WO1996014417A1

WO1996014417A1 - Dna encoding a thermostable dna polymerase enzyme

Info

Publication number: WO1996014417A1
Application number: PCT/US1995/014418
Authority: WO
Inventors: Deborah A. Nickerson; Reinhold D. Mueller; Piotr M. Skowron; Neela Swaminathan; Richard F. Piehl
Original assignee: Molecular Biology Resources, Inc.
Priority date: 1994-11-04
Filing date: 1995-11-03
Publication date: 1996-05-17
Also published as: AU4147796A

Abstract

The present invention is directed to a DNA encoding a thermostable, full-length, Thermus flavus DNA polymerase I of approximately 94,000 daltons, and to methods for producing and purifying a recombinant polypeptide encoded by the DNA. The recombinant peptide is useful in DNA sequencing, Thermal Cycle Labeling, Polymerase Chain Reaction, and other molecular biological applications.

Description

DNA ENCODING A THERMOSTABLE DNA POLYMERASE ENZYME

BACKGROUND OF THE INVENTION A. Field of the Invention The present invention relates to an isolated and purified DNA that encodes a thermostable DNA polymerase. Additionally, the present invention relates to a recombinant and thermostable DNA polymerase and to fragments thereof, all having enhanced polymerase activity, and to methods for producing the DNA polymerase and fragments. The present invention further relates to recombinant fragments having decreased exonuclease activity. The thermostable recombinant polymerases of the present invention are useful because they are capable of providing enhanced polymerase activity in bio-applications, such as in the polymerase chain reaction (PCR), in DNA amplification and in thermal cycle labeling (TCL).

B. Background

The burgeoning field of biotechnology was revolutionized by recombinant DNA technology, and DNA polymerase enzymes are an indispensable tool used in many modern molecular in vitro recombinant DNA biological applications, such as in DNA sequencing; DNA cycle sequencing; Polymerase Chain Reaction (PCR) and its many variations (see, e.g., Erlich et al. , Current Communications in Molecular Biology: Polymerase Chain Reaction. Cold Spring Harbor Press, Cold Spring Harbor (1989); Innis et al. , PCR protocols: A guide to methods and applications. Academic Press, San Diego (1990)); Thermal Cycle Labeling (TCL) (Mead and Swaminathan, U.S. Patent App. Ser. No. 08/217,459, filed March 24, 1994; PCT App. No. US94.03246, filed March 24, 1994); Random Primer Labeling (RPL); Ligase Chain Reaction (LCR) (Wiedmann et al., PCR Methods and Applications 3: S51-S64 (1994)); and other applications. To date scientists have reported more than 40 different DNA polymerases, and have reported DNA sequence information for some DNA polymerase genes. Amino acid sequence information has been deduced from the reported genes, and comparison of amino acid sequences has resulted in the placement of reported polymerase genes into four major families: namely, A, B, C, and X. Family A contains £. coli DNA polymerase I, an enzyme involved in repair of DNA and in replication during fast growth. Family B includes E. coli DNA polymerase II. Family C polymerases include £. coli DNA polymerase III, the major replication enzyme. The fourth group, Family X, contains enzymes such as the eukaryotic DNA polymerase β and eukaryotic terminal transferases (Ito and Braithwaite, Nucleic Acids Res. 19: 4045-4057 (1991)). The breakdown of DNA polymerases into families has been helpful for the understanding of fundamental biological processes and for the selection of enzymes for particular molecular biological applications. DNA polymerase I (pol I) (Family A) enzymes have proved to be very useful for DNA sequencing applications, PCR, TCL, and other applications known in the art. Structure-function relationship studies indicate that known DNA pol I molecules share a similar modular organization. A 5 ' -> 3 ' exonuclease function is located in the N-terminal one-third of the enzyme. The remainder of the molecule forms one domain which is further classified into functional sub-domains. Adjacent to the 5 ' - 3 ^' exonuclease domain lies a 3 ' -* 5 ' exonuclease sub-domain, followed by a polymerase sub-domain (Blanco et al., Gene 700:27-38 (1991)).

