WO2000042199A1 - Biologically active reverse transcriptases - Google Patents

Abstract

The invention provides modified reverse transcriptase polypeptides (Types I, II, and III), along with polynucleotides encoding such polypeptides, vectors containing such polynucleotides and host cells transformed with those polynucleotides. The modified RTs typically exhibit improved stability and/or improved solubility, relative to naturally occurring reverse transcriptases. The modified RTs are also found in a variety of forms, such as monomers as well as both homo- and hetero-multimers. The modified RTs may be used in any one or more of the methods known to benefit from reverse transcriptase activity, such as cDNA synthesis, and amplification techniques such as PCR and RAMP.

Description

BIOLOGICALLY ACTIVE REVERSE TRANSCRIPT ASES

FIELD OF THE INVENTION

In general, the invention relates to the field of molecular biology. In particular, the invention relates to reverse transcriptases.

BACKGROUND OF THE INVENTION

The defining activity of a reverse transcriptase (RT) is its ability to synthesize a cDNA strand using an RNA template. This activity has been exploited in a wide variety of techniques fundamental to progress in the academic and commercial arenas. For example, reverse transcription is useful in the production of cDNA molecules and libraries, sequence-specific probes having a variety of labels, sequencing techniques, and any of several amplification techniques. These amplification techniques include Reverse Transcription-Polymerase Chain Reaction (RT-PCR; Myers et ai, Biochemistry 50:7661-7666 (1991) and U.S. Patent Nos. 5,310.652 and 5,407.800), Nucleic Acid Sequence-Based Amplification (NASBA; Kievits et al, J. Virol. Methods 15:273-286 (1991) and U.S. Patent Nos. 5, 130,238 and 5,409,818), Self-Sustained Sequence Replication (3SR; Guatelli et al., Proc. Natl. Acad. Sci. (USA) 57: 1874-1878, 1990) and Rapid Amplification (RAMP; PCT/US97/04170). Other amplification techniques take advantage, at least in part, of the DNA-dependent DNA polymerase activity of some RTs. Amplification techniques falling within this category include, e.g. . the Polymerase Chain Reaction (i.e. , PCR; Saiki et al.. Science 239:487-491 (1989) and U.S. Patent Nos. 4,683, 195, 4.683,202 and 4.800, 159), the Inverse Polymerase Chain Reaction, the Multiplex Polymerase Chain Reaction, Strand Displacement Amplification (i.e. , SDA; Walker et al. , Proc. Natl. Acad. Sci. (USA) 89:392-396 (1992). Walker et al. , Nucl. Acids Res. 20(7): 1691-1696 (1992), and U.S. Patent Nos. 5,270, 184. and 5,455, 166), and the Multiplex Strand Displacement Amplification (U.S. Patent No. 5.422,252 and 5,470,723).

Reverse transcriptases are found in a variety of retroviruses. or RNA tumor viruses. Techniques for producing RT from these native sources involve isolation of virus particles which contain about thirty RT molecules per virion. The RT is released from the virions by lysis of the virion coat. Released native RTs may then be purified using conventional techniques. However, the procedure involved in the production of these viruses is labor- intensive and costly (1 ,000 infected chicks produce 10-20 grams of virus, which is approximately 25,000-40,000 units/gram of virus). Additional problems with RT production from natural sources are the high natural mutation rates which, in part, result in restricted host ranges such as specific strains of chickens. An alternative source of RTs is recombinant production, which in turn is dependent on an understanding of RT expression by the various retroviruses. In general terms, retroviruses bind to receptors on susceptible cells and insert the retroviral core particle into the cytoplasm of the host. Two major events occur in the life cycle of retroviruses. First, the single-stranded RNA genome is converted to double-stranded DNA by reverse transcriptase. Second, this DNA copy is inserted into the genome of the host cell (Varmus, et al. , In Mobile DNA (ed. Berg, et al. ,) pp 53-108, (1989), Washington D.C : AM. Soc. Microbiol. 972 pp; Brown, Curr. Top. Microbiol. Immunol. 157: 19-48 (1990); Goff, Cancer Cells 2: 172-178 (1990a); Goff, J. Acquired Immune Defic. Syndr. 3:817-31 (1990b); Boeke, et al. , Curr. Opin. Cell. Biol. 3: 502-507 (1991), an event typically mediated by a virally encoded integrase activity. Following integration, this proviral DNA can be transcribed by the host RNA polymerase to make viral RNA which is then transported back to the cytoplasm for synthesis of various viral proteins. Virus assembly takes place in the cytoplasm followed by release of budded viruses from the cell for another round of infection (Whitcomb, et al. , Ann. Rev. Cell Biol. 8: 275-306 (1992)). Any defect in the reverse transcription or integrase functions will result in a defective virus that cannot replicate. As an example, Avian Myeloblastosis Virus (i.e. , AMV) is a defective virus that requires a helper virus such as Myeloblastosis-Associated Virus ( . e. , MAV) for viral propagation.

Integrase ensures a stable association of viral and host DNAs. Integration is site- specific with respect to the viral DNA but is essentially random with respect to the host. This observation indicates that there is a DNA binding region in the integrase domain that is necessary for the binding of viral and host DNAs, in a manner independent of host sequence, during the integration process.

Although encoded by the cognate genes, the integrase domain is not found within mature MMLV-RT (i.e. , Moloney- Murine Leukemia Virus Reverse Transcriptase, a Type I RT) or mature HIV-RT (i.e. , Human Immunodeficiency Virus Reverse Transcriptase, a Type II RT). However, the integrase domain is found as an integral part of the mature avian RT (a Type III RT). The presence of this integral integrase domain, along with thermostability. are two features of avian RTs that distinguish this class of RT from other RTs. Investigations of the integrase domain of avian RTs have revealed that it functions in DNA binding and in polymerization, or multimerization.

Some evidence for a DNA binding function comes from alignment of the deduced amino acid sequences of retroviral integrases. Three potential functional domains have been identified. An N-terminal region is characterized by an HHCC (Histidine, Cysteine) zinc finger-like domain which stabilizes the structure of the integrase (approximately, amino acids 579-629 of SEQ ID NO: 2). The central region of these integrases contains a catalytic domain which shares homology with bacterial transposases involved in the breaking and joining of nucleic acid molecules (approximately, amino acids 630-807 of SEQ ID NO: 2). This region has acidic amino acid residues which have been proposed to be involved in the binding of required metals (Mg^{~ ~} or Mn^{+ +}). Khan et al. , Nucl. Acids Res. 19:851-860 (1991), reported DNA binding activity in this central region. The C- terminal region of these integrases is not conserved at the sequence level and its function is unknown (approximately, amino acids 808-858 of SEQ ID NO:2). However, deletion analyses indicate that this region contains strong sequence-independent DNA binding activity as well.

The integrase polypeptide functions as a multimer, or polymer. The N-terminal zinc finger-like domain and the C-terminal deletion derivative have less tendency to dimerize. Hickman et al. , J. Biol. Chem. 269:29,279-29,287 (1994). Sedimentation analyses suggest that integrase occurs as a mixture of monomers, dimers and tetramers.

The genome of the retroviruses codes for several genes, namely gag, pol, env, and the cellular oncogenes, tat, ars/trs, nef, rev etc. The pol gene codes for a polypeptide with reverse transcriptase (RT) activity. The RT enzyme has several activities, such as RNA- dependent DNA polymerase, DNA-dependent DNA polymerase, ribonuclease (RNase H), integrase, endonuclease and, possibly, protease activities. In the laboratory, reverse transcriptase is mainly used for its RNA-dependent DNA polymerase activity, which elongates an oligonucleotide primer, such as a tRNA, annealed to a template RNA or DNA strand to synthesize a DNA strand that is complementary to the template strand (cDNA) (Copeland, et al. , J. of Virology 36: 1 15-119 (1980); Berger, et. al.. Biochemistry 22: 2365-2372) (1983)).

Generally, there are three types of RT. Moloney-Murine Leukemia Virus (MMLV) is a monomeric RT, while HIV-RT and avian RTs are heterodimers. The HIV-RT heterodimer consists of a 66 kDa β polypeptide and a 51 kDa a polypeptide. The avian RT heterodimer consists of a larger 95 kDa β polypeptide and a 63 kDa polypeptide. The polypeptides from HIV-RT and the avian RTs differ in that the HIV-RT polypeptide lacks RNase H activity. The β polypeptide of HIV-RT and the β polypeptide of avian RTs differ in that the HIV-RT β polypeptide lacks the integrase activity of avian RT β polypeptides.

AMV-RT occurs in nature in multiple molecular forms, such as monomers, homodimers and heterodimers. However, the major active native form is a heterodimer of two structurally related polypeptide chains, an subunit of 63 kDa and a β subunit of 95 kDa. These mature subunits are the products of post-translational processing of a precursor protein of 180 kDa (Gag + Pol). The 180 kDa protein is cleaved to a 95 kDa β subunit. The β subunit may be further cleaved to a 63 kDa subunit and a 32 kDa endonuclease subunit. The and β subunits have identical N-termini. (Roth, et al. ,J. Biol. Chem. 260:9326-9335 (1985); Gerard, et «/. ,DNA 5: 271-279 (1986)). Beyond a difference in form (monomer v. heterodimer), the avian RTs differ from

MMLV-RT in other ways. In contrast to MMLV-RT, the avian reverse transcriptases exhibit high processivity and yield, as well as biological activity (e.g. , polynucleotide polymerase activity) over a wider range of temperatures extending up to at least 70° C. This ability to polymerize at higher temperatures is useful when working with RNA templates that have secondary structures. Additionally, this temperature stability has been exploited in amplification technologies such as NASBA and RAMP. Non-avian RTs, including those RTs having RNase H activity, have relatively low processivity and yield. For example, it has been estimated that approximately 50 times more MMLV RT is required than AMV-RT for cDNA synthesis. In addition to Avian Myeloblastosis Virus, the avian retroviruses include Avian

Sarcoma Leukosis Virus (ASLV), Rous Sarcoma Virus (RSV), Avian Sarcoma virus (ASV), Avian Tumor Virus (ATV) and their helper viruses such as MAV, Avian Sarcoma helper virus UR2AVRT, Rous-Associated Virus (RAV), and others. The homology among the avian reverse transcriptases at the DNA level is between 90-98 % and, at the amino acid level, the homology is 95-100% .

Although the nucleotide sequences of many avian viruses are known (Schwartz et al. , Cell 32:853-869 (1983); see also Genbank Accession Nos. M24159, M37980, J02342, J02021 , and J02343), cloning and expression of an active and stable RT in commercially useful amounts has not been achieved.

When the DNA sequence of the pal gene of AMV and MAV were compared, approximately 111 bp from the 3' end of MAV was found to be replaced by host DNA sequences in AMV. Kan et al . Virology 145: 323-329 (1985). The rest of the DNA coding for the RNA- and DNA-dependent DNA polymerase and RNase H activities was intact. This deletion involved the coding region for the integrase domain of the β polypeptide, which causes AMV to be defective in the propagation of the virus, thereby creating a requirement for helper virus MAV to produce infectious progeny virus. Hence, the integrase domain is critical for producing infectious particles. Nevertheless, both the avian retroviruses and their helper viruses encode reverse transcriptases having RNA- and DNA-dependent polymerase and RNase H activities.

AMV Reverse Transcriptase (i.e. , AMV-RT) has been characterized and conditions for the synthesis of full-length cDNA products have been investigated. Berger et al. , Biochemistry 22:2365-2372 (1983). However, the length and yield of cDNA produced by AMV-RT have reportedly been limited by either a nuclease integral to AMV-RT or associated contaminants. See, U.S. Patent No. 5,017,492. In efforts to maximize cDNA length and yield, attention has turned to MMLV-RT. MMLV-RT is a reverse transcriptase that is relatively thermosensitive and exhibits relatively low reverse transcriptase activity. Efforts to improve the stability, and hence activity, of MMLV-RT reportedly met with some success in the form of C-terminal truncations of MMLV-RT. U.S. Patent No. 5,017,492; see also U.S. Patent Nos. 5.244,797, 5,405,776, and 5,668,005. Beyond these modifications, the '492 Patent reports that some C-terminal amino acid changes enhanced MMLV-RT activity, albeit at the cost of a reduction in processivity. Notwithstanding these improvements, MMLV-RT is relatively thermosensitive and inefficient in catalyzing cDNA synthesis.

The avian RTs are structurally distinct from MMLV-RT. At the primary structure level, avian RT, e.g. , AMV-RT, shares no more than 28% amino acid sequence similarity to MMLV-RT (no more than 50% similarity at the polynucleotide level). Moreover, the native AMV-RT is a heterodimer composed of a 63 kDa alpha peptide and a 95 kDa beta peptide while MMLV-RT is an 80 kDa monomer. Not surprisingly, these enzymes differ in their thermostability. The thermophilic AMV-RT is active over a broad temperature range extending, at least, to 70°C. Consequently, these avian RTs can often copy RNA templates capable of forming relatively strong secondary structures In contrast, MMLV- RT is a mesophilic enzyme Also, relative to AMV-RT, approximately 50-fold more MMLV-RT is lequired foi cDNA synthesis Purthermoie, AMV-RT and MMLV-RT differ in other properties such as processivity, metal co-factor requirements, error rate (i e , rate of incorrect nucleotide incorporation), and tRNA primer preferences These drawbacks in using MMLV-RT, in turn, increase the cost of effectively using MMLV-RT Therefore, a need continues to exist in the art for a reverse transcriptase that can be produced economically and that exhibits one or more improvements in terms of processivity, stability, solubility, and thermal range, leading to increased lengths and yields of polynucleotide products, while minimizing the cost of the reverse transcriptase

SUMMARY OF THE INVENTION

The present invention relates to the discovery that reverse transcriptase polypeptides which have been modified, e g , b alteπng existing integrase domains or by adding integrase domains that is modified themselves, are characterized by one or more improved properties, which include increased activity, stability, and solubility, as well as increased ease and versatility in producing such polypeptides The reverse transcriptase polypeptides of the invention may be derived from any source, including, but not limited to, Moloney-Muπne Leukemia Virus (a Type I reverse transcriptase or RT), HIV (Type II RTs), and avian retroviruses (Type III RTs) One aspect of the invention is drawn to RT polypeptides that are truncated internally and/or at their C-termini, yet retain RNA-dependent DNA polymerase activity, the defining characteπstic of reverse transcriptases The truncated polypeptides may also have and preferably do have, DNA-dependent DNA polymerase activity Preferred polypeptides according to the invention exhibit RNase H activity For those truncated polypeptides corresponding to full-length reverse transcriptases having an integral integrase activity (e g , avian retroviral RTs or modified Type I and Type II RTs that retain an integrase domain, unlike natural forms of these RTs), the truncation preferably extends into the integrase domain, effectively eliminating integrase activity from the truncated polypeptide Such truncated polypeptides exhibit improvements in one or more of the following properties compared to their full-length counterparts RNA-dependent DNA polymerase activity, expression levels, stability, and solubility These improvements result in more cost-effective

RTs for use in a wide variety of DNA synthesis, amplification and sequencing technologies

The invention also provides a chimeric RT polypeptide resulting from the effective addition of a protein domain to the C-terminus of the truncated RT, resulting in a non-native chimeric polypeptide (/ e , a polypeptide not found in nature) These protein domains provide a DNA binding capability, a metal binding capability, a structure stabilizing capacity, or a polymerization (/ e , multimeπzation) capability and preferably several capabilities With these added or enhanced, capabilities, the chimeric polypeptides of the invention exhibit improvements in RNA-dependent DNA polymerase activity, protein expression levels, protein stability, and/or protein solubility, with chimeric polypeptides of the invention frequently showing improvement in all four properties Preferred protein domains include a plurality of histidine residues (/ e , His tags), and either the N-terminal domain (providing a DNA binding capacity, preferably resulting from a zinc finger domain) or the C-terminal domain (providing a polymerization domain) of the integrase region of a native RT More specifically, the invention provides reverse transcriptase polypeptide fragments (i.e., portions of full-length RT polypeptides), modified reverse transcriptase polypeptides, and analogs and variants thereof Preferably the polypeptides of the invention aie thermostable avian RTs that have improved RNA- and DNA-dependent DNA polymerase activities, resulting in increased lengths and yields of synthesized polynucleotide products Typically, the polypeptides of the invention lack the catalytic activity of the integrase domain provided by the C-terminal region of the full-length polypeptides (e.g., nucleotides 1719-2571 of SEQ ID NO 1 (Type III), nucleotides 2464-3012 of SEQ ID NO 40 (Type I), and nucleotides 1840-2708 of SEQ ID NO 42 (Type II)) The absence of catalytic activity provided by the integrase domain is expected to result in polypeptides that are more soluble and expressed at higher levels, hence, such polypeptides are more amenable to economical purification in commercially useful quantities In addition to this benefit, the chimeric polypeptides of the invention are expected to facilitate nucleic acid binding or polymerization (homo-polymerization or hetero-polymerization), and preferably both activities, which contribute to the improved performance of the polypeptides The improved RT performance, in turn, translates into improvements in the many techniques dependent on RT activity, such as cDNA production and cDNA library preparation as well as a variety of polynucleotide amplification and sequencing technologies These amplification techniques include RT-PCR, NASBA, 3SR, and RAMP The improved DNA-dependent DNA polymerase activities of the polypeptides of the invention are useful in, e.g., PCR, the Inverse Polymerase Chain Reaction, the Multiplex Polymerase Chain Reaction, SDA, and Multiplex SDA The sequencing technologies include the many variations on the Sanger dideoxy sequencing technique

One aspect of the invention is an isolated polynucleotide encoding a polypeptide according to the invention. In general terms, the invention comprehends polynucleotides encoding polypeptides having RT activities, those polynucleotides typically lacking approximately 200-1 , 122 bp of the 3 ' ends of the corresponding native RT genes. For example, a full-length avian RT gene (i.e.. MAV pot) is 2,692 bp (SEQ ID NO: 1) and the invention contemplates MAV-derived polynucleotides of approximately 1 ,570-2,492 bp in length. More generally, the polynucleotides of the invention may result from truncations to RT-encoding polynucleotides derived from any source, including: AMV, MAV, RSV, ASLV, ATV, MMLV and HIV. In particular, the invention contemplates an isolated polynucleotide encoding a polypeptide having RNA-dependent DNA polymerase activity, the polypeptide consisting of any one of the following sequences: an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 857 of SEQ ID NO:2; an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 1 ,054 of SEQ ID NO: 39; an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 548 to 1 , 198 of SEQ ID NO:41 ; an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 901 of SEQ ID NO:43; and variants, analogs and fragments of any of the above-described polypeptides having RNA-dependent DNA polymerase activity, the aforementioned polypeptides (i.e. , polypeptides and variants, analogs, and fragments thereof) optionally having an N-terminal methionine. An exemplary polynucleotide has a sequence set forth in any one of SEQ ID NOs 1 , 7, 9, 38, 40, and 42. The polynucleotides preferably comprise a start codon specifying methionine at the 5' end. Other truncated polynucleotides of the invention have internal deletions, preferably removing at least part of an integrase domain. For example, polynucleotides according to the invention comprise the sequence set forth in SEQ ID NO:40, with part or all of nucleotides 2464-3012 deleted, or comprise the sequence set forth in SEQ ID NO:42, with part or all of nucleotides 1840-2708 deleted, or comprise the sequence set forth in SEQ ID NO: l , with part or all of nucleotides 1719-2571 deleted (e.g. , deletion of nucleotides 1860-2310, 1920-2310, or 1980-2310 of SEQ ID NO: l). Such polynucleotides encode polypeptides that lack an effective integrase activity in that the polypeptides do not promote detectable polynucleotide integration.

