the absorbance at 260 nm and 280 nm (ratio ~2). RNA integrity was

1606 verified by agarose gel electrophoresis and ethidium bromide staining. The 28S and 18S

1607 rRNAs, represented in discrete individual bands, had a 2: 1 intensity ratio. RNA samples with

1608 a visible degree of degradation were not further processed. In parallel, an equimolar mixture

1609 of promoter-less TAG plasmids were transfected and analyzed for mRNA expression using

1610 the array. This control detected the possible presence of cryptic promoter activity in the

1611 TAGs. The promoter-less TAG plasmids yielding above-background signals were discarded. 1612

1613 7. Labeling, hybridization, and detection

1614 Radioactive cDNA probes were synthesized from total RNA. The total RNA was

1615 purified with Trizol (Invitrogen) and the concentration of the RNA was determined by the

1616 OD260 reading. One to five microgram of total RNA was mixed with MA5-a oligo (5'-

1617 TAGTCACTTCGATCGCTGAGG-3 ') ([SEQ ID NO. I]), and the nucleotides dATP, dTTP,

1618 dGTG, and 32P-dCTP. The reaction was incubated at 8O⁰C for 3 minutes and then cooled to

1619 42⁰C. Then added were 1OX reverse transcription buffer (NEB), RNAse inhibitor, and M- 1620 MuLV reverse transcriptase (NEB). The reaction was mixed and incubated at 42⁰C for 60

1621 minutes, then denatured at 9O⁰C for 10 minutes.

1622 The radioactive probes were hybridized to the membrane using Ultrahyb-oligo

1623 hybridization buffer (Ambion, Inc.) at 6O⁰C overnight. After washing the membrane twice

1624 with 2X SSC/1% SDS and twice with IX SSC/ 1% SDS at 60⁰C, the bound probes were

1625 detected by autoradiography, using for example, Kodak Biomax Light Film (Carestream

1626 Health, Inc., New Haven, CT). The density of each spot was quantified with computer

1627 software, for example, Kodak ID Image Analysis Software (Carestream Health, Inc., New

1628 Haven, CT)

1629 In an alternate embodiment, biotin-labeled cDNA probes were synthesized from the

1630 total KNA. The probes were synthesized using the AmpoLabeling-LPR method developed

1631 by SuperArray Bioscience Corporation This method increased the sensitivity of cDNA

1632 arrays by amplifying the cDNAs obtained by reverse transcription by up to 30 rounds of

1633 Linear Polymerase Replication (LPR) A 300 nucleotide long region from the 5' end of the

1634 luciferase mRNAs, encompassing the 60 nucleotide TAGs, was reverse transcribed and

1635 amplified in the presence of biotin-labeled dUTP The total RNA was annealed with primer

1636 complementary to the MA4 segment, in a thermal cycler at 7O⁰C for 3 minutes, cooled to

1637 37^CC and incubated at 37⁰C for 10 minutes The annealed product was reverse transcribed 1638 using MMLV reverse transcriptase in presence of RNasin Ribonuclease Inhibitor. After

1639 mactivation of the reverse transcriptase and RNA hydrolysis at 85⁰C, the cDNAs were

1640 amplified by LPR with primer 5'-GGCTCGGCCTCTGAGCTAAT^' ([SEQ ID NO 2])

1641 located immediately upstream of the TAG, in the presence of biotin-16-dUTP, and a

1642 thermostable DNA-dependent DNA polymerase, using the following program: 85⁰C for 5

1643 minutes, then 30 cycles of 85⁰C for 1 minute, 5O⁰C for 1 minute, 72⁰C for 1 mmute, followed

1644 by 72⁰C for 5 minutes. The probe was then checked for biotin incorporation by making serial

1645 dilutions of the probe synthesis reaction, spotting 1 μl ahquots on a HyBond nylon membrane

1646 and detecting the probe using the ECL chemiluminescent detection kit. Probes that were

1647 detectable at 1000-fold dilutions or higher were used in the hybridizations

1648 The hybridization of the biotinylated probes to the membranes was performed using

1649 the Ultrahyb-oligo hybridization buffer (Ambion Inc.), at 6O⁰C overnight. After washing the

1650 membrane twice with 2xSSC, 1%SDS and twice with IxSSC, 1%SDS at 60C, the bound

1651 probes were detected by chemiluminescence usmg a streptavidm-alkalme phosphatase