In addition to classifying DNA polymerase enzymes into the above families, it is also useful to classify such polymerases as mesophilic (purified from mesophilic organisms) or thermophilic (purified from thermophilic organisms) in origin. DNA polymerases of mesophilic organisms were discovered earlier and have been more extensively studied than their thermophilic counterparts. As early as the 1950' s, isolation and purification protocols for DNA polymerase I from mesophilic bacteria (e.g., E. coli) and some of their phages were developed and have since been modified. See, e.g., Bessman et al. , J. Biol. Chem. 255: 171-177 (1958);

Buttin and Kornberg, J. Biol. Chem. 241:5419-5427 (1966). The DNA polymerases studied most extensively are the DNA polymerase I enzymes isolated from E. coli and the bacteriophage T7 DNA polymerase.

The DNA polymerases of mesophilic origin are useful in many biological applications, such as in certain DNA sequencing applications.

However, many important applications (e.g., polymerase chain reaction (PCR) applications and thermal cycle labeling (TCL)) require thermal cycling to repeatedly denature template DNA and/or RNA and their extension products.

Because mesophilic DNA polymerases do not withstand the high temperatures or thermal cycling of these applications, thermostable DNA polymerases enjoy significant advantages over mesophilic DNA polymerases in such applications.

The discovery and study of such thermostable DNA polymerases ~ from thermophilic bacteria — has been a much more recent phenomenon. See, e.g., Uemori et al., J. Biochem. 113: 401-410 (1993);

Uemori et al., Nucleic Acids Res. 21: 259-265 (1993)); Lawyer et al. J. Biol.

Chem. 264: 6427-6437 (1989); Kaledin et al., Biokhimiya 45:644-651 (1980);

Chien et al., J. Bacteriol. 727: 1550-1557 (1976); Gelfand et al , U.S. Patent Nos. 4,889,818 and 5,079,352; Burke et al., U.S. Patent No. 5,108,892.

Perhaps the best-studied thermostable DNA polymerase, derived from Thermits aquaticus, is called Taq pol I. A number of routes have been taken in attempts to clone the Taq DNA pol I gene. (See, e.g., Lawyer et al.

(1989); Gelfand et al. , U.S. Patent No. 5,079,352 (1992) (purification to approx. 200,000 units/mg reported); Lawyer et al. , PCR Methods and

Applications 2:275-287 (1993) (purification to 292,000 units/mg reported);

Engelke et al , Anal. Biochem. 191: 396-400 (1990); Sagner et al., Gene

97: 119-123 (1991)). As explained above, in addition to possessing useful DNA polymerase activity, a number of DNA polymerase I holoenzymes possess exonuclease activities, which for many biological applications are undesirable. Therefore, modified DNA polymerase enzymes having reduced exonuclease activities are desirable. Through deletion of the 5 ' one-third of DNA polymerase I genes, or the proteolytic cleavage and subsequent removal of the portion of the holoenzyme encoded thereby, scientists have created DNA pol I fragments retaining polymerizing activity, but having reduced 5 ' -* 3 ' exonuclease activity. (See, e.g., Joyce and Grindley, Proc. Natl. Acad. Sci. 80: 1830-1834 (1983) (the Klenow-Fragment of the E. coli DNA polymerase enzyme); Lawyer et al. , J. Biol. Chem. 264:6421-6431 (1989), Gelfand et al. , U.S. Patent No. 5,079,352 (1992), Lawyer et al , PCR Methods and Applications 2:275-287 (1993) (the Stoffel fragment of the T. aquaticus (Taq) DNA polymerase enzyme, reportedly purified to a specific activity of 369,000 units/mg); and Barnes, Gene 772:29-35 (1992) (the KlenTaq DNA polymerase).)

In addition to Taq DNA polymerases, other thermophilic DNA polymerases reportedly have been cloned and expressed in E. coli. Uemori et al. reportedly expressed DNA polymerases from Bacillus caldotenax (J. Biochem. 775:401-410 (1993)) and Pyrococcus fitriosus (Nucleic Acids Res. 27:259-265 (1993)).