Other polynucleotides according to the invention encode chimeric polypeptides, such polynucleotides comprising a polynucleotide encoding a polypeptide having RNA- dependent DNA polymerase activity and an adjacent polynucleotide encoding a terminal modification of that polypeptide, thereby encoding a chimeric polypeptide. Preferred polynucleotides encode a chimeric polypeptide having one or more amino acids attached to the C-terminus of a polypeptide having RNA-dependent DNA polymerase activity. Such polynucleotides may contain one of the above-described coding regions fused (in frame) at its 3' end to a region encoding one or more amino acids. For example, the 3' end of a coding region may be fused to one or more codons for a charged amino acid such as histidine, lysine, arginine, aspartate, or glutamate. Alternatively, the 3' end of the coding region may be fused to a region encoding a polypeptide, preferably having four to fifty (e.g. , six) amino acids and preferably comprising a domain selected from the group consisting of a DNA binding domain, an RNA binding domain, a metal binding domain, a polymerization domain, and a structure stabilizing domain. Examples of such domains include, but are not limited to, disulfide bond forming cysteine residues, a zinc finger domain, an acidic amino acid domain, and a basic amino acid domain, a bulk) amino acid domain (e.g. , W or W-H, single-letter amino acid identifications), a PPG domain, a GPRP or a PRPG (i.e. , inverse GPRP) domain, a leucine zipper motif or domain, and an NSl binding site, among others. Examples of suitable domains include, but are not limited to, the N-terminal domain of the MAV-RT integrase region which provides a DNA binding domain and the C-terminal domain of the integrase region which provides a polymerization domain. Further, the polynucleotides encoding chimeric polypeptides having a plurality of C-terminal amino acids may encode the same amino acid a number of times. Such polynucleotides may encode basic (e.g. , Histidine) amino acids at the C-terminus. Also preferred are polynucleotides that have a stop codon (e.g. , TAA, TAG, or TGA) at the 3 ' end of a coding region of a chimera according to the invention. An exemplary polynucleotide encoding a chimeric polypeptide has a sequence selected from the group consisting of a sequence set forth in any one of SEQ ID NOs 11-19.

Still other polynucleotides of the invention encode a chimeric polypeptide having one or more amino acids attached to the N-terminus of a polypeptide having RNA- dependent DNA polymerase activity. In addition, the invention contemplates polynucleotides that encode more than one modification, such as an N-terminal peptide addition and a C-terminal peptide addition or a C-terminal peptide addition coupled to an internal deletion of at least part of an integrase domain.

The invention also provides a vector comprising any of the aforementioned polynucleotides. A preferred vector comprises a polynucleotide operably linked to a promoter. Another aspect of the invention is directed to a host cell transformed with a polynucleotide of the invention, such as prokaryotic (e.g., Escherichia coli) or eukaryotic cells (e.g. , insect cells). In a related aspect, the invention comprehends a method of transforming host cells comprising the following steps: introducing a vector according to the invention into a host cell; incubating the host cells; and identifying host cells containing the vector, thereby identifying a transformed host cell.

Still another aspect of the invention is a method of producing an isolated reverse transcriptase polypeptide comprising the step of transforming a host cell with a vector as described above, incubating the host cell under conditions suitable for expression of a polypeptide, and recovering the polypeptide, thereby producing an isolated reverse transcriptase polypeptide according to the invention.

In another aspect, the invention provides the polypeptides encoded by the polynucleotides described above. These polypeptides include polypeptide fragments (e.g. , β RT fragments containing part, but not all, of the C-terminal integrase domain) and chimeric polypeptides, as described above, as well as variants and analogs thereof. In general terms, the invention contemplates all types of reverse transcriptase fragments and chimeras (and variants and analogs thereof) including, but not limited to, the three classes of RTs exemplified by MMLV-RT, HIV-l-RT, and avian RTs. Exemplary chimeric polypeptides contain an N-terminal methionine or a C-terminal peptide providing useful functions (e.g. , expression enhancement, nucleic acid binding domains, metal binding domain, structure stabilizing domains, or polymer- forming domains). Other chimeric polypeptides according to the invention may result from modification of RTs derived from, e.g. , the following sources of Types I-III: ASLV, ATV, MMLV, HIV-1 , and HIV-2. A preferred addition to an RT is a C-terminal peptide comprising a plurality of amino acids such as basic amino acids, a nucleic acid binding domain, a metal binding domain, or a polymerization domain. Preferably, the C-terminal peptide provides more than one functionally significant domain. Also preferred is one or more C-terminal cysteine residues, which, at a minimum, provide a capacity to induce polypeptide homo-, or hetero- , polymerization, such as dimerization. Typical polypeptides of the invention are relatively soluble and are capable of being expressed at high levels, resulting in relatively high levels of RT activity expected to facilitate economical purification.

Yet another aspect of the invention is an improvement in a method for copying a target nucleic acid by extending a target nucleic acid-bound primer, the improvement comprising: contacting the target nucleic acid and primer with a polypeptide according to the present invention. The method preferably produces one or more copies of the target nucleic acid and the polypeptide may be a polymer. Any method for copying a target nucleic acid using a polymerase is comprehended by the invention, including, but not limited to, cDNA synthesis, Polymerase Chain Reaction, Polymerase Chain Reaction- Reverse Transcription, Inverse Polymerase Chain Reaction, Multiplex Polymerase Chain Reaction, Strand Displacement Amplification, Multiplex Strand Displacement Amplification, Nucleic Acid Sequence-Based Amplification, Sequence-Specific Strand Replication and Rapid Amplification. Another aspect of the invention is directed to improved methods for sequencing a target nucleic acid by extending a target nucleic acid-bound primer, the improvement comprising: contacting the target nucleic acid and primer with a polypeptide according to the present invention. Yet another aspect of the invention is a kit for copying a target nucleic acid comprising one or more nucleotides and a polypeptide according to the invention. Preferred polypeptides include those polypeptides encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and polynucleotide derivatives thereof encoding C-terminal amino acids or polypeptides at their 3' ends.

Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following drawing and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 photographically depicts Western blot analysis of RT expression products of insect cells.

Fig. 2 illustrates recombinant RT fractionated on an 8% SDS-PAGE gel and stained with Coomassie Blue. Fig. 3 presents an autoradiograph of gel-fractionated cDNAs produced by an RT polypeptide according to the invention.

Fig. 4 graphically presents temperature profiles for cDNA production using native and recombinant RTs (Fig. 4A), temperamre profiles of nRT and rRT catalyzing RT-PCR (Fig. 4B), temperature profiles for RT-mediated RAMP (Fig. 4C), pH profiles for nRT and rRT in RT assays (Figs. 4D and 4E), magnesium ion profile for nRT and rRT in RT assays (Fig. 4F), and other divalent cation profiles for nRT and rRT in RT assays (Fig. 4G).

Fig. 5 illustrates the relative DNA-dependent DNA polymerase activities of native RT and recombinant RT. Fig. 6 shows a graphic comparison of the relative RNase activities of native RT and recombinant RT at 37 °C (Fig. 6A); Fig. 6B shows a temperature profile for the RNase H activity of rRT. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides truncated reverse transcriptase polypeptides (i.e. , fragments), and analogs and variants thereof. Preferably, these polypeptides exhibit improved levels of RNA-dependent DNA polymerase activity, frequently extending over a wide range of temperatures up to 70°C and beyond. Also preferred are internally or terminally truncated polypeptides having sequences compatible with improved levels of expression. A preferred polypeptide according to the invention has a temperature optimum of 45°-55°C. Also preferred is a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO: 43. Some of these polypeptides correspond to C-terminal truncated forms of avian reverse transcriptases, such as the full-length Myelogenetic Avian Virus-Reverse Transcriptase (i.e. , MAV-RT). A preferred polypeptide of the invention lacks an effective integrase catalytic activity and is expressed at elevated levels, providing a source of soluble, and recoverable, polypeptide in active form. Exemplary integrase domains include a Type I domain (nucleotides 2464-3012 of SEQ ID NO: 40). a Type II domain (nucleotides 1840-2708 of SEQ ID NO:42) and a Type III domain ((nucleotides 1734-2571 of SEQ ID NO: l), any of which may be modified by internal or terminal deletion(s) or by substitution or chemical modification. Because integrase and RT function sequentially in the viral life cycle, it is possible that RT and integrase act in a complex. Thus, without wishing to be bound by theory, the added functions of nucleic acid binding and polymerization provided by the integrase domain of avian RTs may result in increased processivity and superior performance of such RTs. Accordingly, non-native chimeric polypeptides of the invention further include the C-terminal addition of a polymerizing domain, such as a plurality of the same, or different, amino acids. Non-native chimeric polypeptides are herein defined as polypeptides not found in nature. Thus, if the parts of the chimera are found in nature, they are not found in the same relationship as exists in the non-native chimeric polypeptide. Preferred C-terminal amino acid additions are basic amino acids, such as histidine, lysine and arginine. These preferred C-terminal additions may promote polymerization by, e.g. , metal chelation; the basic amino acids also may provide or enhance the nucleic acid binding capacity of the polypeptide. A preferred number of C-terminal amino acid additions is 4-50, more preferably six amino acids. As one alternative to a plurality of basic amino acids, one or more cysteine residues may be added to the C-terminus of the polypeptide. Other alternatives are C-terminal peptides of 4-50 amino acids having a polymerizing capacity or a DNA binding capacity, and preferably both capacities. In addition, to RNA-dependent DNA polymerase activity, the polypeptides may also have DNA-dependent DNA polymerase activities or RNase H activity.

The invention also comprehends polypeptide variants, which have substantially the same amino acid sequence as one of the polypeptides described above. "Substantially the same" means that the sequence of the polypeptide may be aligned with one of the sequences disclosed herein, using any of the approaches known in the art (e.g. , DNASIS, Hitachi Software Engineering America, Ltd. , San Bruno, CA) such that the sequences are at least 90%, and preferably 95% or 98% , similar throughout the aligned region. For example, the invention contemplates the conservative substitution of asparagine for aspartate at any one or more of amino acid positions 450, 505, or 564 of SEQ ID NO:2 to produce variant MAV-RT polypeptides lacking RNase H activity; that same substitution at any one or more of amino acid positions 497, 552, or 603 of SEQ ID NO:43 produces variants of HIV-RT polypeptides lacking RNase H activity. Other residues which may be changed by conservative substitution to generate RNase H ^" variants of MAV-RT include amino acid positions 484, 549, and 572 of SEQ ID NO:2. More generally, the invention comprehends polypeptides having substantially the same amino acid sequences, regardless of whether the differences involve conservative substitutions or not. For example, the residues identified above may be changed in a non-conservative manner. In addition, other residues known to be involved in RNase H activity may be altered by substitution or deletion. These residues include, but are not limited to, amino acids at positions 441-578 of SEQ ID NO: 2 (AMV-RT and MAV-RT; see also, RSV-RT); positions 427-1 ,055 of SEQ ID NO:39 (HIV-2-RT); positions 625-911 of SEQ ID NO:41 (MMLV-RT); and positions 427-902 of SEQ ID NO:43 (HIV-l-RT). The invention also comprehends polypeptide analogs, which are defined herein as polypeptides that either contain known equivalents for one or more of the conventional amino acids or have been derivatized in a manner understood in the art (e.g. , glycosylation, pegylation, phosphorylation), or both.

Another aspect of the invention is drawn to polynucleotides encoding the aforementioned polypeptides. A preferred polynucleotide consists of the sequence set forth as SEQ ID NO: l , SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19. SEQ ID NO: 38, SEQ ID NO: 40. or SEQ ID NO: 42. Also contemplated by the invention are polynucleotides substantially the same as the polynucleotides having one of the above- identified sequences. In the context of polynucleotides, "substantially the same" means that the polynucleotide has a sequence that is at least 90% homologous to one of the above- described polynucleotides.

Beyond the polynucleotides, the invention provides vectors containing at least one of these polynucleotides. Further, these vectors may be functional in prokaryotic cells, eukaryotic cells, or both cell types. A preferred vector is a Baculovirus vector such as pBacPak9 (Clontech Inc. Palo Alto, CA). The invention also provides prokaryotic and eukaryotic host cells transformed with the above-identified polynucleotides. A preferred host cell is an Sf9 insect cell transformed with a Baculovirus-based recombinant molecule of the invention. Other insect cell lines, such as SF21 HighFive may also be used. In another aspect, the invention provides methods of using the polynucleotides to produce RTs according to the invention. In particular, the polynucleotides are transformed into a prokaryotic or eukaryotic host cell under conditions that allow expression of the encoded RT polypeptide and, following an incubation period, the polypeptide is isolated.

In yet another aspect of the invention, methods of using the RT polypeptides are provided. These methods realize the benefits of speed and yield from using highly active and thermostable RT polypeptides to copy target nucleic acids (e.g. , cDNA synthesis, cDNA library construction), amplify, or sequence a target nucleic acid. Suitable amplification methodologies include, but are not limited to, PCR, RT-PCR, Inverse PCR, Multiplex PCR, SDA, Multiplex SDA, NASBA. 3SR, and RAMP. Suitable sequencing methodologies include the original enzymatic sequencing technology disclosed by Sanger and co-workers, or any of the numerous variations of that technique that have been developed since that disclosure.

Various aspects of the invention are described in the following Examples, wherein Example 1 describes the cloning of a coding region encoding the full-length MAV-RT; Example 2 describes the sequencing of the full-length MAV pol gene encoding reverse transcriptase; Example 3 discloses the cloning of selected polynucleotides according to the invention; Example 4 details the large-scale purification of the expressed recombinant RT; Example 5 describes SDS-PAGE and Western blot analyses of expressed proteins; Example 6 discloses an assay for RNA-dependent DNA polymerase activity; Example 7 illustrates assays characterizing the native reverse transcriptase (nRT) and recombinant reverse transcriptase (rRT) in terms of optima for temperature, pH, MgCl₂, and other divalent cation concentrations; Example 8 discloses use of RTs in methods for copying and/or amplifying target nucleic acids; Example 9 describes a DNA-dependent DNA polymerase assay used to characterize nRT and rRT: Example 10 reports a comparison of the RNase H activities of nRT and rRT; and Example 11 describes the cloning and expression of additional polynucleotides according to the invention.

Example 1 The pol gene of MAV, encoding the full-length RT precursor polypeptide, was cloned from pMAV, a pBR322 derivative containing the pol, gag and partial env gene of MAV. Data derived from a partial restriction map of the insert fragment of pMAV is shown in Table I. Based on the map data, i pol coding region, along with some 5' and 3' non-coding sequences, was excised and ligated into several prokaryotic and eukaryotic vectors, as described below. Several recombinants were obtained from these vectors. Anti-RT monoclonal antibodies were used to analyze the expression of RT (see Example 5).