1652 conjugate and following the manufacturer's protocol (CDP-Star Universal Detection Kit,

1653 Sigma). The image was acquired with a Kodak image station 440 for 1 hour (Figure 3A, 1654 Figure 4A, and Figure 5A). The density from each spot was quantified using the Kodak ID

1655 Image Analysis software. The data presented in Figures 3A and 3B and Figures 4A and 4B

1656 show that: a) all the "blank" reporter-TAG plasmids which lack promoter sequences (# 10,

1657 19, 26, 28, 30, 35, 39, and 47 in Table 1) give very low intensity signals, a fact, which

1658 suggests the absence of intrinsic promoter activity from the plasmid backbone; b) with the

1659 series of defensin promoters (#74-85), the clone expressing the highest mRNA level (# 79) is

1660 also the one expressing the highest level of hiciferase. The data presented in Figures 5 A and

1661 5B show that: a) as expected, the viral CMV promoter appeared to be the strongest, a fact,

1662 which is well-documented in the scientific literature (U.S. Patents Nos. 5, 168,062 and

1663 5,385,839; Cayer et al J Immunol Methods. 2007 Apr 30;322(l-2): 118-27; Sakurai et al Gene

1664 Ther. 2005 Oct;12(19): 1424-33; Fabre et al. I Gene Med. 2006 May;8(5):636-45.); b) The

1665 GAPDH (glyceraldehyde-3-phosphate dehydrogenase ) promoter was able to drive very high

1666 expression levels, which is consistent with observation made by others (Hirano T et al,

1667 Biosci Biotechnol Biochem. 1999;63(7): 1223-7; Punt PJ et al.Gene. 1990; 93(1): 101-9;

1668 Nagashima T et al., Biosci Biotechnol Biochem. 1994;58(7): 1292-6); c) the ferritin light-

1669 chain promoter was about 40% stronger than the Ferritin heavy chain promoter, a fact that

1670 supports findings made by Cairo et al. in rat liver (Biochem J. 1991; 275 (Pt 3):813-6); d)

1671 Promoters OM3 (TAG61) and Def6 (TAG77) produced the strongest hybridization signals in

1672 their respective groups (OM and Defensin promoters), a fact, which correlates with the

1673 luciferase activities determined previously (Ma et al., Nucleic Acids Res. 1999;27(23):4649-

1674 57; Ma et al. J Biol Chem. 1998 Apr 10;273(15):8727-40.). Taken altogether, these data

1675 validate the present disclosure compared to other methods.

1676 The following examples are offered by way of illustration, and not by way of

1677 limitation.

1678

1679 EXAMPLES

1680 Example 1. Construction of 100 pTAG-reporter plasmids

1681 One hundred pTAG-plasmids featuring a multiple cloning site (MCS), attP sequences,

1682 a ccdB gene, a T7 promoter, a unique 60 bp-long reporter TAG, a specific MA4 segment, a

1683 3-frame translation stop codon, a hemoglobin RNA stabilization fragment and a poly -A

1684 signal were constructed. The construction was performed in 6 steps (Figure 6). First, a

1685 partial MCS was inserted, between the Sfil sites of plasmid pGL4 (Promega, Madison, WI).

1686 All the cloning sites from the original pGL4 plasmid were deleted and replaced with EcoRI,

1687 Kpnl, Sad, Nhel, Xhol, BgIII sites, and followed by two sets of SfuVBgII sites separated by a 1688 CG dinucleotide. The two sets of Sfil sites allowed for the directional insertion of TAG

1689 sequences. The dinucleotide CG between the Sfil sites created a unique restriction site

1690 (Smal/Xmal), which revealed useful to facilitate plasmid digestion with Sfil, either by

1691 insertion of a -170 bp-long spacer fragment to dissociate both Sfil sites, or by digestion of

1692 the plasmid sequentially with Smal and then Sfil.

1693 In the second step, a second partial MCS was inserted between the Xhol and BgIII sites of

1694 pGL4-12. The resulting plasmid (pGL-1256) contained BgIII, Apal, Nrul, Kpnl, Xhol Sacl,

1695 BgIII, Nhel, EcoRV, and MIuI sites following the existing MCS. As a result, pGL-1256

1696 contained two structurally different but functionally equivalent MCS surrounding the Apal

1697 and Nrul sites, a feature useful for cloning promoter sequence candidates in the TAG-

1698 plasmids.