DNA polymerases from other bacteria of the genus Thermits have been reported. A method of recovering a thermostable DNA polymerase from cultured Thermits thermophilus is reported in U.S. Patent No. 5,242,818 to Oshima et al. (1993). The purported purification of native Thermits flavus DNA polymerase with an apparent molecular weight of 66,000 daltons was described by Kaledin et al , Biokhimiya 46: 1576-1584 (1981)). In one application, Kainz et al , Anal. Biochem. 202:46-49 (1992), reported the amplification of a 10.9 kb fragment and a 15.6 kb fragment from phage lambda DNA with Hot Tub (T. flavus) polymerase (Amersham, Arlington Heights, IL), but not with Taq polymerases. The rapid filter assay of Sagner et al., Gene 97: 119-123 (1991) has allowed Akhmetzjanov and Vakhitov to identify a purported T. flavus (strain and origin unidentified) DNA polymerase I gene and to determine the DNA sequence of this gene (Nucleic Acids Res. 20:5839 (1992)). There is no report of the expression of an active DNA polymerase encoded by the purported Thermits flavus DNA polymerase I (Tfl DNA pol I) gene characterized by Akhmetzjanov and Vakhitov. Native T. flavus (Tfl) DNA polymerase I is commercially available, e.g. , from Molecular Biology Resources, Inc. (Milwaukee, WI, Catalogue #1112-01).

The different reports of thermostable DNA polymerases and their derivatives suggest these enzymes possess different, unpredictable properties that may be advantageous or detrimental, depending on the biological application in which the DNA polymerase is to be employed. For example, Thermits thermophilus DNA polymerase I was reported to have a significant reverse transcriptase activity. In the same reaction tube, in successive steps, the reverse transcriptase function allows the production of double stranded DNA from RNA and then the DNA polymerase function is used to amplify this cDNA. Myers and Gelfand, Biochemistry 50:7661-7666 (1991).

The KlenTaq DNA polymerase is an example of an enzyme fragment with important properties differing from the Taq holoenzyme. The KlenTaq DNA polymerase reportedly has a roughly two-fold lower PCR- induced relative mutation rate than Taq polymerase holoenzyme. However, more units of KlenTaq are needed to obtain PCR products similar to those generated with Taq DNA pol I.

Similarly, Lawyer et al. (1993) reported that T. aquaticus DNA polymerase I fragments possessed greater thermostability and were active over a broader Mg⁺⁺ -range than the corresponding holoenzyme. Because of its broader range of magnesium ion concentration, the Stoffel fragment has been used in multiplex PCR, where more than two primers must anneal to the template. The thermostability of the Stoffel fragment makes this enzyme a better choice when GC-rich templates are amplified. It is desirable to purify and isolate additional DNA polymerase enzymes and derivatives, to take advantage of the unique but unpredictable properties that such molecules may have.

There remains a need in the art for new, thermostable DNA polymerase enzymes for use in the expanding universe of molecular biological applications. More particularly, there exists a need for thermostable DNA polymerase enzymes having high purity, high DNA polymerase specific activity, low levels of exonuclease activity, and possessing high fidelity (low mutation frequencies) and high processivity when used in DNA amplification protocols. An object of the present invention is to provide polymerase enzyme preparations of greater purity, quantity, DNA polymerase specific activity, and processivity than has heretofore been possible. A further object is to eliminate the need and expense of culturing of large volumes of thermophilic bacteria at high temperatures that is associated with preparing thermostable polymerase enzyme preparations. Yet another object is to provide a recombinant polymerase possessing reduced exonuclease activities, as compared to the currently available native holoenzyme.