Table I

Feature Relative Position (bp)

EcoRI 69

Pstl 200

Start codon (pol) 253

Bglll 1988

Kpnl 2748

Stop codon (pol) 2943

Xhόl 3013

Pstl 3155 All non-coding 5' (i. e. . upstream) nucleotides were removed to increase the expression of RT. Also, the open reading frame of the natural RT gene starts with an "ACT" (Thr), which is not a frequently used start codon in prokaryotes. The codon that is most frequently used is "ATG" (Met). "ATG" can serve as a start codon for efficient expression of RT in both prokaryotes and eukaryotes. Therefore, an "ATG" was added 5' to the natural "ACT" start codon in order to allow efficient expression of the protein in prokaryotes and eukaryotes (ATG ACT GTT GCG CTA CAT CTG GCT ATT CCG CTC AAA TGG AAG CCA AAC CAC ACG CCT GTG TGG ATT TTC CAG TGG CCC, etc. ; compare the sequences provided in SEQ ID NOs 2 and 3). Construction of Prokaryotic Recombinant Vectors pH contains a strong and tightly regulated lambda P_R promoter, a temperature sensitive λ cl repressor, an E. coli origin of replication, and Amp^r for selection. Because this vector encodes a temperature-sensitive repressor, a special E.coli strain was not required for regulation of expression. The entire coding region of the MAV-RT (EcoRl-Xhol fragment, obtained by restriction digestion or PCR with suitable primer pairs) , as characterized by the restriction map data of Table I, was inserted into the multiple cloning site (MCS) of pH. Briefly, the vector was restricted with EcoRI and Sail. A 1 : 1 ratio of insert to vector was ligated in the presence of 1 mM ATP in ligation buffer (100 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 20 mM DTT) and T4 DNA ligase using a convention protocol. Sambrook et al. , in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (2d Εd. 1989). The ligation mix was incubated at 16°C for 2-4 hours. The ligated mix was transformed into electro-competent E.coli cells in 1 mm cuvettes using a BioRad electroporator at 1.8 KeV and 200 ohms. The transformed cells were plated on LB-ampicillin plates and single colonies were picked for overnight growth and mini-prep analyses. The recombinants were then confirmed by sequence analyses. Subsequently, the 5' noncoding regions of selected recombinants were removed by site- directed mutagenesis where appropriate. The pH vector containing the full-length RT gene was named pHSΕMl and the vector having the 5' non-coding region deleted was called pHSΕMUΕ33 (i.e. , pHRT). The RT protein was expressed and analyzed by SDS-PAGE and RT assays were performed as described in Examples 5 and 6. Other prokaryotic vectors were also successfully used (e.g. , pET21d and pTZ18U, which have the T7 promoter and the lacZ promoter, respectively). Construction of Eukaryotic Recombinant Transfer Vectors

A baculovual expression system consisting of a transfer vector, a wild-type virus AcMNPV (Autogiaphd cahfornica nucleai polyhediosis virus) oi a derivative of ACMNPX (/ e , BacPakό (Clontech Inc )) was used to obtain recombinant transfer vectors containing the RT gene

The AcMNPV genome is a double-stranded circular DNA of 134 kb The size of the virus makes it difficult to directly manipulate the viral genome itself Therefore, transfer vector pBacPak9 was used to generate recombinant molecules in accordance with the invention, such as pMBacRT, pBacMIBA, pBacMIKA, pBacMIBAhis and pBacMIKAhis (see below) These recombinant molecules, containing exogenous and typically foreign RT coding regions, were used to introduce the sequence into the viral genome for expression and propagation Vector pBacPak9 has a strong polyhedron promoter which is induced in insect cells late in the replication cycle of the virus Hence, foreign genes, including lethal genes, expressed with this late promoter are not toxic to the growing cell The polyhedron gene is not necessary for the maintenance of the virus and was therefore replaced by the foreign gene (i e , MAV-RT pol gene)

The 2 81 kb Pstl fragment from pHSEMl, containing the full-length RT gene, was inserted into the Pstl site of the MCS of pBacPak9, and the recombinants were called pBpHPC3,4 (/ e. , pBacRT) Insertions of the gene were confirmed by mimprep analyses and sequencing The 5' non-coding region (see, SEQ ID NO 1) was removed by site- directed mutagenesis, as described in Sambrook et al , (1989) The resulting recombinant vector was called pBpHPCM10. i l , 17 (/ e , pMBacRT) In pMBacRT, the RT gene is flanked by viral DNA sequences of BacPakό, a derivative of AcMNPV When pMBacRT was introduced into insect cells along with BacPakό DNA, the plasmid recombined with the BacPakό DNA to yield recombinant, infectious progeny virus (Ml-5 and Ml-6, collectively Ml-5, 6) containing the RT gene

In general, when SF9 tissue culture cells are infected with lecombinant virus, the viral particles entered the cells and the viral DNA is uncoated in the nucleus Viral DNA replication occurs approximately 6-24 hours post-infection Duπng the late phase of the viral infection, approximately 48-72 hours after virus infection, all transcription is shut off except the genes having the polyhedron and plO promoters, which are transcribed at very high levels Hence, the RT gene under the control of the polyhedion promoter in the recombinant virus was expressed at high levels late in the infection cycle. This recombinant AcMNPV was propagated in the budded form only.

Example 2 A primer walking sequencing strategy implementing Sanger's enzymatic sequencing technique was used to confirm the sequence of the MAV-RT pol gene. Sambrook et al. , (1989). The sequencing template was the insert of pHSEMl . Primers were designed to be homologous or complementary to an end of a previously determined sequence. These primers were then used to progressively extend the identification of pol gene sequence until the sequence of the entire coding region had been determined. The polynucleotide sequence of the MAV-RT gene and the flanking sequences are set forth as SEQ ID NO: l . Amino acid sequences encoded thereby are set forth in SEQ ID NO:2. Of the 3, 155 bp presented in SEQ ID NO: l , 2,498 bp codes for the beta fragment (nucleotides 253-2751 of SEQ ID NO: 1) of MAV-RT; the alpha fragment of MAV-RT is encoded by nucleotides 253-1990 of SEQ ID NO: 1. These coding regions are expected to encode polypeptides containing amino acids 1-895 of SEQ ID NO: 2 (full- length RT; see also SEQ ID NO:3), amino acids 1-833 of SEQ ID NO:2 (β-like polypeptide; see also, SEQ ID NO:5) and amino acids 1-579 of SEQ ID NO:2 (α-like polypeptide; see also, residues 1-578 of SEQ ID NO:4). The β-like polypeptide is a fragment of the native MAV-RT β polypeptide. The α-like polypeptide is larger than the native MAV-RT α polypeptide and smaller than the native MAV-RT β polypeptide, with the native α polypeptide sequence extending from the N-terminus of the α-like polypeptide. For brevity, the α-like and β-like polypeptides are referred to as the α and β polypeptides, respectively.

Example 3 As described in Example 1 , plasmids pHSEMl and pBacRT were constructed to contain 2.95 kb and 2.81 kb inserts, respectively. These fragments contained the entire reverse transcriptase gene along with 5' and 3' non-coding regions. The 5 ' non-coding region of each construct was then removed by site-directed mutagenesis, a well-known technique in the art. In particular, the primer FSDRT (5'-TGTACTAAGGAGGTG- TTCATGACTGTTGCGCTACAT-3' ; SEQ ID NO:20) was used with pHSEM l as a template to generate pHRT (pHSEMUE33). Primer RSDBAC2 (5'-GCCAGATGT- AGCGCAACAGTCATATTTATAGGTTTTTTTATTAC-3'; SEQ ID NO:21) was used with pBacRT as a template to generate pMBacRT (pBPHPC3M10, pBPHPC3Ml l , pBPHPC3M 17, or, respective!) , pMBaclO, pMBacl l and pMBacl 7).

The full-length RT coding region was used as a starting material in constructing deletion derivatives that lacked the 3' end of the MAV-RT coding region to varying extents. Relative to the full-length gene (MI-5.6, see below), the 3 ' (C-terminal) deletion extending to the Kpnl site (MIKA) increased the RT expression level, as evidenced by SDS-PAGE. Relative to the full-length gene (MI-5,6), deletion of the region extending from the Bglll site to the 3' terminus (MIBA) increased the RT expression level, activity and solubility, as evidenced by SDS-PAGE and activity assays (see below). Relative to the alpha fragment of MAV-RT, the beta fragment has an additional 254 amino acids at the C-terminus, which provides an integrase activity. This region of the polypeptide contributes to the insolubility of the polypeptide and reduces its recovery from cell extracts, as shown by the relative insolubility of a ( +) integrase form of RT (e.g., the MIKA gene product, see below) compared to a (-) integrase form (e.g. , the MIBA gene product). Because the integrase domain is only needed for the retroviral life cycle and not for the RNA- or DNA-dependent DNA polymerase activities, this region was deleted in MIBA (α fragment equivalent). Note that the α fragment of MIBA (amino acids 1-578 of SEQ ID NO:2) is larger than the naturally occurring α fragment of MAV-RT (amino acids 1-573 of SEQ ID NO:2). Without wishing to be bound by theory, this deletion was expected to result in an increase in the solubility, and hence recovery, of the protein.

Using the full-length RT recombinants, additional clones were constructed to express polypeptides having C-terminal deletions in order to increase the levels of expression and to stabilize the RT activity (RNA-dependent DNA polymerase activity). Convenient restriction sites such as Bgl II (spanning nucleotides 1 ,986-1 ,991 of SEQ ID NO: l) and Kpnl (spanning nucleotides 2,745-2,750 of SEQ ID NO:l) were used to eliminate the 3' end of the coding region of the RT gene (see, Table I). The 3' deletion derivatives, encoding RT polypeptide fragments having C-terminal deletions, were obtained by Bglll-Ps or Kpnl-Pstl restrictions of pMBacRT and pHRT, respectively (Bglll and Kpnl sites in the MAV-RT coding region; Pstl site in the vector). Recombinant molecules containing the Bgl II-Pstl 3 ' terminal deletion were designated pBacMIBA and pHBRT (pH33ΔBP6) and recombinant molecules containing the Kpnl-Pstl deletion were designated pBacMIKA and pHKRT (pH33ΔKP5). The deletion derivatives pBacMIBA and pBacMIKA had approximately 1.2 and 0.4 kb deletions from the 3' end of the full-length gene (see, SEQ ID NO: l), respectively. The fragment bounded at its 3' end by the Bglll site (SEQ ID NO:6) was used to express an alpha fragment equivalent of RT and the fragment bounded by the Kpnl site (SEQ ID NO: 8) was used to express the beta fragment equivalent of RT (the β fragment equivalent of MIKA contained amino acids 1-832 of SEQ ID NO:2; native MAV-RT β contains amino acids 1-858 of SEQ ID NO:2).

Miniprep and sequencing analyses were done to confirm the identities of the recombinant clones described above. Recombinant viruses obtained from co-transfection with virus BacPakό and transfer vector pBacMIBA or pBacMIKA were called MIBA and M IKA, respectively.

Recombinants encoding 3 ' terminal amino acid tags

Without wishing to be bound by theory, the constructs that deleted the integrase domain of RT, such as MIBA and pBacMIBA, were not expected to retain the DNA binding, structure stabilizing, and polymerization functions attributable to the integrase domain. To re-introduce these functions, without the deleterious impact on solubility and host cell viability associated with the native integrase domain, codons specifying amino acids (His) were added to the 3' end of the modified RT coding regions. The basic nature of the added amino acids may have been responsible for increased binding to the negatively charged nucleic acids, enhancing the stability of the polypeptides. The increased binding may, in turn, have been responsible for the increase in activity found with the his-tagged RTs, relative to their untagged counterparts. In addition, the his tags may have contributed to the tendency of the his-tagged RTs of the invention to form polymers, perhaps through his-mediated chelation of metal ions such as Ni^{" '}. A his-tagged RT (MIBAhis) was found in homo-polymeric form (molecular weight greater than 200 kDa), as determined using non-denaturing PAGE and molecular sieve chromatography with Superose 12HR 10/30 (separation range of 1-300 kDa; Pharmacia-Upjohn). Thus, the invention contemplates RT polypeptides lacking an effective integrase domain, but having the capacity to bind DNA and/or polymerize. These additional functionalities may be provided by adding, preferably at the C-terminus of the modified RT, such structures as known DNA binding domains, zinc-finger or zinc-finger-like domains, polymerization domains, acidic amino acids, basic amino acids, or one or more cysteines. Such modified RTs may be ultimately derived from avian or non-avian sources. His-tag additions to the C-termini of the RT polypeptides were achieved by recombinant expression of coding regions fusing RT coding regions to His codons. In particular, the fusions were constructed b> adding oligonucleotides containing 6 histidine codons at the 3' end of the RT gene using ligase, as in the case of the construction of pBacMIKAhis, or by PCR amplification with oligonucleotides that specified 6 histidine codons, as in the case of the construction of pBacMIBAhis.

The construction of pBacMIKAhis was accomplished with oligonucleotides FNhis (SEQ ID NO:33) and RNhis (SEQ ID NO:34), each of which contained internal histidine codons and compatible Notl restriction sites at each end. Following their conventional syntheses, the oligonucleotides were annealed and ligated to the 3' terminus of RT in pBacMIKA cut with Notl. For the construction of pBacMIBAhis or pBacMIKAhis using PCR, primers FRT (SEQ ID ΝO:22) and either MlBARSDhis (SEQ ID NO:23) or MlKARSDhis (SEQ ID NO:24) were used with pHSEMl as the template. Blunt-ended and phosphorylated PCR products containing the 3 ' deletions and histidine tag-encoding regions were inserted into the Smal site in the MCS of pBacPak9. The his-tag derivatives of the transfer vectors were called pBacMIBAhis and pBacMIKAhis and the viruses obtained by co-transfection of Sf9 cells with the aforementioned transfer vectors and BacPakό were called MlBAhis ((-) integrase) and MlKAhis ((+) integrase), respectively. Introduction of the His codons led to increased activity of the encoded polypeptides in eukaryotes, as measured by SDS-polyacrylamide gel electrophoretic analyses and RT assays (see below). As shown below, the his-tag additions increased the stability (perhaps by providing a DNA binding site), activity, polymerization capabilities and ease of purification of RTs such as MlBAhis.

The 5' end of the MAV pol gene was also modified. Beyond deletion of the 5' non-coding sequence of pol (see the description of pHRT and pMBaclO above), the widely recognized Met initiation codon ("ATG") was introduced immediately upstream of the natural start codon (the Thr codon "ACT" at nucleotides 253-255 of SEQ ID NO: 1) of the MAV pol gene.

In general, the above-described cloning strategy reflected efforts to eliminate the integrase domain of avian RT and thereby avoid the insolubility and lethality problems associated with that protein domain. Deletion of 192 bp from the 3' terminus of the full- length MAV-RT gene (SEQ ID NO: 1) by terminating the coding region at the Kpnl site (Table I) produced the "MIKA" clone series. These clones coded for a β polypeptide that is smaller than the naturally occurring β polypeptide. These clones exhibited enhanced RT expression and the expressed polypeptides exhibited enhanced activity levels (compare below, the expression of Ml-5, 6 [full-lengthj to MIKA [β polypeptide]). Larger deletions extending from the 3' end of the full-length MAV-RT gene were constructed using a convenient Bglll site to generate the M IBA clone series. These clones encoded an α subunit of RT that was larger than the naturally occurring α polypeptide. The MIBA clones exhibited increased expression and activity, in comparison to the expression and activity of full-length MAV-RT; moreover, MIBA was more soluble than naturally occurring MAV-RT. The invention also contemplates polynucleotides and polypeptides resulting from a recognition that some advantageous properties of the integrase, e.g. , DNA binding and polymerization, could be re-introduced into avian RTs without re-introducing the deleterious (i.e. , insolubility and lethality) characteristics of the avian RT integrase domain. One approach is to attach RT integrase domains or non-RT integrase domains known in the art to the (-) integrase polypeptides or attach the coding regions of these domains to the polynucleotides encoding these (-) integrase polypeptides. Another approach is to add amino acid tags to the (-) integrase RT polypeptides (or corresponding codons to (-) integrase polynucleotides) as disclosed herein. A preferred tag is a basic amino acid tag such as a His tag. As disclosed below, a His tag was attached at the C- terminus of an α polypeptide equivalent (MlBAhis). This clone exhibited relatively high levels of expression, activity and solubility. Thus, the invention provides avian RTs improved in terms of expression and activity levels, and in terms of solubility and ease of purification, while retaining the processivity and thermostability characteristic of avian RTs. Accordingly, the invention contemplates the construction of analogous polynucleotides and recombinant molecules encoding RT polypeptides of unnatural length from other sources, such as MMLV, HIV. RSV, ASLV, ATV, and others. Further, the invention extends to polynucleotides encoding these RTs of modified length, or full length RTs, provided that the polynucleotides additionally encode polymerizing or nucleic acid binding domains, and preferably both domains, at their 3' termini. Examples of polynucleotides encoding a non-avian RT of unnatural length are polynucleotides encoding an RT portion or fragment having the amino acid sequence set forth at any one of the following: positions 1-765 of SEQ ID NO: 39 (an HIV-2 RT sequence), positions 1-800 of SEQ ID NO:41 (an MMLV-RT sequence), and positions 1-625 of SEQ ID NO:43 (an HIV-1 RT sequence). These polynucleotide sequences have some correspondence to the sequence of the polynucleotide encoding the MAV-derived MIBA polypeptide and are expected to function in a manner analogous to polynucleotides encoding MIBA. Of course, a polynucleotide encoding the full-length β polypeptide of HIV-2 (SEQ ID NO:38), or encoding equivalent polypeptides from MMLV or HIV-1 (SEQ ID NO:40 or SEQ ID NO:42, respectively), along with a 3' terminal sequence encoding a polymerizing and/or nucleic acid binding domain, are also contemplated by the invention.

With respect to polypeptides, the invention comprehends the polypeptides encoded by the above-described polynucleotides, as well as polypeptides that have a C-terminal polymerizing and/or nucleic acid binding domain that has been added by means other than expression. For example, an RT polypeptide having a Cys residue or a His residue attached at the C-terminus by chemical condensation falls within the scope of the present invention. In addition, effective elimination of an integrase domain, such as is found in avian RTs, may be effected by altering a suitable coding region by inserting, deleting, or substituting (transitions and/or transversions), one or more nucleotides. Thus, the invention contemplates RT polypeptides that are the same length as naturally occurring RT polypeptides. These RT polypeptides may have the same amino acid sequence as naturally occurring RTs, provided that the RTs of the invention have a polymerizing and/or nucleic acid binding domain at their C-termini. Alternatively, RTs of the same length as natural RTs may have sequences that differ from the natural RTs, thereby effectively eliminating integrase activity. The RTs of the invention may also be shorter, or longer, than naturally occurring RT polypeptides. The shorter RT polypeptides of the invention eliminate some, or all, of the C-terminal sequence of a naturally occurring RT which, in the case of avian RTs, contains the integrase domain. RTs of the invention that are longer than naturally occurring RT polypeptides contain the sequence of that naturally occurring RT and, in addition, sequence of an adjacent peptide region. Additionally, these polypeptides of unnatural length may have a polymerizing and/or nucleic acid binding domain added at their C-termini. Example 4

The RT constructs described in Example 3 were transformed into prokaryotic and eukaryotic host cells and the expression of RT polypeptides was analyzed. A prokaryotic host cell, Escherichia coli DH5αF', was transformed with pHRT, pHBRT or pHKRT, using a technique standard in the art. Cells subjected to the transformation protocol were plated on LB plates (10 g tryptone, 5 g yeast extract, 5 g NaCl, 1 ml of IN NaOH, 1.5 g agar, ddH-,0 in a total volume of 1 liter) containing 50 μg/ml ampicillin for selection of transformed host cells. Single colonies were picked, expanded in small culture (i.e. , 5 ml), episomal DNAs were rapidly isolated from an aliquot of cells, and the purified DNAs were analyzed for the presence of a recombinant molecule of the expected size. Dideoxynucleotide-based sequencing of these DNAs confirmed that the first ATG (i.e. , the initiation codon) was in-frame with the remainder of the RT coding region.