1699 In the third step, the sequence encoding the luciferase reporter gene (Ncol-Xbal

1700 fragment) was replaced with an 80-mer oligonucleotide which contained a specific 25 bp-

1701 long sequence (MA4), a three-frame translation stop codon, and a RNA stabilization

1702 sequence derived from human alpha globin gene. The MA4 facilitated the synthesis of TAG-

1703 specific probes from mRNAs.

1704 In the fourth step, the resulting plasmid 1256MA4 was digested with EcoRV and MIuI,

1705 which allowed for insertion of an oligonucleotide that contained the bacteriophage T7 RNA

1706 polymerase promoter sequence. The presence of the T7 promoter allowed for synthesis of

1707 biotinylated RNA probes by in vitro transcription, a method which increased the sensitivity of

1708 the assay by at least one order of magnitude.

1709 In the fifth step, the Gateway® sequences attP - ccdB - chloramphenicol-resistance gene

1710 were amplified by PCR using plasmid pDONR-201 as template (Invitrogen Inc., Carlsbad,

1711 CA) and the following primers: sense-tcgggccccaaataatgattttattttgactgatag [SEQ ID NO. 3]

1712 and antisense-atgggcccaaataatgattttattttgactgatagtgacctgttc [SEQ ID NO. 4]. The PCR

1713 product was inserted into the Apal site of plasmid 1256MA4T7, generating plasmid

1714 1256MA4T7att.

1715 Finally, plasmid 1256MA4T7att was digested with BgII and 60 bp-long ds

1716 oligonucleotides (TAG) were directionally inserted into the plasmid. In total, we created 100

1717 reporter plasmids— pTAG-Reporter 1 to 100. These plasmids were used to generate the 92

1718 promoter-TAG plasmids. The remaining 8 pTAG-Reporter plasmids were used as blank.

1719 These 100 pTAG-Reporter plasmids are used for cloning putative promoters into the

1720 MCS, using either conventional methods (restriction digestion and ligation), or the

1721 GATEWAY® technology with attB-modified PCR products. 1722

1723 Example 2. Manual and Robotic Production of Macro-Array Membranes

1724 First, three nylon membranes BπghtStar-Plus (Ambion Inc., Austin, TX), Tropilon-Plus

1725 (Applied Biosystems, Foster City, CA), and Nytran SuperCharge (Whatman PLC, Middlesex,

1726 UK) were compared for their ability in being printed with short oligonucleotides. The 63 bp-

1727 long oligonucleotides complementary to the TAGs present on the TAG-reporter plasmids

1728 were manually spotted on the membranes , and hybridized with the biotm end-labeled sense

1729 TAG oligonucleotides. BrightStar-Plus (Ambion Inc., Austin, TX) was selected for use in

1730 subsequent experiments as this membrane produced the best results in terms of low

1731 background, sharpness of the signal spots, and the observation the rough surface of the

1732 BrightStar-Plus membrane produced stronger signals than the smooth surfaces of the other

1733 two membranes, without increasing the background. The nylon membranes were cut (2 x 4

1734 cm) to fit 5-mL glass hybridization tubes and the 8-well hybridization plates (SuperArray

1735 Inc., Frederick, MD).

1736 Next, the amount of oligonucleotides to be spotted on the membrane was optimized

1737 Stock solutions for all the reverse strand TAG oligonucleotides were made by reconstituting

1738 the lyophilized products in TE pH 7.5 to 100 μM. Serial dilutions of 2OX, 6OX, 180X, 540X

1739 and 1620X were made. Using a 2 μL Pipetman, the diluted oligonucleotides (0.2 μl) were

1740 spotted manually, in duplicate, on the membrane. Following hybridization of the membrane

1741 with biotin end-labeled sense-strand TAG oligonucleotide probes, detection of the signals

1742 was performed by chemiluminescence using the Southern-Star kit (Applied Biosystems,

1743 Foster City, CA). The 20-fold dilutions produced a strong and clean signal spots, and were

1744 selected.

1745 The same diluted oligonucleotides (n = 100) (Figure 2) were printed using a Biorobotics

1746 MicroGrid array spotting robot (Genomic Solutions, Ann Arbor, MI) at the microarray

1747 facility of the University of Idaho Environmental Biotechnology Institute (Moscow, ID).