SUMMARY OF THE INVENTION

The present invention relates to the cloning and expression of a gene encoding a thermostable DNA polymerase, the purification of a recombinant thermostable DNA polymerase encoded by the gene, and applications for using the polymerase. The gene of the Thermits flavus DNA polymerase I (Tfl DNA pol I), was cloned and expressed in Escherichia coli. The purified recombinant T. flavus DNA polymerase enzyme is shown to be thermostable and have a molecular weight of about 90,000 to 100,000 daltons. The DNA sequence of the Tfl DNA pol I gene, including flanking sequences, was determined and the coding sequence of the recombinant enzyme was mapped within this gene. A Tfl DNA Pol I gene fragment also was expressed in E. coli, the purified recombinant protein products ("exo^" fragment") lacking 274 and 275 amino acids from the N-terminus of the Tfl DNA pol I holoenzyme. This Tfl exo^" fragment has very low 3 ' -> 5 ' and 5 ' -> 3 ' exonuclease activities. Numerous properties of and applications for the recombinant enzymes are described. In one aspect, this invention provides purified polynucleotides

(e.g. DNA sequences and RNA transcripts thereof) encoding a thermostable polypeptide having DNA polymerase activity. Preferred DNAs include the Thermits flavus DNA pol I gene comprising nucleotides 301 to 2802 of SEQ ID NO: 1 ; the Thermits flavus DNA pol I exo^" fragment gene comprising nucleotides 1 to 1791 of SEQ ID NO: 3; the DNA comprising nucleotides 112-1791 of SEQ ID NO: 3; a portion of the insert of plasmid pTFLRT4 (ATCC Accession No. 69633), said portion encoding a thermostable polypeptide having DNA polymerase activity; a portion of the insert of plasmid p21EHcMl. l, (ATCC Accession No. 69632), said portion encoding a thermostable polypeptide having DNA polymerase activity; fragments or portions of these DNAs that encode thermostable polypeptides having DNA polymerase activity; and variants of these DNAs that encode thermostable polypeptides having DNA polymerase activity.

In another aspect, this invention provides DNA sequences such as those described above operatively linked to a promoter sequence, a cloning vector, an expression vector, or combinations thereof.

In related aspects, the invention provides novel plasmids and vectors. For example, the invention provides a plasmid pTFLRT4, having ATCC Accession No. 69633; and a plasmid p21EHcMl. l , having ATCC Accession No. 69632. The invention also provides a vector that includes nucleotides 301 to 2802 of SEQ ID NO:l, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity; and a vector that includes nucleotides 112 to 1791 of SEQ ID No:3, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity. In related aspects, the invention provides a vector having at least one insert consisting essentially of nucleotides 301 to 2802 of SEQ ID NO: 1, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity. The invention further provides a vector having at least one insert consisting essentially of nucleotides 112 to 1791 of SEQ ID NO: 3, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity.

The present invention is also directed to host cells, such as prokaryotic and eukaryotic cells, that have been stably transformed with DNAs vectors, or plasmids of the invention. Another aspect of the invention is directed to such transformed host cells that are capable of expressing a thermostable polypeptide encoded by the DNAs, the peptide having DNA polymerase activity.

In another aspect, this invention provides purified thermostable polypeptides having DNA polymerase activity. Preferred peptides include a Thermits flavus DNA polymerase I holoenzyme substantially free of other Therm s flavus proteins; a polypeptide having the amino acid sequence of SEQ ID NO: 2; a fragment of a Thermus flavus DNA polymerase I holoenzyme, including a fragment with reduced exonuclease activity as compared to the holoenzyme, and also including a fragment having the amino acid residues 1-560 or 2-560 of the amino acid sequence shown in SEQ ID NO: 5; a fragment encoded by the insert of plasmid p21EHcMl. l, having ATCC Accession No. 69632; fragments of the above peptides that retain DNA polymerase activity; and variants of the above peptides that retain DNA polymerase activity. In another aspect, this invention provides methods for purifying a thermostable polypeptide having DNA polymerase activity including the steps of transforming a host cell with a DNA of the present invention to create a transformed host cell; cultivating the transformed host cell under conditions that promote expression of a thermostable polypeptide encoded by the DNA, the polypeptide having DNA polymerase activity; and purifying the thermostable polypeptide with a monoclonal antibody that is cross-reactive with the thermostable polypeptide. In one preferred method, the cross- reactive monoclonal antibody has specificity for a Thermus aquaticus DNA polymerase and/or for a Thermus flavus DNA polymerase.