Another aliquot of those small cultures containing cells transformed with pHRT, pHBRT, or pHKRT was used to inoculate flasks containing 10 ml of LB-ampicillin and grown at 30°C until an OD^, of 0.6 was reached. Flasks containing these cells were then quickly shifted to 42 °C to de-repress the λP_R promoter and express the recombinant protein. After an hour at 42°C, cells were pelleted and analyzed for expression of protein by SDS-PAGE, Western blot analyses, and RT activity assays, as described below.

In general, about 10% of the expressed protein was recovered in soluble form and 90% of the expressed protein was found in inclusion bodies, as revealed by pelleting lysed cells at 12,000 x g for 5-15 minutes. RT activity was also found when expressing both the full-length and the deletion derivatives of the MAV pol coding region from other recombinant vectors, such as pTZ18U and pET21d, that contained similar insert fragments encoding full-length or C-terminally deleted MAV-RT. A eukaryotic host cell suitable for use in practicing the invention is the Sf9 insect cell. Several polynucleotides were separately introduced into Sf9 cells using the Baculoviral expression system. O'Reilly et al. , in Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press (1994). The polynucleotides (i.e. , pMBacRT, pBacMIBA, pBacMIBAhis, pBacMIKA, and pBacMIKAhis) were purified by the standard alkaline lysis method, as described in Sambrook et al. , (1989). The DNA was then centrifuged through a CHROMA SPIN+TE-400 column (Clontech Laboratories, Inc. , Palo Alto. CA.) at 500 x g for 7 minutes in a swinging bucket rotor. (HN-SII centrifuge from IEC. Inc.) This purified DNA was then used to transform eukaryotic cells.

Sf9 insect host cells were prepared for transformation using an established procedure. The Sf9 cells from an exponentially growing cell culture were initially counted using a hemocytometer and diluted to 5xl0⁶ cells/ml of TNM-FH Insect Cell Medium (Product No. T-1032; Sigma Chemical Co. , St. Louis, MO.) with 10% fetal bovine serum (FBS) and antibiotics (50 units/ml nystatin, 50 units/ml penicillin, and 50 μg/ml of streptomycin). Subsequently, 1.5 ml of this culture was added to each well of several 12- well tissue-culture plates. The cells were allowed to attach to the plate for a period of 1 hour. The medium covering the cells was then removed and 2 ml of TNM-FH medium without serum was added. The serum-free medium was swirled over the cells and again the medium was removed. This process was repeated one more time to remove all traces of fetal bovine serum (i.e. , FBS) and antibiotics. The cells were then incubated in TNM- FH medium for 30 minutes while the transfection mixture was prepared. The 50 μl transfection mixture contained 500 ng of DNA, 500 ng of Bsu36l- digested BacPakό viral DNA, and ddH₂O. This mixture was gently mixed with 50 μl of transfection reagent (Clontech, Inc.) and incubated at room temperature for 15 minutes to allow the transfection reagent to form a complex with the DNA, as recommended by the supplier of the transfection reagent. Medium covering the Sf9 cells was removed and 300-500 μl of TNM-FH medium was added to each well. To this medium, the transfection reagent-DNA mixture was added drop-wise while gently swirling the dish. The cells were then incubated at 27 °C for 5 hours before adding 2 ml of TNM-FH medium containing 10% FBS and the antibiotics identified above. DNA-cell contact was continued at 27 °C for 60-72 hours. Medium from these plates was then collected and used as primary virus stocks.

Primary virus stocks were subsequently subjected to plaque purifications by standard methods, as described in King et al. , in The Baculovirus Expression System: A Laboratory Guide (eds. Chapman and Hall, NA. 1992), to produce clonal stocks. The clonal stocks were amplified using a 1 : 1 virus to insect cell ratio to produce large quantities of recombinant viruses.

The viruses from the clonal stocks were used to infect insect cells and ultimately analyze RT expression in a eukaryotic environment. Based on the titer obtained from the plaque assays, an infection was set up using a ratio of 5 viruses per Sf9 cell. After 60 hours, the medium and cells were collected. The cells were pelleted, resuspended in cell lysis buffer ( 10 mM Tris HCl, pH 8.0. 50 mM NaCl, 5 % glycerol, 0.5 % Triton X-100, and protease inhibitors (50 μg/ml Benzamidine HCl. 0. 1 mM 4-(2-aminoeth l)-benzene- sulfonylfluoride, and 1 μg/ml pepstatin A)) and lysed by sonication. These samples were subsequently subjected to SDS-PAGE, Western blot analyses, and RT activity assays.

For large-scale expression studies, Sf9 cells were initially grown in T25 tissue culture flasks under the conditions described above. Sf9 cells adhering to the T25 tissue culture flasks were gently dislodged and adapted to suspension cultures as described by King et al , 1992. These suspension cultures were expanded in spinner flasks to a volume of 1-3 liters. When the insect cells reached a density of lxlO⁶ cells per ml, they were infected with a concentrated stock of recombinant viruses at a ratio of 5: 1 viruses per insect cell. A variation of a standard protocol was used to infect these cells. A large volume of amplified viral stock (MIBA, MIKA, MlBAhis, and MlKAhis, or M 1-5,6) was concentrated using one-half volume of 40% PEG 8000 and one-sixth volume of 5 M NaCl. Precipitated viruses were collected at 12,000 x g for 30 minutes using a Sorvall RC5C centrifuge (Dupont, Newtown, CT). The pelleted viruses were resuspended in lx PBS (10 mM K»PO₄, pH 7.5, and 150 mM NaCl) at 1/20 of the culture volume and stored at - 20^CC. Before infection, the viruses were filtered through a 0.2 μ filter.

After a 48 hour period of infection, 1 ml aliquots of infected cells were collected for RT assays to monitor RT expression levels. Cells were harvested at the peak of RT expression (generally around 60 hours post-infection), as determined from previous trials. Cells were pelleted at 5,000 x g for 30 minutes and stored at -80°C.

Polypeptides expressed in insect cells were also characterized by SDS-PAGE and Western blot analyses. Results of a Western blot assay using a mixture of anti-RT monoclonal antibodies 1D8, 2E10, 6F1 , 4C4, 9H 10 and 9C2 are shown in Fig. 1 (lane 1 : prestained molecular weight markers of 123 kDa, 90 kDa, 64 kDa, 50 kDa, and 38 kDa; lane 2: native AMV-RT(nRT) ( lane 2), lane 3 : M lBAhis, and lane 4: MlKAhis. Further analysis of the antigenic properties of MlBAhis and native RT revealed that monoclonal antibody 6F1 recognized native RT but failed to recognize the MlBAhis polypeptide. Thus, at least one epitope found on native RT is not found on MlBAhis, indicative of structural differences between the proteins.

The results further indicate that both MIBA and MIKA expressed ten-fold more RT than Ml-5, 6, which encodes full-length RT. When cell pellets were assayed for RNA- dependent DNA polymerase activity, M IBA was expressed at 10.000 units per liter of insect cell culture, whereas MIKA and Ml-5, 6 were each expressed at 1 ,000 units per liter of insect cell culture. Though M IKA expressed as well as M I BA when analyzed on Western blots, active MIKA recovered from the cell pellet was ten-fold less than MIBA. Most of the expressed MIKA remained insoluble in the pellet. Although the corresponding his-tagged proteins (MlBAhis and MlKAhis) were expressed at levels similar to their MIBA and MIKA counterparts as revealed by Western blotting, the activities of the his- tagged proteins were higher. MlKAhis was expressed at 2,000 units per liter of insect cell culture and MlBAhis was expressed at 200,000-400,000 units per liter of insect cell culture.

The Baculoviral system is preferred for expression of RT and fragments thereof. A relative comparison of RT expression in prokaryotic and eukaryotic cells, as measured by reverse transcriptase assays of purified recombinant and crude protein, revealed that His-tagged RT polypeptides from eukaryotic insect cells were most active and stable, while untagged polypeptides expressed in prokaryotic cells were less active and stable.

Recombinantly expressed polypeptides of the invention were purified using conventional protocols, with metal-affinity chromatography included for the isolation of His-tagged polypeptides. Host cells containing recombinant molecules (i.e. M 1-5,6, M IBA. M IKA, MlBAhis and MlKAhis) encoding an RT or fragment thereof were centrifuged and the cell pellet was solubilized in 20 ml cell lysis buffer (20 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.5 % Triton X, and 5 % glycerol) per gram of cell pellet. The resuspended cells were sonicated with five 30-second bursts at 50% power on ice with 30 seconds of cooling between each round of sonication. Sonicated cells were then stirred at a low speed on a magnetic stirrer at 4°C for one hour to complete cell lysis. The lysed samples were centrifuged at 12,000 x g for 30 minutes. The pellet was discarded and the supernatant was subjected to column chromatography.

RTs lacking his tags were purified according to conventional protocols, which included removal of cell debris by centrifugation and subjection of supematants to chromatographic purification procedures known in the art. The soluble extract containing his-tagged RTs were mixed with a commercially available Ni⁺ ' affinity column (Ni-NTA resin from Qiagen, Inc. , Chatsworth, CA), thereby using the his tags for their known purpose of facilitating purification via metal affinity chromatography. The extract and affinity resin were gently rocked on ice for 1 hour in a 50 ml plastic test tube. The resin was then packed in a column and washed with two column volumes of wash buffer (20 mM Tris HCl, pH 8.0, 250 mM NaCl, 0.5 % Triton X-100, and 5 % glycerol) and two column volumes of buffer A (20 mM Tris-HCl, pH 8.0. 250 mM NaCl. 0.5 % Triton X- 100, 5% glycerol and 50 mM imidazole). (Of course, the extract could have been applied to a pre-formed affinity column and purified using conventional column chromatography, as would be understood in the art.) The protein bound to the column was eluted by setting up a linear gradient from buffer A to buffer B (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.5% Triton x-100, 5% glycerol and 250 mM imidazole).

Fractions from the nickel affinity column that had RT activity were analyzed by SDS-PAGE to determine the purity of the protein, as shown in Fig. 2. Fig 2 presents an electrophoretogram of an 8% SDS-PAGE gel stained with Coomassie Blue. The lanes of the gel shown in Fig. 2 contain molecular weight markers of 94 kDa, 64 kDa, 43 kDa, 30 kDa and 20 kDa (lane 1) and aliquots of fractions obtained from the nickel affinity column ( lanes 2 to 4) The fractions that were greater than 95 % pure were pooled and dialyzed against storage buffer (200 mM KPi, pH 7.2, 5 mM DTT, 0.2% Triton X-100 and 50% glycerol). Additionally, conventional purification steps may be incorporated into the protocol to achieve greater purity, as would be understood in the art.

Protein concentrations were determined using the Bradford protein assay (BioRad Laboratories, Inc., Hercules, CA). Generally, the specific activity of rRT (MlBAhis) was calculated to be approximately 30,000-100,000 units/mg, which is similar to the specific activity of nRT (30-100,000 units/mg).

Example 5 The purified rRT prepared from cultures expressing MlBAhis at 400,000 units/liter of culture, a level well beyond a commercially feasible production limit, was found to be greater than 95 % pure as judged by electrophoretic fractionation using 10% SDS-PAGE. The apparent molecular weight of the monomer is 60 kDa, which compares well with the calculated molecular weight of approximately 59.5 kDa. The recombinant protein was analyzed on a 12.5 % polyacrylamide non-denaturing gel for the presence of monomers and polymers (e.g. , dimers) using the Pharmacia Phast System. The protein sample was prepared in either of two ways. One aliquot was completely denatured by heating at 100°C for three minutes in treatment buffer (0.125 mM Tris-HCl, pH 6.5, 4% SDS, 20% glycerol, 10% β-mercaptoethanol). Another aliquot was partially denatured at 70°C in treatment buffer without 2-mercaptoethanol. Under completely denaturing conditions. rRT was observed to migrate at approximately 66 kDa (BSA marker) and the partially denatured samples had additional bands ranging from 60-200 kDa, indicating that rRT formed polymers. Protein size determinations were confirmed using molecular sieve chromatography with Superose 12HR 10/30 (separation range of 1-300 kDa), as described above, which revealed that the majority of the rRT eluted between beta amylase (approximately 200 kDa) and apoferritin (443 kDa). Thus, the rRT was predominantly in a polymeric form. Without wishing to be bound by theory, the addition of C-terminal histidine residues may have provided a polymerization capacity, perhaps by complex formation via metal (e.g. , nickel) chelation, to substitute for the loss of that capacity attributable to the integrase domain, which had been deleted. Thus, the invention contemplates RT polypeptides having C-terminal attachments in the form of compounds capable of promoting polymer formation. Suitable compounds would include, but are not limited to, a plurality of basic or acidic amino acids, as well as Cys residues capable of disulfide bond formation.

Expressed rRT was also characterized immunochemically. Monoclonal antibodies against AMV reverse transcriptase were prepared using techniques well known in the art. See Harlow et al. , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988). Briefly, spleen cells from a mouse that had been immunized with RT were fused with mouse myeloma cells to make hybridomas. These hybridomas were allowed to grow into colonies in 96-well plates; supematants from these wells were then tested to find hybridomas that appeared to make anti-RT antibodies. Further testing confirmed these results.

To prepare spleen cells for hybridoma production, a BALB/C mouse (female, ten weeks old, obtained from Harlan Sprague Dawley, Madison, WI) was immunized by several intraperitoneal injections with AMV-RT (Molecular Biology Resources, Inc.) using a conventional immunization schedule. To prepare RT for injection, the storage buffer was removed from purified RT by diluting the enzyme in phosphate-buffered saline (PBS) and reconcentrating it using a Centricon 30 concentrator (Amicon Corp.). The concentrated RT was then diluted again in PBS and emulsified with an equal volume of an adjuvant. For the initial injection, the adjuvant was complete Freund's adjuvant (Sigma Chemical Co.); for the booster injections, the Ribi Adjuvant System (Ribi Immunochem Research, Inc. Hamilton, VT) was used. The dose of RT was approximately 20 micrograms per injection. The injections were made over a period of eight months, with successive intervals of five weeks, four weeks, three weeks, eleven weeks, eight weeks, and three weeks. The fusion was performed five days after the final boost.

For the fusion experiment, the mouse was sacrificed and spleen cells were isolated and fused with myeloma cells (P3X63-AG8.653, ATCC CRL 1580), using procedures well known in the art. See Harlow et al. In particular, the cells were fused in 50% polyethylene glycol, resuspended in a selection medium (i.e. , HAT medium), and distributed into the wells of fourteen 96- well plates. After three weeks of growth, approximately 350 wells contained hybridoma colonies. Hybridomas making anti-RT antibodies were identified by ELISA. For this procedure, the wells of 96-well polystyrene ELISA plates were first coated with purified RT (2 micrograms RT/ml in 100 mM Tris-HCl, pH 8.5, 0.05 % NaN₃: overnight incubation at room temperature), then washed with TBST (Tris-buffered saline, pH 8.5, 0.05 % Triton X-100) to remove excess RT. For the assay itself, the wells were filled with 95 microliters of TBST plus 5 microliters of hybridoma culture supernatant. The plates were incubated at room temperature for two hours, then washed with TBST to remove unbound immunoglobulin. To detect wells with anti-RT antibodies, peroxidase-conjugated goat anti-mouse IgG (heavy-chain specific; Jackson ImmunoResearch, West Grove, PA) was diluted 5, 000-fold into TBST and added to the wells of the ELISA plates. After the wells had been incubated for one hour at room temperature, the unbound peroxidase conjugate was removed by thorough washing of the plates with TBST. Wells positive for RT were visualized colorimetrically following addition of the substrate 3-methyl-2- benzothiazolinone hydrazone/3-dimethylaminobenzoic acid/hydrogen peroxide to detect immobilized HRP. Hybridomas from positive wells were repeatedly cloned by limiting dilution until all wells with growth were ELISA-positive.

Supematants from wells that tested positive by ELISA were further screened by irnmunoprecipitation of RT using techniques well known in the art. See Harlo et al. The immunoprecipitation assay relies on the presence of protein A (which binds IgG) on the surface of Staphylococcus aureus cells (SAC, Sigma Chemical Co.). Since protein A does not bind strongly to mouse IgG, a pellet of centrifuged SAC cells was first treated with rabbit anti-mouse IgG antibodies. The pellet from 10 microliters of a 10% suspension of these cells was then incubated with 50 microliters of hybridoma culture supernatant for 2 hours at room temperature. The resultant SAC cells were centrifuged. washed, and resuspended in diluted RT. The RT cell suspensions were incubated for 3 hours at 4°C and centrifuged. The resultant supematants were removed and tested for depletion of RT activity using a standard radiochemical assay.

Six hybridoma lines tested positive in both the ELISA and immunoprecipitation assays. These lines were designated 1D8, 2E10, 4C4, 6F1 , 9C2 and 9H10. All six monoclonal antibodies had gamma- 1 , kappa isotypes.

The form of active rRT (/^'. e. , monomer or polymer) was confirmed using ELISA in a sandwich format with anti-RT monoclonal antibodies. Initially, monoclonal antibody was immobilized in DNA bind plates. Costar Corp. , Cambridge MA. The plate was then blocked with BSA to prevent non-specific binding. The wells were then incubated with purified rRT (i.e. , MlBAhis). Excess or unbound protein was removed by washing with phosphate-buffered saline. The wells were then incubated with the same monoclonal antibody linked to biotin for detection. If the rRT existed as a monomer, the biotin-linked monoclonal antibody should not bind to it. However, the biotin-linked monoclonal antibody did bind to the rRT, indicating that the rRT had formed a polymer.

To determine the purity of the samples containing reverse transcriptase, recombinant protein expressed from each of a variety of clones (e.g. , MlBAhis) and found in either the solubilized cell pellets or protein fractions from the different chromatographic columns used in purification were subjected to SDS-PAGE. Samples were electrophoresed on 8% polyacrylamide gels containing 6% stacking gels, followed by Coomassie Blue R- 250 staining using standard protocols (Sambrook et al. , 1989). The recombinant protein was found to be greater than 95 % pure.