1748 Each oligonucleotide was printed as a quadruple spot. Both types of membranes were air-

1749 dried at room temperature for 10 min and then UV-crosslinked twice using a Stratalinker

1750 1800 (Stratagene) at 120 mj/ sec, then baked at 70 ⁰C for 2 hours. The printed membranes

1751 were then sealed in parafilm and stored at 4 ⁰C. The size of the membrane was designed to fit

1752 into convenient small containers such as 2-mL microcentrifuge tubes and 8-well plates. 1753

1754 Example 3. Cloning of 92 Human and Viral Promoter Sequences into the TAG-Reporter

1755 Plasmids 1756 Ninety-two human and viral promoter sequences (TABLE 1) were cloned into the TAG-

1757 reporter plasmids using the Gateway® system. They included 12 defensin promoters and 15

1758 Oncostatin M promoters, 57 genomic DNA fragments from both EPD and chromosome 21,

1759 which have been studied experimentally for promoter activity, and 8 well-known promoters

1760 (SV40, CMV, wild-type and mutant RSV, GAPDH, HSP, FerL, and FerH).

1761 First, the promoter sequences were amplified by PCR, using human chromosomal DNA

1762 or plasmids as templates, and primers carrying attB sequence extensions. The PCR products

1763 were inserted into the pTAG-reporter plasmids in place of the ccdB and chloramphenicol-

1764 resistance genes by in vitro recombination using the BP clonase (Invitrogen, Carlsbad, CA).

1765 The recombinant plasmids were introduced into E. coll Top 10 using the heat-shock

1766 procedure, and amplified. Recombinant clones lacking promoter inserts were obtained at a

1767 frequency of about 1:200. To ascertain the correct clones, the plasmid DNAs of each clone

1768 were prepared and analyzed by agarose gel electrophoresis separately. Plasmid DNAs were

1769 quantified by spectrophotometry. Finally, equimolar amounts were pooled at a final

1770 concentration of 0.4 μg DNA/μL.

1771 In the context of screening plasmid libraries of putative promoters, E. coli clones are

1772 arrayed in 96-well plates. The bacteria (not their plasmid DNA) are pooled and amplified in

1773 the same flask. Their plasmid DNA is purified in a single preparation, before being

1774 transfected into the same cell population. 1775

1776 Example 4. Testing the Promoter Detective Method with 92 Promoter-TAG Plasmids

1777 The method was performed with the 92 promoter-TAG and 8 blank reporter-TAG

1778 plasmids. Different amounts (4, 16, 64 μg) of equimolar mixtures of these plasmids were

1779 transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After

1780 14 and 25 hours culture at 37 ⁰C, cells were harvested. Total RNA was extracted and

1781 purified using the TRIzol-based method (Invitrogen, Carlsbad, CA). Biotin labeled cDNA

1782 probes were synthesized from the total RNA. The probes were synthesized using the

1783 AmpoLabeling LPR method (SuperArray Bioscience Corp., Frederick, MD). The sensitivity

1784 of cDNA arrays was increased by amplifying the cDNAs obtained by reverse transcription by

1785 up to 30 rounds of Linear Polymerase Replication (LPR). A 300 nucleotide long region,

1786 encompassing the 60 nucleotide TAGs, was reversed transcribed and amplified in the

1787 presence of biotin labeled dUTP. The 2.5 μg total RNA was annealed with primer

1788 complementary to the MA4 segment, in a thermal cycler at 7O⁰C for 3 minutes, cooled to

1789 37⁰C and incubated at 37⁰C for 10 minutes. The annealed product was reverse transcribed 1790 using MMLV reverse transcriptase and RNA hydrolysis at 85⁰C, the cDNAs were amplified

1791 by LPR with primer 5'-GGCTCGGCCTCTGAGCTAAT-3 ' [SEQ ID NO 2] located

1792 immediately upstream of the TAG, in the presence of biotin 16 dUTP, and a thermostable

1793 DNA dependent DNA polymerase, with the following program: 85⁰C for 5 minutes; then 30

1794 cycles of 85°C for 1 minute; 5O⁰C for 1 minute; 72⁰C for 1 minute; followed by 72⁰C for 5

1795 minutes The probe was then checked for biotin incorporation by making serial dilutions of

1796 the probe synthesis, spotting 1 μl aliquots onto a HyBond nylon membrane (Amersham,