In another preferred method, commercially available chromato¬ graphy columns are used to purify the expressed polypeptide.

In another aspect, this invention provides methods of purifying a thermostable polypeptide having DNA polymerase activity. One such method includes the steps of expressing the thermostable polypeptide in a host cell, the polypeptide having an amino acid sequence encoded by a DNA of the present invention; lysing the cell to create a suspension containing the thermostable polypeptide, as well as host cell proteins and cell debris; contacting a soluble portion of the suspension with an antibody that is immunologically cross-reactive with the thermostable polypeptide under conditions wherein the antibody binds to the thermostable polypeptide to form an antibody-polypeptide complex; isolating the antibody-polypeptide complex; and separating the thermostable polypeptide from the isolated antibody- polypeptide complex to provide a purified thermostable polypeptide. Preferably, such a method further includes the steps of heating the suspension to denature the host cell proteins; and centrifuging the suspension to remove the cell debris and denatured host cell proteins. In more preferred methods, the immunologically cross-reactive antibody is a monoclonal antibody, such as a monoclonal antibody that is immunologically cross-reactive with Thermus aquaticus DNA polymerase I and/or Thermus flavus DNA polymerase I. This preferred method is exemplified herein using the monoclonal antibody purified from a hybridoma designated hybridoma 7B12.

In another aspect, this invention provides methods of using the DNA constructs of the invention to produce recombinant thermostable polypeptides having DNA polymerase activity. One such method involves using a DNA encoding a DNA polymerase enzyme to generate active fragments of the DNA polymerase enzyme, including the steps of: deleting a portion of the DNA to create a modified DNA; expressing the modified DNA to produce a DNA polymerase enzyme fragment; purifying the DNA polymerase enzyme fragment; assaying the DNA polymerase enzyme fragment for DNA polymerase activity; and selecting a DNA polymerase enzyme fragment having DNA polymerase activity; wherein the DNA is selected from among the DNAs described herein.

In another aspect, this invention provides methods for using the proteins of the invention in biological applications, such as DNA sequencing; amplification of DNA and/or RNA sequences; polymerase chain reaction (PCR); thermal cycle labeling (TCL); universal thermal cycle labeling (UTCL); ligase chain reaction (LCR); and other applications or processes that would be apparent to those skilled in the art. In yet another aspect, this invention provides kits for using the proteins of the invention in various biological applications, such as kits for labeling DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURES IA and IB graphically depict the cloning strategy: (IA) for the gene encoding the Tfl DNA pol I holoenzyme; and (IB) for the DNA encoding the exo" fragment of T. flavus DNA polymerase I. The abbreviations used are: B: BamHI, Rl: EcoRI, RV: EcoRV, He: Hindi, P lacZ: promoter of the lacZ gene, S: Sail, and X: Xbal. Jagged lines (

£-» represent vector DNA; straight horizontal ( ) lines represent Tfl insert DNA; dark and light shaded rectangles depict Tfl DNA pol I gene sequences. The graphical depictions are not drawn to scale, and not all available restriction sites are shown in all steps.

FIGURE 2 depicts the DNA sequence and the deduced amino acid sequence for the Tfl DNA pol I holoenzyme coding sequence and for 5 ' untranslated and 3 ' untranslated sequences. The circled amino acid (Glu₂₃₉) is the first amino acid believed to be translated during translation of plasmid p21EHcMl .1 , encoding the Tfl exo^" fragment. The boxed amino acid (Leu^s) is the amino acid determined to be the first amino acid of the purified and isolated major Tfl exo^' fragment. An asterisk (*) indicates the stop codon TAG.