Using the Pharmacia Phast System, the recombinant (MlBAhis) and native reverse transcriptase, as well as appropriate standards supplied with the system (i.e. , IEF 3-9), were subjected to isoelectric focusing electrophoresis. (Pharmacia-Upjohn, Piscataway, NJ.) The experimentally determined pi values of the rRT and rRT were 6.0. The theoretical pi of rRT, calculated from its amino acid sequence, was 6.8.

For analyses of total expression, host cells containing one of several recombinant DNAs (i.e. , pMBacRT, pBacMIBA, pBacMIKA, pBacMIBAhis, and pBacMIKAhis) were induced to express recombinant protein. The induced cells were pelleted at 12,000 x g for 5 minutes. The cell pellet was then resuspended in SDS sample buffer (Sambrook et al. , 1989) or cell resuspension buffer (20 mM Tris HCl, pH 8.0, 250 mM NaCl, 0.5 % Triton X-100. and 5% glycerol) to assess the solubility of the protein. Resuspended cells were pulse-sonicated three times at a setting of 3 (Virsonic 100 from Virtis Company, Inc. , Gardiner, NY) for 20 seconds each (500 mM Tris HCl, pH 6.5, 14% SDS, 30% glycerol, 9.3 % DTT. and 0.012% bromophenol blue). Small aliquots of the samples in SDS sample buffer were loaded on duplicate gels and electrophoresed. One of the duplicate gels was stained with Coomassie Blue and the other gel was used to transfer protein to a 0.2 μ nitrocellulose membrane using a Bio-Rad transfer apparatus for Western blot analysis. Bio Rad Laboratories, Inc. , Hercules, CA. Detection of expressed protein in fractionated crude lysates was possible using specific, monoclonal anti-RT antibodies (a mixture of monoclonal antibodies 4C4, 1D8, 2E10, 6F1 , 9H10, and 9C2; Molecular Biology Resources, Inc. , Milwaukee, WI) to detect the recombinant protein.

In practice, the nitrocellulose membrane containing the transferred protein was contacted with a blocking buffer (5% casein hydrolysate, 150 mM NaCl, 10 mM Tris HCl, pH 8.0) for 30 minutes followed by incubation with a 1 : 1000 dilution of anti-RT monoclonal antibody in blocking buffer. After overnight incubation, blots were rinsed in wash buffer (10 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.5 % Tween 20) and incubated with a 1 :5000 dilution of alkaline phosphatase-conjugated goat anti-mouse antibody in blocking buffer for 1 hour. Subsequently, the blots were rinsed 3x with wash buffer and lx with AP buffer (100 mM Tris HCl, pH 9.5, 5 mM MgCl₂ and 100 mM NaCl). RT was indirectly detected by performing a colorimetric phosphatase assay using a standard substrate mixture of NBT (nitroblue tetrazolium; 75 mg/ml in dimethylformamide) and BCIP (5-bromo-4-chloro-3-indolyl phosphate, 50 mg/ml in dimethylformamide), which forms a blue precipitate when dephosphorylated by any immunologically immobilized phosphatase. The anti-RT antibody recognized two bands, one at approximately 61 kDa and one at 92 kDa, in the lane containing native RT. In the lane containing recombinant, His-tagged RT expressed from MlBAhis (alpha fragment equivalent), a single band at approximately 60 kDa was found; in the lane containing recombinant, His-tagged RT expressed from MlKAhis (beta fragment equivalent) a single 91 kDa band was found.

Assays were also performed to determine the intrinsic/extrinsic exonuclease, endonuclease, (i.e. , nicking) DNase, and RNase activities of the rRT. An assay for 3'— > 5' exonuclease activity was performed using radiolabeled Taql fragments of lambda DNA as a substrate. The 3' ends of 7α<7l-digested lambda DNA fragments (265 μg) were labeled with 60 μCi [ H]-dCTP (57.4 μCi/mmole) and 60 μCi [³H]-dGTP (8.9 μCi) using 40 units of exo^" Klenow fragment of DNA polymerase in a standard labeling reaction. Sambrook et al , (1989). The 3'— > 5' exonuclease assay was performed in a final volume of 10 μl containing 50 mM Tris HCl, pH 7.6, 10 mM MgCl₂, 1 mM DTT. 0.015 μg of labeled Taql fragments of λ DNA, and either 2.5 or 10 units of RT enzyme. One unit of RT enzyme is the amount of enzyme required to incorporate 1 nmol of dTTP into an acid- insoluble form in 10 minutes at 37°C under the stated assay conditions (see. Example 6). Each sample was incubated at 37°C for 1 hour. The reaction was terminated by the addition of 50 μl of yeast tRNA and 200 μl of 10% trichloroacetic acid. After incubation for 10 minutes on ice, the samples were centrifuged for 7 minutes in a microcentrifuge. The supernatant (200 μl), which contained the released label, was removed and added to 6 ml of scintillation fluid and counted in a scintillation counter. The results showed that the rRT released 0.13 % of the label, an acceptably low level of 3'— > 5 'exonuclease activity.

The rRT was also subjected to a 5'— > 3' exonuclease assay, using radiolabeled Haelll fragments of λDNA. The λ fragments were 5' end-labeled using 60 μCi [γ-³³P] dATP (2,000 Ci/mmol) and 40 units of T4 polynucleotide kinase in a conventional procedure. Sambrook et al , (1989). Except for the use of 5' end-labeled Haelll fragments as substrate, this assay was performed in accordance with the description of the 3'— > 5' exonuclease assay above. The purified rRT released - 0.36% of the label into an acid-soluble form, an acceptably low level of 5'— > 3' exonuclease activity. Double-stranded and single-stranded DNase assays were also performed using the protocol for the 3'-> 5' exonuclease assay, again with the exception of the type of labeled substrate being used. For each of the DNase assays, intact lambda DNA (0.5 μg) was labeled with 30 μCi [α- ³PJ dATP (2,000 Ci/mmol) using the random primer extension technique understood in the art. Each assay used 0.015 μg of labeled λ DNA. For single- stranded DNase assays, the labeled λDNA fragments were further subjected to heat denaturation (3 minutes at 100°C followed immediately by chilling on ice) to prepare the substrate. Again with the exception of the type of substrate employed, each of the DNase assays were conducted as described above in the context of the 3'— > 5' exonuclease assay. The rRT released 0.5 % of the label in the double-stranded DNase assay: 0.02% of the label was released in the single-stranded DNase assay. Both results indicate acceptably low levels of DNase activities. The purified rRT was also subjected to an endonuclease, or nicking, assay by examining the extent to which the rRT converted a supercoiled substrate in the form of pBR322 to a relaxed form, as visualized by agarose gel electrophoretic fractionation. The assay for endonuclease activity was performed in a final volume of 10 μl containing 50 mM Tris HCl, pH 7.6. 10 mM MgCl₂, 1 mM β-mercaptoethanol, 0.5 μg pBR322. and 2.5, 5 or 10 units of enzyme. Each sample was incubated at 37°C for 1 hour. Two microliters of 0.25% bromophenol blue, 1 mM EDTA and 40% sucrose were added to stop the reaction. After a brief centrifugation, 6 μl of the sample were electrophoresed on a 1.0% agarose gel in IX TBE. Sambrook et al. , (1989). The results showed that less than 10% of the supercoiled substrate was converted to a relaxed form, an acceptably low level of nicking activity.

The rRT was also characterized in terms of its RNase activity. In particular, this assay was designed to measure general RNase activity and, specifically, not an RNase H activity. Substrate was prepared using run-off transcription from a T7 promoter in the presence of [α-³ P] UTP. In particular, the plasmid pPV2 (a pTZ-based vector containing a ColEl ori; an ampicillin selectable marker; T7, T3 and lac promoters; and a 695 bp insert from plum pox virus) was linearized with RvwII. The run-off transcription reaction was performed with 1 μg of linearized pPV2, 30 μCi of [α-³³P] dATP (2,000 Ci/mmol), and 10 units of T7 RNA polymerase using a conventional procedure. The RNase assay was then performed in the presence of single-stranded RNA substrate (0.15 μg) and rRT (2.5, 5 or 10 units). Released label was again recovered as acid-soluble material using the TCA precipitation procedure described above. Scintillation counting showed that 1 % of the radiolabel was released, indicating an acceptably low level of RNase activity.

Example 6

The RNA-dependent DNA polymerase activities of native RT and recombinant RT (purified expression product of MlBAhis) were compared. One unit of enzyme was compared in RT assays with either poly rA:dT₁₂._lg (20: 1) or mRNA as substrate. Product quantity was determined by either glass filter precipitation or binding to DE52 filters; product quality was monitored by autoradiography of a 1.2% TBE agarose gel containing fractionated reaction products.

The reverse transcriptase activities of the native and recombinant proteins were compared using a modification of a procedure described by Meyers et al , Biochemistry 30:7661-7666 (1991). The reaction mixture contained lx reaction buffer ( 50 mM Tris- HCl, pH 8.3, 40 mM KC1, 10 mM MgCl₂), 1 mM DTT, 0.4 mM poly rA:dT₁₈, 0.5 μCi [α-³²P] TTP (3,000 Ci/mmol), 0.5 mM dTTP, 1 unit of enzyme (one unit of RT enzyme is the amount of enzyme required to incorporate 1 nmol of dTTP into an acid-insoluble form in 10 minutes at 37 °C under the stated assay conditions), and ddH₂O to 50 μl total volume. Reaction mixtures without enzyme were pre-incubated at 37 ^CC for 1 minute prior to the addition of enzyme. Reactions were then incubated at 37 °C for 20 minutes, and terminated by adding 2 μl of 0.5 M EDTA followed by applying 40 μl of each reaction mixture to separate DE52 filter membranes. The filters were washed three times with 5 % Na₂HPO₄ for 5 minutes each, then rinsed with ddH₂O followed by 95 % ethanol. The filters were air dried, placed in scintillation fluid, and immobilized radioactivity was quantitated. A variation of the filter assay was used to compare the quantity and quality of reaction products. Messenger RNA, 891 bp control and 7.5 kb mRNA, were obtained from GIBCO BRL, Gaithersburg, MO. The following substitutions in the assay described above were made: 1 μg of mRNA primed with 0.5 mM oligo dT₁₂._lg primer, instead of poly rA:dT_lg; and mixed dNTPs (0.5 μM each of dGTP, dATP, TTP and dCTP, and 0.02 μCi [α-³²P]-dATP (6,000 Ci/mmol)), instead of [α-³²P] dTTP. Reactions were initiated by adding 5 units of RT to the reaction mixture. After 1 hour of incubation at 37° C, a 20 μl sample was removed and mixed with 5 μl of stop solution (95% de-ionized formamide, 10 mM EDTA, 0.05% xylene cyanol FF, and 0.05 % bromophenol blue) and loaded onto a 1.2% TBE agarose gel along with a 1 kb ladder of standards (Chimerx, Madison, WI). Gel samples were electrophoresed at 100 volts for approximately 2 hours and dried. Dried gels were autoradiographed at -70°C for 3 days and developed to visualize bands. The results are presented in Fig. 3, which presents autoradiographic data showing size- fractionated reverse transcriptase products using poly A-tailed mRNA as a template and oligo dT₅₀ primers. In particular, the template was 891 nucleotides (lanes 2 and 4) or 7,500 nucleotides (lanes 1 and 3); nRT was used in reactions analyzed in lanes 3 and 4, while rRT was used in reactions analyzed in lanes 1 and 2. Both the native and recombinant RTs produced products of 891 bp and 7.5 kb, depending on the size of the mRNA template.

Example 7

The properties of native MAV-RT and recombinant RT were compared. In particular, optima for temperature, pH, magnesium ion concentration, and other divalent cation (i.e., calcium, copper, manganese and zinc) concentrations were determined. a) Temperature optima The RNA-dependent DNA polymerase activity of native MAV-RT and recombinant MAV-RT (i.e. , MlBAhis) were compared in RT assays conducted at different temperatures.

The relative RT activities of the enzymes were compared between 37 °C and 70 °C at pH 8.0. The activity assays were performed in a 50 μl reaction mixture, containing 50 mM Tris HCl, pH 8, 40 mM KC1, 10 mM MgCl₂, 1.34% trehalose, 2% maltitol, 1 mM DTT, 0.5 mM poly rA:dT₁₈, 0.5 mCi [³H]-dTTP (70-90 Ci/mmol), 0.5 mM dTTP, 5 U enzyme (rRT or nRT), and ddH₂O. Duplicate reactions were incubated at each temperature for 10 minutes. Products were quantitated by determining the [³H]-dTTP incorporated using a scintillation counter. The results are presented as counts per minute as a function of temperature in degrees Celsius, as shown in Fig. 4A (black: rRT (i.e., MlBAhis); hatched: nRT). These results reveal that the optimum temperature for both nRT and rRT in RT assays was 55 °C.

The temperature profiles of nRT and rRT (i.e. , MlBAhis) in RAMP assays were also determined. RAMP reactions were conducted as described in PCT/US97/04170, incorporated herein by reference in its entirety. In particular, the target nucleic acid being amplified was Cryptosporidium mRNA from one oocyte. As described in detail in Example 8, this mRNA target was reverse transcribed into cDNA at different temperatures using 20 units of native RT or 15 units of recombinant RT. The results are presented absorbance at 450 nm as a function of temperature in degrees Celsius, as shown in Fig. 4C (hatched line: standard; closed circles: rRT (i.e. , MlBAhis)). The actual absorbance values at the various temperatures are shown below the figure (upper row: standard; lower row: rRT). b) pH optima

The relative RT activities of nRT and rRT in reactions at various pH values were also compared. Two sets of comparative reactions were designed: one set incubated at a conventional temperature of 37° C, the other set incubated at a 60° C temperature suitable only for thermostable enzymes.

The pH values of selected buffers were adjusted at room temperature. Activity assays were performed in a 50 μl reaction mixture, containing 40 mM KC1, 10 mM MgCl₂, 1 mM DTT, 0.5 mM poly rA:dT_]8, 0.5 mCi [Η]-dTTP (70-90 Ci/mmol), 0.5 mM dTTP, 5 units enzyme, ddH₂O, and 50 mM Tris-HCl (pH 6, 7, 8, 8.3, 9, or 9.5). Reactions were incubated at 37° C or 60° C for 10 minutes. Products were quantitated by determining the [³H]-dTTP incoφorated as counts per minute using a scintillation counter, with the activities of nRT and rRT (i.e. , M lBAhis) under the various pH conditions being shown in Figs. 4D and 4E (black: rRT (i.e. , M lBAhis); gray-hatched: nRT). The data in the Figures establish that the optimum pH for nRT and rRT is pH 8. c ) Mg^{+ +} ion optima The RT assay described in Example 7(b) was modified to determine the influence of MgCl₂ concentration on the activities of the native and recombinant RTs. The reaction buffer contained 50 mM Tris-HCl, pH 8.3, and MgCl₂ ranging in concentration from 0-100 mM; all other reaction components were as described in Example 7(b). The reactions were incubated at 37°C. Incoφorated [³H]-dTTP was measured by scintillation counting, with the results presented as counts per minute. The optimum MgCl₂ concentration was found to be 5 mM for both nRT and rRT, as shown in Fig. 4F (black: rRT (i.e. , MlBAhis); gray-hatched: nRT). d) Other divalent cation requirements

The reaction described above in the context of determining Mg^{+ ^} concentration optima was modified to determine the influence of different divalent cations on RT activity. The reaction buffer included 50 mM Tris-HCl, pH 8.3, and 10 mM of the chloride salt of a divalent cation (MgCl₂, CuCl₂, MnCl₂, ZnCl₂, or CaCl₂). Independent experiments were performed and a curve was constructed. Fig. 4G shows the activities of the enzymes as counts incoφorated as a function of the cation used in the reaction (black: rRT (i.e., MlBAhis); gray-hatched: nRT). As shown in Fig. 4G, maximal activity of both nRT and rRT (i.e. , MlBAhis) was achieved using magnesium as the divalent cation.

Example 8

Conceptually, RT-PCR consists of a pre-amplification reaction followed by an amplification reaction. The pre-amplification reaction involves the use of reverse transcriptase to synthesize the first strand of cDNA using a CAT (i.e. , chloramphenicol acetyltransferase) mRNA as template. The CAT mRNA was provided in the Superscript kit from GIBCO-BRL, and the reaction was performed according to the supplier's recommendations. Following this reaction, the RNA from the RNA-DNA hybrid was removed by RNase H to free the first strand for use as a template in a Polymerase Chain Reaction (PCR).

The pre-amplification reaction mixture initially consisted of 50 ng of control mRNA (i.e. , CAT mRNA), 500 ng of oligo dT₁₂.₁₈, and ddH₂O to bring the mixture to a total volume of 12 μl. This mixture was incubated at 70 °C for 1 minute. Subsequently, 2 μl of lOx PCR buffer, 2 μl of 25 mM MgCl₂, 1 μl of dNTP from a combined stock solution containing 10 mM each of dGTP, dATP, TTP and dCTP, and 2 μl of 0.1 M DTT were added to the mRNA/oligo dT mixture. One set of reactions was incubated with 20 U of nRT and the other set of reactions was incubated with 20 U of rRT (i. e. , M 1 B Ahis) . One tube from each set was incubated at one of several temperatures and each reaction proceeded for one hour. The reactions were terminated by incubation at 90 °C for 2 minutes. Reactions were then cooled on ice and 1 μl of RNase H was added to each tube and incubated at 37 °C for 20 minutes. For the amplification reactions, each reaction mixture was assembled in a thin-wall tube containing: 5 μl of lOx PCR buffer, 3 μl of 25 mM MgCl₂, 1 μl of dNTP from a combined stock solution containing 10 mM each of dGTP, dATP, TTP and dCTP, 1 μl each of 10 μM amplification primer 1 and 10 μM amplification primer 2 as supplied in the superscript kit, 1 μl of Taq DNA polymerase and Pyrococcus woesii (i.e. , Pwo) DNA polymerase mix, (Boehringer Mannheim Corp. , Indianapolis, IN) 2 μl of the cDNA mixture from the first-strand synthesis reaction and ddH₂O to 50 μl total volume. Reaction products were analyzed by subjecting 5 μl of the reaction to fractionation on a 1.2% TBE agarose gel and determining the intensity of the bands, in ng of DNA, using an imager equipped with a DC40 camera and Kodak Digital Sciences ID™ software. The quantity of DNA synthesized by rRT was comparable to the quantity synthesized by nRT.