1797 Little Chalfont, UK) and detecting the probe using the ECL chemiluminescent detection kit.

1798 Probes detectable at 1000-fold dilutions or higher were used in the hybridizations

1799 The hybridization of the biotinylated probes to the membranes was performed using

1800 the Ultrahyb-oligo hybridization buffer (Ambion Inc.), at 6O⁰C overnight. After washing the

1801 membrane twice with 2xSSC, 1%SDS and twice with IxSSC, 1%SDS at 60C, we detected

1802 bound probes by chemilummescence using a streptavidin-alkalme phosphatase conjugate and

1803 following the manufacturer's protocol (CDP-Star Universal Detection Kit, Sigma). The

1804 image was acquired with a Kodak image station 440 for 1 hour (Figure 4A) The density

1805 from each quadruple spot was quantified using the Kodak ID Image Analysis software. The

1806 results indicate a) all the "blank^"' reporter-TAG plasmids which lack promoter sequences (#

1807 10, 19, 26, 28, 30, 35, 39, and 47 in Table 1) give very low intensity signals, a fact, which

1808 suggests the absence of intrinsic promoter activity from the plasmid backbone; b) with the

1809 series of defensin promoters (#74-85), the clone expressing the highest mRNA level (# 79) is

1810 also the one expressing the highest level of luciferase 1811

1812 Example 5 Testing the promoter detection method with 35 promoter-TAG plasmids

1813 The method was tested with a set of 35 promoter-TAG plasmids Twenty μg of an

1814 equimolar mixture of these plasmids were transfected into U937 cells by electroporation.

1815 After 7 hours culture at 37 ⁰C, cells were harvested. Total RNA was extracted and purified

1816 using the TRIzol-based method (Invitrogen. Carlsbad, CA), and quantified by

1817 spectrophotometry (Abs260 nm)

1818 Radioactrv e cDNA probes were synthesized as follows. One microgram total RNA in

1819 6.3 μL H₂O was mixed with 0.7 μL of 100 μM MA5-a oligonucleotide (5'-

1820 TAGTCACTTCGATCGCTGAGG-S ') ([SEQ ID NO. I]), 1.1 μL of 5 mM each of

1821 dATP/dTTP/dGTG, and 1.9 μL ³²P dCTP. The reaction mixture was heated to 80 ⁰C for 3

1822 minutes and then cooled down to 42 ⁰C. Then 1.5 μL 10x reverse transcription buffer (New

1823 England Biolabs), 0.75 μL RNAse inhibitor, and M-MuLV reverse transcriptase (New 1824 England Biolabs) were added, and the reaction was performed at 42 ⁰C for 60 minutes. The

1825 probes were then denatured at 90 ⁰C for 10 minutes.

1826 The hybridization of the radioactive probes to the membranes was performed using

1827 the Ultrahyb-oligo hybridization buffer (Ambion Inc.), at 60 ⁰C overnight After washing the

1828 membrane twice with 2x SSC, 1% SDS and twice with Ix SSC, 1% SDS at 60 ⁰C, bound

1829 probes were detected by autoradiography using a Kodak Biomax Light film The density of

1830 each spot was quantified using the Kodak ID Image Analysis software (Figures 5A and 5B)

1831 where the autoradiogram was obtained by hybridizing radioactive TAG-cDNA probes to a

1832 membrane spotted with complementary TAG strands. The intensities of the various spots

1833 were compared, relative to the signal obtained with the CMV promoter. As expected, the

1834 viral CMV promoter appeared to be the strongest, a fact, which is well-documented in the

1835 scientific literature (U S Patents Nos 5,168,062 and 5,385,839, Cayer et al J Immunol

1836 Methods. 2007 Apr 30,322(1-2): 118-27; Sakurai et al Gene Ther. 2005 Oct;12(19) 1424-33;

1837 Fabre et al J Gene Med 2006 May;8(5) 636-45.). The GAPDH (glyceraldehyde-3-

1838 phosphate dehydrogenase ) promoter was able to drive very high expression levels, which is

1839 consistent with observation made by others (Hirano T et al, Biosci Biotechnol Biochem

1840 1999,63(7):1223-7; Punt PJ et al Gene. 1990, 93(l): 101-9; Nagashima T et al , Biosci

1841 Biotechnol Biochem. 1994,58(7) 1292-6). Also, the ferritin light-chain promoter was about

1842 40% stronger than the Ferritin heavy chain promoter, a fact that supports findings made by