FIGURE 3 is a comparison of deduced amino acid sequences from the Thermus flavus DNA polymerase I of this invention (MBR TFL); Thermus aquaticus DNA polymerase I (TAQ) reported in Lawyer et al, J. Biol. Chem. 264:6427-6437 (1989)); and purported Thermus flavus DNA polymerase I (A&V TFL) described in Akhmetzjanov and Vakhitov, Nucleic Acids Res. 20:5839 (1992). The sequences were aligned to maximize homology. Conservative differences between the amino acid sequences are indicated with asterisks (*) and non-conservative differences are indicated with arrowheads (^Λ).

FIGURE 4 depicts double-stranded DNA sequence of the T. flavus DNA pol I gene, including 5' untranslated and 3' untranslated sequences. Lower case letters indicate untranslated sequences, upper case letters represent the coding sequence. The start codon (ATG) of Tfl DNA pol I is at positions 301-303, and the stop codon is at positions 2803-2805. The positions in the sequence that correspond to synthetic primers used in sequencing reactions have been indicated with boxes. The sequence of the 2-4 fragment is underlined with an arrow ( < >). FIGURE 5 depicts the relative DNA polymerase enzymatic activity, at different buffered pH levels, of native Thermus flavus holoenzyme (nTfl Holo: empty squares); recombinant Thermus flavus holoenzyme (rTfl Holo: diamonds); Thermus flavus exo^' fragment (Tfl exo^": circles); T. aquaticus DNA pol I (AmpliTaq: crossed boxes); and the Taq enzyme Stoffel fragment (Stoffel: triangles).

FIGURE <ΪA depicts the relative DNA polymerase enzymatic activity, at different concentrations of MgCl₂, of native Thermus flavus holoenzyme (nTfl Holo: empty squares); recombinant Thermus flavus holoenzyme (rTfl Holo: diamonds); Thermus flavus exo fragment (Tfl exo : circles); and T. aquaticus DNA pol I Stoffel fragment (Stoffel: triangles).

FIGURE 6B depicts the relative DNA polymerase enzymatic activity, at different concentrations of MnCl₂, of native Thermus flavus holoenzyme (nTfl Holo: empty squares); recombinant Thermus flavus holoenzyme (rTfl Holo: diamonds); Thermus flavus exo' fragment (Tfl exo^": circles); and T. aquaticus DNA pol I Stoffel fragment (Stoffel: triangles).

FIGURE 7A depicts the relative DNA polymerase enzymatic activity, at different temperatures, of native Thermus flavus holoenzyme (nTfl

Holo: open boxes); recombinant Thermus flavus holoenzyme (rTfl Holo: diamonds); Thermits flavus exo^' fragment (Tfl exo : circles); and T. aquaticus

DNA pol I Stoffel fragment (Stoffel: triangles).

FIGURE 7B photographically depicts the relative quantities of

PCR amplification product generated after 25, 30, and 35 reaction cycles, using 10 units of Tfl exo^" fragment (E) or Stoffel fragment (S) as the PCR DNA polymerase. The far right lane depicts the PCR amplification product generated after 35 reaction cycles using 1.1 unit of Tfl exo^" fragment.

FIGURE 8 depicts enzymatic stability in thermal cycling (relative DNA polymerase enzymatic activity after different numbers of PCR cycles), of native Thermus flavus holoenzyme (nTfl Holo: empty squares); Thermus flavus exo' fragment (Tfl exo^": circles); T. aquaticus DNA pol I (AmpliTaq: crossed squares); and 7. aquaticus DNA pol I Stoffel fragment (Stoffel: triangles).

FIGURE 9 photographically depicts the purity of purified E. coli DNA polymerase I (Eco Pol I, control), recombinant Thermus flavus holoenzyme (rTfl Holo), and Thermus flavus exo" fragment (Tfl exo ) on a 12.5% SDS-PAGE gel stained with silver.