The results showed that the temperature optimum for RT-PCR was 60° C using either nRT or rRT, as shown in Fig. 4B (results are presented as ng of PCR products produced as a function of temperature in degrees Celsius, with open squares indicating rRT (i.e. , MlBAhis) and solid squares indicating nRT). The quantity of gene-specific products was greater at 60°C than at 37°C. The optimum temperature for RNA-dependent DNA polymerase activity for both nRT and rRT was 55 °C (see, Example 7a and Fig. 4A). The differences in temperature optima are probably due to the need for both DNA-dependent DNA polymerase and RNase H activities (having different temperature optima) in RT- PCR. Rapid Amplification (i.e. , RAMP) is an amplification technique disclosed in

International Application Serial No. PCT/US97/04170. A RAMP reaction was also performed using an RT according to the invention and a first strand of cDNA from a Cryptosporidium oocyte mRNA as a template, along with a nicking enzyme (i.e., BsiHKCl) and Bst DNA polymerase. The Bst DNA polymerase provided both polynucleotide synthesis activity and strand displacing activity.

The reaction consisted of 35 mM K»PO , 0.7 mM Tris-HCl, pH 7.9, 1.4 mM dCTP, 0.5 mM each of dATP, dGTP and dTTP, 35 mg of Bovine Serum Albumin, 10.2 mM MgCl₂, 3.4 mM KC1, 0.7 mM DTT, 2% Maltitol, 1.34% Trehalose, 0.5 mM of Amplification Primer 1 (5'-ACCCCATCCAATGCATGTCTCGGGTCGTAGTCT- TAACCAT-3'; SEQ ID NO:31) and Amplification Primer 2, (5'-CGATTCCGCTC- CAGACTTCTCGGGTGCTGAAGGAGTAAGG-3'; SEQ ID NO:32) and 1 % glycerol. To each reaction, 15 units of rRT (i.e. , MlBAhis) or 20 units of nRT were added, along with 36 units of Bst DNA polymerase and 250 units of BsiHKCl in a total volume of 10 μl.

The amount of product synthesized in each reaction was measured by a plate assay. The plate assay consisted of a gene-specific capture primer (5'- AAACTATGCCAACTAGAGATTGGAGGTTGTTT-3'; SEQ ID NO: 30) bound to the wells of a microtiter plate and used to capture the product. The captured product was then detected by an oligonucleotide (HRP-conjugated P2 Comp; SEQ ID NO:37) linked to Horse Radish Peroxidase. The amount of bound HRP was detected by a colorimetric assay standard in the art.

The amount of product synthesized by the rRT was two-fold more than the quantity synthesized by nRT between temperatures of 55° C to 64° C, as shown in Fig. 4C. The difference in temperature optima between the RT assays and the amplifications may be due, in part, to the differences in the relative RNase H activities at the assessed temperatures. The lowest RNase H activity was seen between 60°-65°C, temperatures that also produced longer cDNA products and greater amplification of templates. The temperature profile of the RNase H activity of rRT is shown in Fig. 6B.

Example 9

In addition to RNA-dependent DNA polymerase activity, MAV-RT has additional enzyme activities, such as DNA-dependent DNA polymerase activity. The DNA- dependent DNA polymerase activity was investigated using a single-stranded M13mpl8 DNA template and a sequence-specific [γ ²P] labeled primer (i.e. , Forward Sequencing Primer or FSP; 5'-CGCCAGGGTTTTCCCAGTCACGA-3'; SEQ ID NO:29). The 10 μl reaction mixture contained 50 mM Tris HCl, pH 8.3, 40 mM KC1, 10 mM MgCl₂, 20 μM of each conventional dNTP, 0.24 pmol of sequence-specific primer FSP and 800 ng of single-stranded M13mpl8 DNA template. Four units of rRT (i.e. , MlBAhis) and 5 units of nRT were compared to a commercially av ailable thermostable DNA polymerase (Sequitherm; 5 units) using the buffer provided in the kit. (Sequitherm Cycle Sequencing kit, Epicenter Technologies, Madison, WI). The DNA-dependent DNA polymerase activities of nRT and rRT were approximately equivalent.

The DNA-dependent DNA polymerase activity was also determined at different temperatures. For these reactions, incoφorated [α-³²P]-dTTP served as a label and a non- radioactive primer was used. The reaction consisted of 200 ng of single-stranded M13mpl8 DNA, 1-5 pmoles of FSP, 50 mM Tris-HCl, pH 8.3, 40 mM KCl, 10 mM MgCl₂, 1 mM DTT, 0.6 μCi of [α-³²P]-dTTP (3,000 Ci/mmol), and 20 μM each of dATP, dGTP, dCTP, and dTTP, in a total volume of 24 μl. A conventional protocol was used for the reactions (Sambrook et al. , (1989)) and the reactions were terminated by adding 2 μl of 10 mM EDTA (0.8 mM final concentration). The incorporated [α-³²P]-dTTP was determined using DE52 membranes and scintillation counting, as described above. Results shown in Fig. 5 indicate that the optimum temperature for DNA-dependent DNA polymerase activity for rRT was 45°C-50°C; for nRT. the temperature optimum was 55 °C. The DNA-dependent DNA polymerase activities of the RTs of the invention broadens the range of applications amenable to use of these polypeptides. In addition to copying DNA as well as RNA, the enzymes may be used in any of the above-mentioned variety of amplification technologies known in the art. In addition, the polypeptides of the invention may be used to sequence RNA or DNA targets using Sanger's enzymatic approach as originally disclosed or any one of the many variations of that technique that have been developed since that time.

Example 10

An rRT (i.e. , MlBAhis) according to the invention (i.e. , MlBAhis) was subjected to an RNase H assay, using a protocol known in the art. Hillenbrand et al, Nucl. Acids

Res. 10:833 (1982). Reactions (25 μl) contained 20 mM HEPES-KOH, pH 8.0 (23°C),

10 mM MgCl₂, 50 mM KCl, 1 mM DTT, 0.24 mM [α-³²P] poly(A)-poly(dT) (1 :2; 15 μCi/ml), and 4 μl of diluted enzyme purified from MlBAhis as described above.

For control reactions, standard stocks of RNase H (Molecular Biology Resources, Inc. Milwaukee, WI) with known activity were assayed in the range of 0.05 to 0.5 units/reaction (one unit of activity is defined as the amount of enzyme required to produce 1 nmol of acid soluble ribonucleotide from [α-³²P] poly A-poly(dT) in 20 minutes at 37 A). Two reactions were run without enzyme to serve as negative controls.

A reaction mixture, less enzyme, was prepared and the reaction started by the addition of enzyme. After 20 minutes of incubation at 37°C, the reaction was terminated by adding 25 μl of cold yeast tRNA as co-precipitant (10 mg/ml in 0.1 M sodium acetate, pH 5.0) followed by 200 μl of 10% trichloroacetic acid. Samples were then placed on ice for at least 10 minutes. The mixtures were centrifuged for 7 minutes at 16.000 x g in an Eppendorf microcentrifuge (Brinkman Instruments, Westburg, NY), and 200 μl of the supernatant fluid was withdrawn and counted in 5 ml of scintillation fluid.

The RNase H activity of the rRT at different temperatures was also tested using the reaction mixture described above. The results are presented as counts per minute of released radiolabeled ribonucleotide for each of two trials, as shown in Fig. 6A (black: rRT (i.e., MlBAhis); gray-hatched: nRT). The data show that rRT had RNase H activity comparable to that of native RT. In addition, rRT activity was assessed at a variety of temperatures and the results presented in Fig. 6B showed that rRT was active over a wide range of temperatures. The optimum RNase H activity for rRT was 50°C. In contrast, RNase H activity was relatively low at temperatures of 37 °C, 60 °C and 65 °C. Because of differences in the temperature optima for RT RNase H activity and the other RT activities, such as the RNA- and DNA-dependent DNA polymerase activities, the various methods relying on RT activity may be optimized by adjusting the temperature to achieve the desired mix of activities. For example, methods involving use of an RNase H activity may be performed at temperatures relatively close to the 55 °C temperature optimum for the RNase H activity of rRT. Methods that benefit from decreased RNase H activity, such as RT-PCR and RAMP, may be performed at 60-65 °C to maintain a low level of RNase H activity.

Example 11

A variety of polynucleotides were constructed that encoded modified RT fragments.

These modified RTs include α and β polypeptides that have been terminally modified by deletion of a naturally occurring terminal region of the peptide to produce α-like and β-like fragments retaining RNA-dependent DNA polymerase activity. Other modified RTs according to the invention involve an α-like or β-like fragment attached at either the N- terminus, C- terminus, or both termini to one or more peptides (those peptides including simple homo-oligomeric peptides, preferably charged or bulky, and peptides containing useful functionalities such as DNA binding, metal binding, structure stabilizing and polymerizing [e.g. , zinc finger domains, leucine zipper motifs, an NSl binding site, GPRP (single-letter amino acid identification) or its inverse PRPG, among others] capacities). Yet other modified RTs according to the invention include fragments that lack a sequence found internally in one of the native polypeptides, α or β.

Techniques used to construct polynucleotides encoding these modified RTs are known in the art and described in Examples 1 and 3 above. Generally, the strategy was to use PCR to construct the desired polynucleotide, which was then cloned and expressed to produce the encoded modified RT. The expression studies were generally conducted as described in Example 4.

Expression of eukaryotic genes in prokaryotes may result in production of misincoφorated, truncated and/or insoluble proteins (misfolding) due to the presence of rare codons in those eukaryotic genes Translation of these rare codons is limited by the regulated expression of tRNAs corresponding to these rare codons Hence, expression of eukaryotic genes having abundant rare codons sometimes results in misincorporation, truncation and/or misfolding One approach to minimizing such problems is to clone the tRNA corresponding to these rare codons and express the clone in E.coli in order to facilitate the expression of eukaryotic genes We have cloned and expressed the ArgU tRNA because the arginine codons (AGG, AGA CGA and CGG) present the largest number of rare codons in AMV-RT Co-expression of AMV-RT and ArgU is expected to improve expression (i.e., activity levels) of AMV-RT Other rare codons such as leucine (CTA) and proline (CCC) will also be cloned and co-expressed Another approach to improved expression of the modified RTs of the invention in prokaryotes is to change the rare codons in modified RT coding regions to frequently used codons Such changes can be readily effected by a variety of techniques known in the art, e.g., site-directed mutagenesis using synthetic oligonucleotides In an E. coli expression system, there would be 90 rare codons (38 arginine, 23 proline, 15 isoleucine, 10 leucine and 4 serine codons) in the AMV-RT gene, all or some of which may be advantageously changed to frequent codons Changing all 90 rare codons to the frequent codons found in abundantly expressed genes could imbalance host cell metabolism, however To accommodate deleterious effects on host cell metabolism arising from modified RT expression levels that are too high, a library of clones may be constructed using, e.g., an M 13-based approach to site- directed mutagenesis involving oligonucleotide primer incorporation. Specifically, pools of synthetic oligonucleotides, each oligonucleotide designed to convert one or a few rare codons to frequent codons, and a template comprising a modified RT coding region may be used to synthesize a collection of modified RTs having a range of 1 -90 rare codon conversions Clones having RT activity may be isolated from this library by conventional screening techniques (e g , binding to radioactive substrate and activity assays, among others).

To facilitate an understanding of the structures of the various polynucleotides and polypeptides disclosed in this Example, Table II below collects pertinent information. All constructions generated by PCR used a suitable, full-length coding region sequence as a template, such as the pol gene sequence found in Ml -5, 6.

Table II

Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

MIBA (His6) 1754 FM1BA Smal 253-269 (SEQ ID

NO:25); 1986-1967 6 His codons

RMlBAhisXhoI extend (SEQ ID

NO:45)

MIBA (HislO) 1766 FM1BA Smal 253-269 (SEQ ID

NO:25); 1986-1967 10 His codons

RM1BA HislO

(SEQ ID NO:46)

MIBA (Hisl2) 1772 FM1BA Smal 253-269 (SEQ ID

NO:25); 1986-1967 12 His codons

RM1BA Hisl2

(SEQ ID NO:47)

MIBA (Leu) 1754 FM1BA Smal 253-269 (SEQ ID

NO:25); 1986-1968 6 Leu codons

RM1BA Leu

(SEQ ID NO:48) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

MIBA (Lys) 1757 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 7 Lys codons RMIBA Lys (SEQ ID NO:49)

MIBA (Arg6) 1754 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1967 6 Arg codons RMIBA Argό (SEQ ID NO: 50)

MIBA (Arg3, 1757 FMIBA Smal 253-269 X4) (SEQ ID NO:25); 1986-1967 3 Arg, 2 Asn, 1

RMIBA Arg3X4 Gin, 1 Tyr (SEQ ID NO:51) codon

MIBA (Asp6) 1754 FMIBA Smal 253-269 (SEQ ID

NO: 25); 1986-1968 6 Asp codons RMIBA Asp6 (SEQ ID NO:52)

MIBA (Asp4) 1748 FMIBA Smal 253-269 (SEQ ID NO: 25); 1986-1968 4 Asp codons RMIBA Asp4 (SEQ ID NO:53)

MIBA (Asp5) 1751 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 5 Asp codons RMIBA Asp5 (SEQ ID NO:54)

MIBA (Asp8) 1760 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 8 Asp codons RMIBA Asp8 (SEQ ID NO:55) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: l)

MIBA 1772 FMIBA Smal 253-269

(Aspl2) (SEQ ID

NO:25); 1986-1968 12 Asp codons RMIBA Asp 12 (SEQ ID NO:56)

MIBA (Glu6, 1754 FMIBA Smal 253-269 Xhol) (SEQ ID NO:25); 1986-1968 6 Glu codons RMIBA Glu6, Xhol (SEQ ID NO:57)

MIBA 1772 FMIBA Smal 253-269

(Glu 12) (SEQ ID NO:25); 1986-1968 12 Glu codons RMIBA Glul2 (SEQ ID NO:58)

MIBK 620 1862 FMIBA Smal 253-269 (SEQ ID NO:25); 2112-2092 RMIBK 620 (SEQ ID NO:74)

MIBK 620 1880 FMIBA Smal 253-269 His (SEQ ID NO:25); 2112-2092 6 His codons

RMIBK 620 His (SEQ ID NO: 60)

MIBK 640 1919 FMIBA Smal 253-269 Xhol (SEQ ID

NO:25); RMIBK 2149-2169 640 Xhol (SEQ ID NO: 76)

MIBK 660 1982 FMIBA Smal 253-269 Xhol (SEQ ID

NO:25); 2210-2232 RMIBK 660 Xhol (SEQ ID

NO: 77) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

M1BK680 2042 FMIBA Smal 253-269

Xhol (SEQ ID NO:25); 2273-2292 RMIBK 680 Xhol (SEQ ID NO:78)

MIBK 760 2282 FMIBA Smal 253-269

Xhol (SEQ ID

NO: 25); 2512-2532 RMIBK 760 Xhol (SEQ ID NO: 79)

MIBK 800 2399 FMIBA Smal 253-269

Xhol (SEQ ID NO: 25); 2628-2649 RMIBK 800 Xhol (SEQ ID NO: 80)

MIBK 640 1937 FMIBA Smal 253-269

His Xhol (SEQ ID

NO:25); 2149-2169 6 His codons

RMIBK 640 His

Xhol (SEQ ID

NO:81)

MIBK 660 2000 FMIBA Smal 253-269

His Xhol (SEQ ID

NO:25); 2210-2232 6 His codons

RMIBK 660 His

Xhol (SEQ ID

NO: 82)

MIBK 680 2060 FMIBA Smal 253-269

His Xhol (SEQ ID

NO:25); 2273-2292 6 His codons

RMIBK 680 His

Xhol (SEQ ID

NO:83) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide Region numbering of SEQ ID NO: 1)

MIBK 760 2300 FMIBA Smal 253-269

His Xhol (SEQ ID NO:25); 2512-2532 6 His codons

RMIBK 760 His Xhol (SEQ ID NO: 100)

MIBK 800 2417 FMIBA Smal 253-269

His Xhol (SEQ ID

NO:25); 2628-2649 6 His codons

RMIBK 800 His Xhol (SEQ ID NO: 84)

MIBA (LZIP2 1757 FMIBA Smal 253-269

Xhol) (SEQ ID NO:25); 1986-1968 Leucine zipper RMIBA LZIP2 (2 copies) Xhol (SEQ ID NO:61)

MIBA (LZIP3 1778 FMIBA Smal 253-269

Xhol) (SEQ ID NO:25); 1986-1968 Leucine zipper RMIBA LZIP3 (3 copies) Xhol (SEQ ID NO: 62)

MIBA (LZIP4 1799 FMIBA Smal 253-269

Xhol) (SEQ ID NO:25); 1986-1968^* Leucine zipper RMIBA LZIP4 (4 copies) Xhol (SEQ ID NO:63)

MIBA (LZIP5 1820 FMIBA Smal 253-269

Xhol) (SEQ ID NO: 25): 1986-1968^* Leucine zipper RMIBA LZIP5 (5 copies) Xhol (SEQ ID NO: 64) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

MIBA (Cyst2) 1742 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 2 Cys codons RMIBA Cyst2 (SEQ ID NO:65)

MIBA (Cystό) 1754 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 6 Cys codons RMIBA Cystό (SEQ ID NO:66)

MIBA 1748 FMIBA Smal 253-269 (GPRP) (SEQ ID NO:25); 1986-1968 GPRP motif RMIBA GPRP (SEQ ID NO:67)

MIBA 1748 FMIBA Smal 253-269 (PRPG) (SEQ ID NO:25); 1986-1968 PRPG motif RMIBA PRPG (SEQ ID NO:68)