1843 Cairo et al. in rat liver (Biochem J. 1991; 275 (Pt 3):813-6). Promoters OM3 (TAG61) and

1844 Def6 (TAG77) produced the strongest hybridization signals in their respective groups (OM

1845 and Defensin promoters), a fact, which correlates with the luciferase activities determined

1846 previously (Ma et al., Nucleic Acids Res. 1999;27(23) 4649-57; Ma et al J Biol Chem. 1998

1847 Apr 10,273(15) 8727-40.) Taken altogether, these data validate the present disclosure

1848 compared to other methods 1849

1850

\$$ l * * * * * TABLE 1

Gene Promoter Refseq or Promoter Refseq or

TAG # symbol size (bp) Accession # TAG # Gene symbol size (bp) Accession #

1 MT1 B 471 M 13484 51 SV 330 N/A

PROC 495 NM 000312 52 CMV 655 N/A

MMP1 477 NM 002421 53 RSV 396 N/A

CEA 508 NM 002483 54 RSV303 396 N/A

GAS 539 NM 000805 55 GAPDH 532 N/A

H3FL 506 NM 003537 56 HSP 464 N/A

RUN3 356 K00777 57 FerL 270 N/A

SLC9A1 509 XM 046881 58 FerH 180 N/A

ADAMTS 1 560 NM 006988 59 OM1 (pGL3BomB1 ) 189 BC011589

10 Blank 60 OM2 (N1 ) 304 BC011589

11 CCT8 528 NM 006585 61 OM3 (3STAT) 300 BC011589

12 CRYZL1 583 NM 005111 62 OM4 (3STATm) 300 BC011589

13 DAF 557 NM 000574 63 OM5 (3STATmm) 300 BC011589

14 GABPA 611 NM 002040 64 OM6 (NI ApI) 304 BC011589

15 IFNAR1 667 NM 000629 65 OM7 (N1 SpI mutation) 304 BC011589

16 KRT1 520 NM 006121 66 OM8 (N1 3STATrnm) 304 BC011589

17 LHB 494 NM 000894 67 OM9 (RI) 194 BC011589

18 NEFL 495 NM 006158 68 OMI O (StUl) 94 BC011589

19 Blank 69 OM 11 (2STATm) 194 BC011589

20 NEG9 407 N/A 70 OM 12 (N1 2STATmrn) 304 BC011589

21 IVL 500 NM 005547 71 OM13 (1 STAT) 109 BC011589

22 APOE 509 NM 000041 72 OM 14 (1 STATm) 109 BC011589

23 C21ORF33 689 NM 004649 73 OM 15 (TATA) 31 BC011589

24 DSCR4 688 NM 005867 74 Def3 (B/3) 619 AA321199

25 FTCD 596 NM 006657 75 Def4 (Aval) 497 AA321199

26 Blank 76 Def5 (Hindi) 321 AA321199

27 ITGB2 647 NM 000211 77 Def6 (Hinfl) 299 AA321199

28 Blank 78 Def7 (Apol) 203 AA321199

29 TFF1 605 NM 003225 79 Def8 (Sau96l (7)) 164 AA321199

30 Blank 80 Def9 (Scrfl (9)) 144 AA321199

31 WRB 639 NM 004627 81 Defl O (Scrfl (TATA)) 144 AA321199

32 AMY2B 488 NM 020978 82 Def11 (Tru9l) 111 AA321199

33 BCKDHA 481 NM 000709 83 Def12 (Tru9ITATA) 111 AA321199

34 CA3 518 NM 005181 84 Def13 (Tru9ITATAm) 111 AA321199

35 Blank 35 Def14 (Tru9ITATAm2) 111 AA321199

36 H4FG 222 NM 003542 86 ALB 517 NM 000477

37 NEG13 376 N/A 87 NEG11 468 N/A

38 NEG18 503 N/A HLCS 645 NM 000411

39 Blank 89 NEG12 522 N/A

40 NEG21 444 N/A 90 NEG1 500 N/A

41 NEG22 418 N/A 91 NEG6 480 N/A

42 NEG23 259 N/A 92 ORM1 499 NM 000607

43 NEG2 285 N/A 93 PKNOX1 593 NM 004571

44 NEG3 460 N/A 94 USP16 581 NM 006447

45 NEG5 488 N/A 95 IGSF5 622 AF121782

46 NEG7 466 N/A 96 NEG10 406 N/A

47 Blank 97 NEG16 202 N/A

48 RNU4C 305 M15957 NEG17 339 N/A

49 SH3BGR 588 007341 99 PCP4 625 NM 006198

50 NEG19 483 N/A 100 TCRD 333 M21624

Claims

LISTING OF CLAIMS We claim:

1. A method for detecting DNA regulatory sequences comprising: a) inserting a promoter sequence candidate into a vector wherein the vector comprises a TAG sequence and wherein the promoter sequence candidate is inserted in a position to drive transcription of the TAG sequence; b) the vector containing the inserted promoter sequence candidate is inserted into a cloning host cell; c) cloning host cells containing different promoter sequence candidates are grown to the same optical density, pooled and the vectors therein are extracted, purified and inserted into a reporter cell line; d) mRNA is extracted from the reporter cell lines wherein the mRNA is directly labeled or is used as template for cDNA or probe synthesis; and e) the labeled mRNA, cDNA or probe is analyzed with an array wherein the array comprises identical or complementary sequence to the TAG sequence.

2. The method of claim 1, wherein the vector is a plasmid.

3. The method of claim 1, wherein the TAG sequence is between about 16 base pairs to about 200 base pairs.

4. The method of claim 1, wherein step (a) further comprises inserting a plurality of promoter sequence candidates into a plurality of vectors wherein each vector is comprised of a unique TAG sequence.

5. The method of claim 1, wherein the cloning host cells are in a single reaction vial, wherein the vectors from within the cloning host cells are purified, and about equal amounts of the purified vectors are transferred into reporter cell lines.

6. The method of claim 1, wherein the cloning host cells are in individual reaction vials, wherein the DNA from the cloning host cells within each individual reaction vial is purified, and wherein the purified DNA from each cloning host cell is pooled in equimolar amounts and the vectors therein are inserted into a reporter cell line.

7. The method of claim 1, wherein the cDNA or probe contains a label.

8. The method of claim 1, wherein the mRNA is directly labeled.

9. The method of claim 1, wherein the mRNA is analyzed with an array, wherein the array comprises complementary sequence to the TAG sequence, and wherein the complementary sequence is the antisense strand.

10. The method of claim 1, wherein the cDNA is analyzed with an array, wherein the array comprises complementary sequences to the cDNA of the TAG sequences, and wherein the complementary sequence is the sense strand.

11. The method of claim 1, wherein the labeled mRNA, cDNA or probe hybridizes to the array and the label of the mRNA, cDNA or probe has a detectable response.

12. The method of claim 1, wherein the vector into which the DNA promoter sequence candidate is inserted into comprises a TAG sequence, one or more multiple-cloning sites, one or more DNA recombination sequences, a negative selection marker, a RNA polymerase promoter sequence, a MA segment, a translation stop codon, a RNA stabilization fragment, and a transcription termination signal, and wherein the DNA promoter sequence candidate is located such that it can drive the transcription of the TAG sequence.

13. The method of claim 12, wherein the RNA stabilization fragment is from an alpha-globin gene.

14. The method of claim 12, wherein the transcription termination signal is a poly-A signal.

15. The method of claim 12, wherein the RNA polymerase promoter sequence is a T7 promoter sequence.

16. The method of claim 12, wherein the DNA recombination sequences are selected from the group consisting of attPl and attP2.

17. The method of claim 12, wherein the TAG sequence is located 3' to the promoter sequence and 5' to the transcription termination site.

18. A vector into which a DNA promoter sequence candidate is inserted into comprising a TAG sequence, one or more multiple-cloning sites, at least one DNA recombination sequence, a negative selection marker, a RNA polymerase promoter sequence, a MA segment, a translation stop codon, a RNA stabilization fragment, and a transcription termination signal, and wherein the DNA promoter sequence candidate is located such that it can drive the transcription of the TAG sequence.

19. The vector of claim 18, wherein the vector is a plasmid.

20. The vector of claim 18, wherein the TAG sequence is between about 16 base pairs to about 200 base pairs.

21. The vector of claim 18, wherein the TAG sequence is located 3' to the inserted promoter sequence and 5' to a transcription termination signal.

22. The vector of claim 18, wherein the RNA stabilization fragment is from an alpha-globin gene.

23. The vector of claim 18, wherein the transcription termination signal is a poly-A signal.

24. The vector of claim 18, wherein the RNA polymerase is a T7 promoter sequence.

5. The vector of claim 18, wherein the DNA recombination sequence is selected from the group consisting of attPl and attP2.