FIGURES 10A, 10B, IOC, and 10D photographically depict portions autoradiographs of sequencing gels showing DNA sequence obtained with the indicated polymerases substituted into the SEQUAL™ or the Cycle SEQUAL™ DNA Sequencing Kit. Abbreviations: recombinant Thermus flavus holoenzyme (Tfl Holo); Thermus flavus exo^" fragment (Tfl exo); T. aquaticus DNA pol I holoenzyme (AmpliTaq); and the Taq enzyme Stoffel fragment (Taq Stoffel).

DETAILED DESCRIPTION OF THE INVENTION

This application describes the isolation and characterization of the gene coding for Thermits flavus (ATCC Accession No. 33923) DNA polymerase I (Tfl DNA pol I) and having homology to the family A enzymes described above. Also described is the expression of this gene in E. coli and the purification and characterization of the recombinant DNA polymerase. The cloning and expression of an active fragment of the Thermus flavus DNA polymerase gene is also described, and the gene fragment and expressed peptides are characterized. Recombinant vectors and host cells are also described. Additionally, methods and kits are described that involve the DNAs and proteins of the present invention. Thus, as the discussion below details, the present invention has several aspects.

As a first step in the generation of the DNAs and polypeptides of the present invention, native T. flavus DNA polymerase I was purified and isolated from T. flavus cells (ATCC Accession No. 33923) and digested with trypsin, and amino acid sequence information was obtained from one of the reaction products (i.e. from a trypsin digest protein fragment). (See Example 1.) Additionally, a Thermus flavus genomic library was constructed in phage λ Dash II and amplified. (See Example 2.)

The amino acid sequence information generated in Example 1 , together with published amino acid sequence information from the Thermus aquaticus DNA pol I gene, was used to create synthetic DNA primers for isolating a portion of the Thermus flavus DNA polymerase I gene. (Example 3.) More particularly, a first primer, designated FTFL2, was synthesized to correspond with known coding sequence from T. aquaticus DNA pol I gene (Lawyer et al., J. Biol. Chem 264: 6427-6437 (1989)), and to bind to the top strand of the T. aquaticus DNA pol I gene. The particular T. aquaticus coding sequence chosen encodes a portion of the T. aquaticus DNA pol I amino acid sequence that is homologous to the native T. flavus DNA pol I peptide that had previously been sequenced (Example 1). A second primer, designated RTFL4, was synthesized to have a sequence that binds to the 3 '-end of the 7. aquaticus gene on the opposite strand. A DNA amplification reaction was performed with primer FTFL2, primer RTFL4, and T. flavus genomic DNA. The amplification reaction yielded a single amplification product, designated the "2-4 fragment. " This fragment was cloned into M13mpl8 vector, amplified in E. coli, and sequenced.

As explained in detail in Example 4, the 2-4 fragment (obtained by the procedures outlined in Example 3) was used to isolate the Thermus flavus DNA pol I gene from the T. flavus genomic library that had been constructed (Example 2). Specifically, the 2-4 fragment was further amplified and used to generate probes via thermal cycle labeling (TCL). The amplified T. flavus genomic library was plated on 2XTY plates and grown until plaques formed. Duplicate plaque lifts were obtained from each plate onto Hybond N filters, and these filters were then screened using the above-described TCL probes using hybridization methods well known in the art. Positive plaques were selected, purified by dilution and by re-screening with the 2-4 probes, and then further characterized. In particular, two clones with inserts of 14-16 kb, designated λ21 and λ51, were chosen for further analyses.

Clones λ21 and λ 1 were used as a starting point from which the complete T. flavus DNA pol I gene was cloned and sequenced. As explained in detail in Example 5 and with reference to FIGURE IA, restriction mapping, subcloning, and partial sequencing led to the determination that a subclone of λ21 designated p21E10 contained about 2/3 of the Tfl DNA pol I gene (3' end), whereas a subclone from λ51 designated p51E9 contained a 5' portion of the gene that overlapped the coding sequence contained in clone p21E10.