MIBA (NSl 1796 FMIBA Smal 253-269 Xhol) (SEQ ID NO:25); 1986-1966 NSl site RMIBA NSl Xhol (SEQ ID NO:98)

MIBA (WH) 1769 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 WH motif RMIBA WH (SEQ ID NO: 69)

MIBA (3PPG 1763 FMIBA Smal 253-269 Xhol) (SEQ ID NO:25); 1986-1968 3 "PPG" motifs RMIBA 3PPG Xhol (SEQ ID NO: 70) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

MIBA (Trp) 1754 FMIBA Smal 253-269 (SEQ ID NO:25); 1986-1968 6 Trp codons RMIBA TRP (SEQ ID NO:71)

MIBA (Nhis 1754 FMIBA Nhis 253-269 6 His codons Smal) Smal (SEQ ID NO:72); RMIBA 1986-1967 Xhol (SEQ ID NO:59)

MIBA (NWH 1769 FMIBA NWH 253-270 WH motif Smal) Smal (SEQ ID NO:73); RMIBA 1986-1967 Xhol (SEQ ID NO: 59)

DNPCRl 1754 FDNPCR1 1577-1622 Mismatch at

(D450N) (D450N) (SEQ position 1600 ID NO:92); of SEQ ID RDNPCR1 NO: l (D450N) (SEQ ID NO:93)

DNPCR2 1754 FDNPCR2 1744-1789 mismatch at

(D505N) (D505N) (SEQ position 1765 ID NO: 94); of SEQ ID RDNPCR2 NO: l (D505N) (SEQ ID NO: 95)

MIBA 1754 FMIBA E484Q 1678-1725 mismatch at

(E484Q) (SEQ ID position 1702

NO:96); RMIBA of SEQ ID E484Q (SEQ ID NO: l NO: 97) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

Core domain 2113 FMIBA Smal 253-269 deletion- (SEQ ID

Fragment la NO: 25); and 2092-2112 RMIBK 620 Xhol (SEQ ID NO: 74); and F 2560-2580 Cint Xhol (SEQ ID NO:85); 2788-2811 R Cint Sail (SEQ ID NO: 86)

Core domain 2170 FMIBA Smal 253-269 deletion- (SEQ ID

Fragment lb NO: 25); and 2149-2169 RMIBK 640 Xhol; and F Cint 2560-2580 Xhol (SEQ ID NO: 85); 2788-2811

R Cint Sail (SEQ ID NO: 86)

Core domain 2233 FMIBA Smal 253-269 deletion- (SEQ ID

Fragment lc NO: 25): and 2210-2232

RMIBK 660

Xhol; and F Cint 2560-2580

Xhol (SEQ ID

NO:85); 2788-2811

R Cint Sail (SEQ

ID NO:86) Core domain 2131 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 2a NO:25); and 2092-2112 6 His codons RMIBK 620 Xhol (SEQ ID 2560-2580 NO: 74); and F Cint Xhol (SEQ 2788-2811 ID NO:85); R Cint His Sail (SEQ ID NO:87) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

Core domain 2188 FMIBA Smal 253-269 6 His codons deletion- 3' (SEQ ID fragment 2b NO: 25); and 2149-2169

RMIBK 640

Xhol; and F Cint 2560-2580

Xhol (SEQ ID

NO:85); 2788-2811

R Cint His Sail

(SEQ ID NO:87)

Core domain 2251 FMIBA Smal 253-269 6 His codons deletion- 3' (SEQ ID fragment 2c NO: 25); and 2210-2232

RMIBK 660

Xhol; and F Cint 2560-2580

Xhol (SEQ ID

NO:85); 2788-2811

R Cint His Sail

(SEQ ID NO:87)

Core domain 2155 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 3 a NO: 25); and 2092-2112 RMIBK 620 Xhol (SEQ ID NO: 74); and F 2443-2463 Cint 731 Sail (SEQ ID 2736-2716 NO:88); RCint 830 Xhol (SEQ ID NO:90)

Core domain FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 3b NO :25); and 2149-2169 RMIBK 640 Xhol; and F Cint 2443-2463 731 Sail (SEQ ID NO:88); 2736-2716 RCint 830 Xhol (SEQ ID NO:90) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

Core domain 2275 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 3 c NO: 25); and 2210-2232 RMIBK 660 Xhol; and F Cint 2443-2463 731 Sail (SEQ ID NO:88); 2736-2716 RCint 830 Xhol (SEQ ID NO: 90)

Core domain 2101 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 4a NO: 25); and 2092-2112 RMIBK 620 Xhol (SEQ ID 2497-2517 NO: 74); and F Cint 751 Sail 2736-2716 (SEQ ID NO: 89); RCint 830 Xhol (SEQ ID NO: 90)

Core domain 2158 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 4b NO: 25); and 2149-2169 RMIBK 640 Xhol; and F Cint 2497-2517 751 Sail (SEQ ID NO:89); 2736-2716 RCint 830 Xhol (SEQ ID NO:90)

Core domain 2221 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 4c NO:25); and 2210-2232 RMIBK 660 Xhol; and F Cint 2497-2517 751 Sail (SEQ ID NO:89); 2736-2716 RCint 830 Xhol (SEQ ID NO: 90) Clone Approx. Oligonucleotide Added Added

Length additions/PCR oligonucleotide oligonucleotide of Primers locations characteristics

Coding (nucleotide

Region numbering of SEQ ID NO: 1)

Core domain 2032 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 5 a NO:25); and 2092-2112 RMIBK 620 Xhol (SEQ ID NO: 74); and F 2566-2586 Cint 771 Sail (SEQ ID 2736-2716 NO:99); RCint 830 Xhol (SEQ ID NO:90)

Core domain 2089 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 5b NO: 25); and 2149-2169 RMIBK 640 Xhol; and F Cint 2566-2586 771 Sail (SEQ ID NO: 99); 2736-2716 RCint 830 Xhol (SEQ ID NO: 90)

Core domain 2152 FMIBA Smal 253-269 deletion- 3' (SEQ ID fragment 5 c NO: 25); and 2210-2232 RMIBK 660 Xhol; and F Cint 2566-2586 771 Sail (SEQ ID NO:99); 2736-2716

RCint 830 Xhol (SEQ ID NO: 90) ^* Oligonucleotides hybridize to an internal region of oligonucleotide RMIBA LZip3 Xhol, which in turn recognizes the indicated region of SEQ ID NO: l . A. Terminally deleted RTs

The full-length RT coding region was truncated by deletions using conventional methodologies described above (e.g. , Example 3). One set of deletion derivatives lacked the 3' end of the MAV-RT coding region to varying extents. Again, relative to the full- length gene (SEQ ID NO: l), the 3' (C-terminal) deletion extending to the Kpnl site (MIKA; see SEQ ID NO: 8) increased the RT expression level, as evidenced by SDS- PAGE. Relative to the full-length gene (SEQ ID NO: 1), deletion of the region extending from the Bglll site to the 3' terminus (MIBA; see SEQ ID NO: 6) also increased RT expression and activity, as evidenced by SDS-PAGE and activity assays (see below). The C-terminally truncated RTs (MIKA and MIBA) have lengths that fall in between the lengths of the native and β polypeptides. Relative to the alpha fragment of MAV-RT, the beta fragment has an additional 254 amino acids at the C-terminus, which provides an integrase activity. This region of the polypeptide contributes to the insolubility of the polypeptide and reduces its recovery from cell extracts, as shown by the relative insolubility of a (+) integrase form of RT (e.g., the MIKA gene product, see below) compared to a (-) integrase form (e.g. , the MIBA gene product). Because the integrase domain is only needed for the retroviral life cycle and not for the RNA- or DNA-dependent DNA polymerase activities, this region was deleted in MIBA (α-like fragment). Note that the MIBA α-like fragment (amino acids 1-578 of SEQ ID NO: 2) is larger than the naturally occurring fragment of MAV-RT (amino acids 1-573 of SEQ ID NO: 2). Without wishing to be bound by theory, this deletion was expected to result in an increase in the solubility, and hence recovery, of the protein.

A series of clones was constructed to express the MIBA and MIKA series of modified RTs, which have C-terminal deletions in order to increase the levels of expression and to stabilize the RT activity (RNA-dependent DNA polymerase activity). Convenient restriction sites in full-length clones such as PMBacRT and pHRT, e.g. , Bgl II (spanning nucleotides 1 ,986-1,991 of SEQ ID NO: l) and Kpnl (spanning nucleotides 2,745-2,750 of SEQ ID NO:l), were used to eliminate the 3' end of the coding region of the RT gene (see, Table I). The 3' deletion derivatives, encoding RT polypeptide fragments having C- terminal deletions, were obtained by Bglll-Pstl or Kpnl-Pstl restrictions of pMBacRT and pHRT, respectively (Bglll and Kpnl sites in the MAV-RT coding region; Pstl site in the vector). Recombinant molecules containing the Bgl H-Pstl 3' terminal deletion were designated pBacMIBA and pHBRT (pH33ΔBP6) and recombinant molecules containing the Kpnl-Pstl deletion were designated pBacMIKA and pHKRT (pH33ΔKP5). The deletion derivatives pBacMIBA and pBacMIKA had approximately 1 . 17 and 0.4 kb deletions from the 3' end of the full-length gene (see, SEQ ID NO: l), respectively. The fragment bounded at its 3' end by the Bglll site (SEQ ID NO:6) was used to express an alpha-like RT fragment (the α-like fragment, MIBA, contained amino acids 1-578 of SEQ ID NO:2; native MAV-RT α contains amino acids 1-572 of SEQ ID NO:2) and the - 56 - fragment bounded by the Kpnl site (SEQ ID NO: 8) was used to express a beta-like RT fragment (the β-like fragment, MIKA, contained amino acids 1-832 of SEQ ID NO:2; native MAV-RT β contains amino acids 1-858 of SEQ ID NO.2).

Miniprep and sequencing analyses were done to confirm the identities of the recombinant clones described above. Recombinant viruses obtained from co-transfection with virus BacPakό and transfer vector pBacMIBA or pBacMIKA were called MIBA and MIKA, respectively.

B. Alpha-like recombinants encoding non-native terminal peptides L. Simple peptide tags One category of fragment modifications was designed to mimic one or more properties of the integrase domain found in the β fragment but missing from the α fragment of Type III RTs. Partial mimicking of the integrase domain, without the deleterious impact on solubility and host cell viability associated with the native integrase domain, was accomplished by adding polynucleotide sequences encoding His tags at the 3 ' ends of the modified RT coding regions.

A His-tag addition to the C-terminus of an RT polypeptide was achieved by recombinant expression of a polynucleotide containing an RT coding region fused in-frame to His codons. In particular, the fusions were constructed by adding oligonucleotides containing 6 histidine codons to the 3 ' end of the RT gene using ligase, as in the case of the construction of pBacMIKAhis, or by PCR amplification with oligonucleotides that specified 6 histidine codons, as in the case of the construction of pBacMIBAhis.

The basic nature of the added His amino acids was expected to increase binding to the negatively charged nucleic acids, enhancing the stability of the polypeptides. The increased stability, in turn, was expected to result in increased activity of amino-acid-tagged RTs, relative to their untagged counterparts In addition, the His tags were expected to chelate metal ions (e.g., Ni^'"), thereby potentiating polymerization of the modified RTs. A His-tagged RT (MlBAhis) was found in homo-polymeric form (molecular weight greater than 200 kDa), as determined using non-denaturing PAGE and molecular sieve chromatography with Superose 12HR10/30 (separation range of 1-300 kDa; Pharmacia-Upjohn). Expression levels of the RT fragments modified by amino acid tagging showed that the structurally unstable alpha fragment was stabilized by addition of peptide tags to the C- terminus of the AMV-RT alpha fragment. Other modified RTs bearing peptides at the C-terminus of the α-like fragment were generated by PCR, as described above The forward and reverse PCR primers had codons corresponding to the - and C-termini of the AMV-RT alpha fragment, along with codons corresponding to the peptide tags to be added A linearized template (pHSEMl) containing the full-length RT gene was used for the PCR amplifications Additional information concerning this class of modified RTs, as well as the polynucleotides encoding them, is found in Table II

The PCR product was restricted with a suitable restriction enzyme and ligated to pBacPak9 that was digested with a compatible enzyme The selected recombinants were sequenced to confirm addition of the appropriate tags

2_ C-terminal peptides exhibiting DNA binding properties

DNA binding motifs of proteins, may have either general affinity (i.e., non-specific binding) or sequence-specific affinity for DNA Several nucleic acid binding domains have been identified and reported to play a role in important cellular functions such as viral packaging, transcriptional and translational regulation, transport between the nucleus and cytoplasm, splicing, and stability, among others Karaya et al , J Biol Chem 266 11621- 1 1627 (1991), Burd, et al , Science 265 615-621 (1994), Weiss, et al , Biopolymers 48(2- 3) 167-180 (1998), Nassal, M , J Virol 66(7) 4107-16 (1992) Ritt, et al , Biochemistry 37 2673-81 (1998) DNA binding domains with general affinity are preferable to target - specific binding domains because of the reduced substrate specificity of modified RTs having such general binding domains

Several basic amino acids are known to enhance the affinity of a protein for nucleic acid templates The positive charges of arginine, lysine, and histidine increase the non-specific affinity of polypeptides containing such residues for nucleic acid, thereby facilitating the search for specific binding sites Several arginine-rich motifs and arginine-lysine-rich motifs have been identified in nucleic acid binding domains The arginine-lysine rich motif ELKIKRLRKKFAQKMLRKARRK is involved in RNA binding, which could enhance the activity of RT In addition, a lysine-rich protein is associated with DNA in the kinetoplast and plays a role in segregation of the kinetoplast DNA Hines, Mol and Biochem Parasitol 94 41-52 (1998) Similarly, acidic amino acid tags are reported to be involved in packaging of viral DNA The packaging may be mediated through metal ions that have affinity for DNA International Patent Publication No WO 98/07869 Additionally, charged amino acids are O 00/42199 _. _ P

- 58 - present on the surface of structural proteins and may play a role in stabilizing secondary structures

The addition of histidine, glutamic acid, and aspartic acid tags enhanced the activity of the alpha fragment 20- 100 fold A peptide tag consisting of six arginine residues improved the activity five-fold However, specific arginine-rich motifs such as RNRNRQY (Arg3X4, found at the C-terminus of the GP67 envelope glycoprotein proposed to be involved in baculoviral DNA packaging) enhanced (i.e., increased or prolonged) activity by 20- to 40- fold Other RNA- and DNA-binding motifs such as RRRDRGRS are expected to yield similar results However, six continuous lysine residues did not increase the activity A higher number of lysine residues or correct spacing of the lysine residues may be required for enhancement of function

The mechanism of enhancement of activity due to these tags could be due to the increased structural stability of the recombinant or stability resulting from direct or metal- mediated nucleic acid binding MIBA 2000-3000 Units/g of insect cells

MIBA his 50000-200,000 U/g

MlBA argό 15,750 U/g

MlBA lysό 2050 U/g

MlBA Arg3X4 57,000 U/g MlBA gluό 170,000 U/g

MlBA aspό 40,000 U/g

MlBA leuό 2250-3900 U/g

Nhis MIBA asp4 95,000 U/g

Nhis MIBA asρ5 1 15,250 U/g Nhis MlBA aspό 236,250 U/g

Most of the sequence-specific DNA binding proteins have a general basic region and a sequence-specific region for binding to DNA There are several sequence-specific DNA binding motifs such as zinc-finger domains (e.g., TFIIIA CX2CX12HX3H) and the basic region of the bZIP family of proteins Similarly, there are arginine-rich domains such as TRQARRNRRRWRARQR and YGRKKRRQRRRP that recognize specific RNA sequences that are also expected to enhance the activity of RT The N-terminus of the RT integrase domain has a zinc-fmger-like (Hx3HX23CX2C) motif This N-terminus binds zinc and has been reported to both induce proper folding of the N-terminus, to be remarkably thermostable as well Burke et al , J Biol. Chem 267 9639-44 (1992) Because the full-length MAV-RT gene has a zinc-finger-like domain, the reverse primer used in some PCR amplifications included this region of the integrase (see Table II)

A beta-like derivative (620 amino acids) containing the zinc-finger-like motif was more active than the non-tagged alpha fragment (578 amino acids) and expressed 30,000 units per gram of cell pellet M1BK620 31,950 U/g

MlBK620 his 50,000-140,000 U/g

The addition of the sequence-specific, zinc-finger-like motif produced a lower level of RT activity than the His-tagged fragment, however These results suggest that a general nucleic acid binding domain (His tag) may enhance RT activity to a greater extent than a sequence- specific domain (zinc-finger-like motif) and, therefore, could replace the sequence-specific zinc-finger-like motif of RT, leading to an increase in activity General nucleic acid binding domains enhance the stability of both the 578- and the 620-amino-acid-length fragments 3_ C-terminal peptide tags having polymerization domains

Disulfide bond-forming domains (i e , cysteine-rich regions) present in immunoglobulin genes are involved in disulfide bond formation between the light and heavy chains Hence, addition of two cysteine residues at the C-terminus was anticipated to promote dimer formation through disulfide bonding Addition of two cysteine residues enhanced the activity of the alpha-like fragment, however, 6 contiguous cysteine residues reduced the activity of the modified RT

MIBA 2000-3000 U/g

MlBA cyst2 190,000 U/g

MIBA cystό 720 U/g The GPRP (fibrin clotting) tetrapeptide is the primary polymerization pocket of the blood clotting protein fibrin This domain is exposed at the amino terminus of fibrin monomers by proteolytic cleavage of the precursor protein The domain then polymerizes by binding to complementary binding sites on other fibrinogen molecules to form clots Because peptides were being added to the C-terminus of α-like constructs, the reverse-sequence tetrapeptide, PRPG, was also examined

Addition of GPRP enhanced the RT activity approximately 50-fold, while addition of PRPG enhanced the activity of RT by approximately 100-fold In other embodiments, the D- isomers of amino acids are used in peptide tags For example, D-isomers are used in generating PRPG peptides for use in preparing modified RTs of the invention IBA 2000-3000 U/g

MIBA GPRP 107,500 U/g MIBA PRPG 243,500 U/g

Histidine residues can also promote dimer formation mediated by metal ions The addition of 6 His residues to the C-terminus of the α-like RT resulted in a 20- to 40-fold increase in activity Additions of different length histidine tags are contemplated MIBA 2000-3000 U/g MlBA his 50000-200000 U/g

NSl is a DNA-binding protein produced by the minute virus of mice The protein has replicational and transcriptional functions Homo-oligomerization of NSl is required for its function and a small region, N-VETTVTTAQETKRGRJQTK-C, of NSl has been identified as the domain involved in oligomerization Pujol et al , J Virol 71 7393-7403 (1997) Addition of this peptide tag to the C-terminus of AMV-RT fragments enhanced RT activity MIBA 2000-3000 U/g

MIBA NSl 380,000 U/g

4_ C-terminal peptide tags having metal binding domains

Histidine tags can be used as metal binding domains, as explained above In addition, modified RTs having C-terminal His tags were constructed and subjected to expression analyses The results, presented above, indicate that peptide tags, having metal binding capacity, enhance RT expression

Zinc fingers also exhibit metal binding capacity and are also involved in DNA binding As described above, the N-terminus of the integrase domain of MAV-RT has a zinc-finger-like (Hx3HX23CX2C) motif This N-terminus binds zinc and has been reported to induce proper folding of the N-terminus It is expected that peptide tags containing one or more zinc-finger- like domains will enhance the activity of modified RTs in which they are found 5_ C-terminal peptide tags having structure-stabilizing domains

Other embodiments of the invention involve the addition of domains designed to structurally stabilize the alpha-like fragment so that it no longer requires a second fragment for structural stability There are several motifs that have been identified and shown to form specific structures, such as alpha helices, beta sheets, and coils, among others, all of which are known in the art Formation of defined structures facilitates the formation of active domains and promotes interactions with other such domains Beta strands and beta sheets frequently promote aggregation in, and precipitation from, solution Desjarlais et al Curr Opin in

Biotechnol 6 460-466 (1995) Hence most of the C-terminal tag additions were capable of forming helices or coils These secondary structure predictions are based on the well-known Chou and Fassman algorithms

The WEAAH (WH) motif, comprising histidine and tryptophan, promotes formation of alpha helices, or defined structures, thereby giving structural stability to the protein

MIBA 2000-3000 U/g

M1BA WH 104,720 U/g Addition of the WH domain may extend the helix at the C-terminus and thereby enhancing the stability of the alpha fragment Regardless of the reason, however, modified RTs containing a WH motif exhibit enhanced RT activity

The "PPG^" triple-helical domain is responsible for binding interactions in the structural protein collagen This motif is responsible for the structural stability and proper assembly of collagen Addition of peptides containing this motif in generating modified RTs according to the invention is expected to enhance the activity of such RTs relative to corresponding RTs lacking such peptides

Addition of tryptophan residues is predicted to extend the α-helix at the C-terminus and to enhance the stability of the alpha-like fragment Tryptophan is a bulky amino acid and could substitute for histidine tags in providing structural stability Comparative assays showed that a domain comprising Trp residues enhanced RT activity approximately 50-fold

MIBA 2000-3000 U/g

MIBA Trp 96,500 U/g

The GPRP and PRPG motifs identified in fibrin as the domains involved in interaction with other clotting proteins enhance the activity of the AMV-RT alpha-like fragment This motif is predicted to form coil-turn-coil structures

MIBA 2000-3000 U/g

MIBA GPRP 107,500 U/g

MIBA PRPG 243.500 U/g The NSl domain primarily forms beta sheets and coils The presence of hydrophobic residues alone is not very desirable because they form beta sheets and are typically buried in the secondary structure of the protein This may affect the natural folding of domains Hence, a motif that had a mixture of coils and beta sheets was chosen for analysis Addition of this domain produced an active α-like fragment that appeared to be stable

MIBA 2000-3000 U/g

MIBA NSl 380,000 U/g The leucine zipper motif is a helix-turn-helix motif which has been reported to dimerize by a coiled-coil interaction This defined structure of the leucine zipper is expected to enhance the stability of the alpha-like fragment in addition to providing dimerization abilities

MIBA 2000-3000 U/g MIBA Lzip23 7170 U/g

MIBA Lzip3 1620 U/g

Addition of a single heptad repeat enhanced the activity by 2-3 fold Addition of two heptad repeats did not improve the activity However, additions of 4-5 heptad repeats produced RTs that had reduced activity levels 6_ N-terminal peptide tags

Consistent with the description in Examples 3 and 4 of N-terminal peptide tags being added to modified RTs that exhibited enhanced expression, several constructs were generated and characterized One modified RT, NhisMIBA, contained a His tag attached to the N- terminus of an α-like fragment Other RTs were modified to contain peptide tags at both termini (Nhis MIBA asp 4, Nhis MIBA asp 5, Nhis M IBA asp 6, and Nhis MIBA WH)

Expression studies conducted as described in Example 4 led to the results shown below

Nhis MIBA 10,000-41,700 U/g

MlBAChis 50,000-200,000 U/g

Nhis MIBA asp 4 95,000 U/g Nhis MIBA asp 5 1 15,250 U/g

Nhis MIBA asp 6 236,250 U/g

Nhis MIBA WH 86,000 U/g

Expression of MlBAChis was measured to provide a relative control for the measurement of

Nhis MIBA expression The results show that activity of RTs modified by a His tag present at either the N-terminus or the C-terminus is increased relative to untagged RTs Other variations, such as the addition of peptide tags to both termini of an RT (e.g., an N-terminal

His tag coupled to a C-terminal Asp-, Glu-, or Trp-His- (i.e., WH) tag), are also contemplated by the invention Large-scale expression studies have shown that similar activity levels of approximately 100,000 units/g insect cells are achieved with MIBA asp (N-terminally modified RT) and Nhis MIBA asp (RT having 6 His residues at the N-terminus and 4-6 Asp residues at the C-teπninus) 2, Peptide tagging of other Type III RTs The strategies described above were also used to modify RTs from other avian sources, such as Rous Sarcoma Virus and Avian Tumor Virus. The C-terminal addition of a six-histidine peptide tag to an alpha fragment of each of these avian RTs substantially increased the RT activity, relative to the non-tagged AMV-RT α-like fragment. MIBA 2000-3000 U/g RSV-RT 43,350 U/g

ATV-RT 71,900 U/g

Therefore, the modification strategies applied to AMV-RT polynucleotides and polypeptides are applicable more generally to dimeric (i.e., Type II and Type III) reverse transcriptase coding regions and polypeptides, and all of these modified RTs fall within the scope of the present invention.

C. Beta-like recombinants Modifications of β RT

Polynucleotides encoding a variety of beta-like modified RTs were constructed using the techniques described in Example 3 and expressed using the techniques described in Example 4, along with Ml-5, 6 encoding the full-length AMV-RT. Expression of the full- length beta fragment resulted in low levels of highly insoluble, full-length protein, in both a eukaryotic (insect cell) and a prokaryotic (E. coli) host. Because expression of the full-length beta fragment resulted in mostly insoluble protein, the native beta polypeptide was modified in an effort to increase its solubility and, hence, activity. One strategy for modifying the β fragment involved deletions of parts of the native β RT. The native beta coding region specifies 858 amino acids and the full-length β-like fragment disclosed herein consists of 832 amino acids. Thus, the β-like fragment lacks the 26 C-terminal amino acids of full-length native β. Expression of the full-length β-like polypeptide, relative to the full-length native β, showed an increase of one-hundred-fold in expression, as evidenced by SDS-PAGE analysis; however, the β-like polypeptide was still highly insoluble (approximately 90% insoluble), resulting in a five-fold increase in activity. M 1 KA 1000 U/Liter of cells

MlKAhis 2,000 U/L Ml-5 200 U/L

Modified RTs having C-termini between 580 and 832 ammo acids (see SEQ ID NO 2) are also contemplated by the invention Because both the 580- and the 620-amino-acid recombinants are soluble, and the 832- and 858-amino-acid recombinants are relatively insoluble, deletions that truncate the C-terminus to a position between 580-832 amino acids are expected to result in modified β-like polypeptides that are soluble In particular embodiments, the β-like polypeptide has a C-terminus at any one of positions 580-832, such as positions 620, 640, 660, 740, 780, or 800 (SEQ ID NO 2), resulting from deletions that eliminate 237, 217, 197, 1 17, 77 and 57 amino acids, respectively, relative to the full-length β RT Construction and expression of a deletion derivative specifying a modified β-like RT of 620 amino acids was accomplished as generally described in Examples 3 and 4, with the expression results presented below MIBA 2000-3000 U/g

MIKA 1000 U/L MIBK 620 3 1,950 U/g

Thus, a truncated β-like RT shows considerable activity, consistent with an increase in solubility relative to the full-length native β RT

Analogous modifications to the corresponding β polypeptides of other avian RTs result in similarly increased RT activity RSV-RT 620 his 33,000 U/g

In addition to 3' deletions resulting in polynucleotides encoding β-like polypeptides having C-termini in the range of positions 580-832, and preferably in the range of 620-800 (SEQ ID NO 2), the invention contemplates polynucleotides having internal deletions relative to the native β gene, as well as the polypeptides encoded by polynucleotides having such internal deletions The central core region of the integrase domain is associated with the DNA cutting and joining properties of the native AMV-RT

The core region of the integrase domain was deleted to varying extents (the region between amino acids 620-770, 640-770 or 660-770 of SEQ ID NO 2), e.g., MIBK Cint lacks amino acids 620-770 of SEQ ID NO 2, using conventional techniques The approach involved the initial construction of first polynucleotide fragments encoding C-terminally truncated β-like fragments using PCR with the full-length AMV-RT pol gene as a template (see Table II) Second fragments containing various lengths the 3 ' of the end of the pol gene (i.e., 3' fragment) were also constructed using PCR These 3' fragments encoded the C- terminal region of the integrase domain, some 3' fragments also contained part, but not all. of the core region of the integrase domain Those of skill in the art will recognize that the first polynucleotide fragments, or 5' fragments, may encode peptide tags at their 5' ends, the 3^" fragments may also encode peptide tags (see e.g., 3' Fragment 2a in Table II), with or without tags encoded by the 5' fragment, and these tag-encoding fragments are readily synthesized using the PCR primers disclosed herein (e.g., F Cint Xhol (SEQ ID NO:85) and R Cint 830 His Xhol (SEQ ID NO 91)) The final step in generating constructs having internal deletions was to ligate truncated β-like coding regions to 3' fragments in proper order and orientation. as determined by the conventional screening of ligation products In one embodiment, amino acids 620-770 were deleted, thereby removing the core region of the integrase domain The C-terminal region of the integrase domain was then placed adjacent to the N-terminal region of that domain

Expression of such constructs in insect cells revealed an increase in solubility (10- 20%)) and activity relative to the full-length, intact β RT, as shown below Other deletions effectively removing part or all of the central region of the integrase domain, such as the deletion of amino acids 620-731, 640-771, 640-731, 660-771, 660-731, 680-771, 680-731, and 740-771 (SEQ ID NO 2) are contemplated by the invention MIKA 1000 U/L Ml-5 200 U/L

Some modified beta fragments have terminal peptide tags Thus, the invention contemplates modified RTs having internal deletions and, optionally, peptide tags at an N- terminus, a C-terminus or both termini In addition, as for α-like modified RTs, the β-like modified RTs may be derived from any Type II or Type III RT, along with polynucleotides encoding them

Any of the modified RTs of the invention may be produced by any process disclosed herein or known in the art, such as in v vo synthesis, m vitro synthesis or chemical synthesis Further, any of these processes may be used to produce active polypeptides in a variety of forms, including monomers, homo-dimers or homo-multimers, hetero-dimers, and hetero- multimers, all of which are comprehended by the invention In particular, expression of the modified beta-like fragment M1BK620 Cint resulted in expression of a heterodimeric form of RT, suggesting that the beta-like fragment was processed as expected, to yield an α polypeptide in association with a modified β-like polypeptide Expression of other modified RTs of the invention, such as other core domain deletions (e.g., β-like fragments lacking amino acids 620-731 , 640-771 , 640-731 , 660-771 , 660-731 , 680-771 , 680-731 , or 740-771 of SEQ ID NO 2) are expected to show activity in other than monomeric form, e.g., in heterodimeric form. In addition, heterodimers or other non-monomeric forms may arise from the interaction of a modified α-like polypeptide and a native β polypeptide, or from a modified α-like polypeptide and a modified β-like polypeptide, regardless of whether the polypeptides were produced by in vivo or m vitro expression, or by chemical synthesis

While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only those limitations appearing in the appended claims should be placed upon the invention.

Claims

What is claimed is:

1. An isolated polynucleotide encoding a polypeptide having RNA-dependent DNA polymerase activity, the polypeptide consisting of

(a) an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 857 of SEQ ID NO: 2;

(b) an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 1054 of SEQ ID NO:39;

(c) an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 548 to 1198 of SEQ ID NO:41; (d) an amino acid sequence beginning at amino acid 1 and terminating at any one of amino acids 428 to 901 of SEQ ID NO: 43; and (e) variants, analogs and fragments of any of subparts (a) to (e) having

RNA-dependent DNA polymerase activity, said polypeptide, variants, analogs, and fragments optionally having an N- terminal methionine.

2. The polynucleotide according to claim 1 step (a) wherein said polypeptide consists of a sequence that begins at about amino acid 1 and ends at about amino acid 578 of SEQ ID NO:2.

3. The polynucleotide according to claim 1 step (a) wherein said polypeptide consists of the sequence set forth as SEQ ID NO:4.

4. The polynucleotide according to claim 1 having a sequence selected from the group consisting of a sequence set forth in any one of SEQ ID NOs 1 , 6-10, 38, 40, and 42.

5. The polynucleotide according to claim 1 wherein said polynucleotide is DNA.

6. The polynucleotide according to claim 1 wherein said polynucleotide encodes a polypeptide that lacks an effective integrase activity.

7. The polynucleotide according to claim 6 wherein said polynucleotide lacks at least part of an integrase coding region.

8. The polynucleotide according to claim 1 further comprising an adjacent polynucleotide encoding at least one terminal modification of said polypeptide selected from the group consisting of an N-terminal modification and a C-terminal modification.

9. The polynucleotide according to claim 8 wherein said modification is a cysteine residue adjacent the C-terminus of said polypeptide.

10. The polynucleotide according to claim 8 wherein said adjacent polynucleotide encodes a polypeptide consisting of a C-terminal modification.

11. The polynucleotide according to claim 10 wherein said C-terminal polypeptide comprises between four and fifty amino acids and wherein said polypeptide comprises a domain selected from the group consisting of a DNA binding domain, an RNA binding domain, a metal binding domain, a structure stabilizing domain, and a polymerizing domain.

12. The polynucleotide according to claim 11 wherein said polypeptide comprises an acidic amino acid domain, a basic amino acid domain, a W domain, a WH domain, a zinc-finger-like domain, a leucine zipper domain, a PPG domain, an NSl domain, a GPRP domain, and a PRPG domain.

13. The polynucleotide according to claim 11 wherein said C-terminal peptide comprises six amino acids.

14. The polynucleotide according to claim 11 wherein said C-terminal peptide comprises amino acids that are the same.

15. The polynucleotide according to claim 11 wherein said C-terminal peptide comprises amino acids that are basic.

16. The polynucleotide according to claim 15 wherein said basic amino acids are histidine.

17. The polynucleotide according to claim 8 having a sequence selected from the group consisting of a sequence set forth in any one of SEQ ID NOs 11- 19.

18. A vector comprising the polynucleotide according to claim 1.

19. The vector according to claim 18 wherein said polynucleotide is operably linked to a promoter.

20. A host cell transformed with a vector according to claim 18.

21. The host cell according to claim 20 wherein said host cell is a eukaryotic cell.

22. The host cell according to claim 20 wherein said host cell is selected from the group consisting of Escherichia coli and an insect cell.

23. An isolated polypeptide encoded by the polynucleotide according to any one of claims 1 to 5.

24. An isolated polypeptide encoded by the polynucleotide according to any one of claims 6 to 17.

25. A method of transforming host cells comprising the following steps:

(a) introducing a vector according to claim 18 into host cells;

(b) incubating said host cells; and (c) identifying host cells containing said vector, thereby identifying a transformed host cell.

26. A method of producing an isolated Reverse Transcriptase polypeptide comprising the following steps: (a) transforming a host cell with a vector according to claim 18;

(b) incubating said host cell under conditions suitable for expression of a polypeptide; and

(c) recovering said polypeptide, thereby producing an isolated Reverse Transcriptase.

27. In a method for copying a target nucleic acid by extending a target nucleic acid-bound primer in the presence of a polymerase, the improvement comprising:

(a) contacting said target nucleic acid and primer with the polypeptide according to any one of claims 23 and 24.

28. The method according to claim 27 wherein said copying produces a plurality of copies of said target nucleic acid.

29. The method according to claim 27 wherein said polypeptide is in a form selected from the group consisting of a monomer and a polymer.

30. The method according to claim 27 wherein said method is selected from the group consisting of cDNA synthesis, Polymerase Chain Reaction,

Polymerase Chain Reaction-Reverse Transcription, Inverse Polymerase Chain Reaction, Multiplex Polymerase Chain Reaction, Strand Displacement Amplification. Multiplex Strand Displacement Amplification, Nucleic Acid Sequence-Based Amplification, Sequence-Specific Strand Replication and Rapid Amplification.

31. In a method for sequencing a target nucleic acid by extending a target nucleic acid-bound primer, the improvement comprising: (a) contacting said target nucleic acid and primer with the polypeptide according to any one of claims 23 and 24.

32. The method according to claim 31 wherein said polypeptide is in a form selected from the group consisting of a monomer and a polymer.

33. A kit for copying a target nucleic acid comprising:

(a) one or more nucleotides, and

(b) a polypeptide encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 1 1, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42 and polynucleotide derivatives thereof encoding C-terminal modifications at their 3 ' ends