A primer walking procedure was used to obtain the complete sequence of the gene. Specifically, primers homologous or complimentary to the ends of previously determined sequences (obtained from p21E10 and from other deletion vectors) were synthesized and used in additional sequencing reactions. By repeating this process the entire length of the gene was sequentially sequenced. The DNA and deduced amino acid sequence for the T. flavus DNA pol I holoenzyme are shown in FIGURE 2, which corresponds to SEQ. ID NO: 1 and 2 in the Sequence Listing. The sequences of each primer used, and the relative location of the primers in the gene sequence, are depicted in Table 2 and in FIGURE 4, respectively. The amino acid sequence of the holoenzyme depicted in FIGURE 2 and SEQ. ID NO: 2 corresponds with nucleotides 301 to 2802 of the DNA depicted in FIGURE 2 and SEQ ID NO: 1.

The foregoing results demonstrate that an aspect of the invention is directed to a purified DNA encoding a thermostable polypeptide having DNA polymerase activity, the DNA comprising nucleotides 301 to 2802 of SEQ ID NO: 1. This DNA may be operatively linked to other DNAs, such as expression vectors known in the art. The invention is also directed to a vector having at least one insert consisting essentially of nucleotides 301 to 2802 of SEQ ID NO: 1, the nucleotides encoding a thermostable polypeptide having DNA polymerase activity. Similarly, the invention is directed to a vector comprising nucleotides 301 to 2802 of SEQ ID NO:l, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity.

With the gene sequence established, the DNA and deduced amino acid sequences of the T. flavus DNA pol I gene were aligned and compared to the DNA and deduced amino acid sequences of the purported Tfl DNA pol I published by Akhmetzjanov and Vakhitov, Nucleic Acids Res. 20:5839 (1992) (83% DNA sequence homology, 85% amino acid sequence homology) and to the deduced amino acid sequence of the Taq pol I gene (86% DNA sequence homology, 87% amino acid sequence homology). The amino acid comparison is depicted in FIGURE 3.

To produce a recombinant T. flavus DNA pol I protein a full- length 7. flavus DNA pol I gene clone was constructed, expressed in E. coli, and purified. As detailed in Example 6 and FIGURE IA, plasmids p51E9 and p21E10 were further restriction mapped and subsequently subcloned to generate plasmid p21BRV2, containing a 1.3 kb insert that includes the 3' region of the Tfl DNA pol I gene, and plasmid p51X16, containing a 2.5 kb BamHI fragment in which the 5' region of the gene was located. Linearization of plasmid p21BVR2 with BamHI and ligation of this linearized plasmid to the BamHI fragment of p51X16 yielded clone pTFL 1.4, containing the entire Tfl DNA pol I gene.

E. coli DH5αF' were transformed with plasmid pTFL 1.4 and grown in a fermentor to recombinantly produce T. flavus DNA pol I holoenzyme. As detailed in Example 6, this recombinant protein was purified from the lysed E. coli with a method that included a heat denaturation of E. coli proteins, precipitations and centrifugations, Sephadex G-25 and Bio-Rex 70 column chromatography, and immunoaffinity chromatography. The calculated DNA polymerase specific activity of T. flavus DNA pol I isolated by this procedure was determined to be 79,500 U/mg protein. In order to increase the yield of recombinant T. flavus DNA pol

I holoenzyme, a second expression clone was constructed in which the lacZ promoter was fused directly to the initiation codon of the Tfl DNA pol I gene. As detailed in Example 7 and FIGURE IA, the promoter was fused to the 5' portion of the gene located using site-directed mutagenesis, and a second generation expression clone, designated pTFLRT4, was generated.

E. coli (strain DH5αF'IQ) were transformed with pTFLRT4 and cultivated, and recombinant T. flavus DNA pol I was isolated therefrom and purified. As detailed in Example 7, the purification protocol includes heat treatment, polyethyleneimine- (PEI-) precipitation, ( H₄)₂SO₄- precipitation, Bio Rex 70 chromatography and immunoaffinity chromatography. The yield was approximately 2,000,000 units of enzyme from 500 g of cells, and the purified enzyme preparation was found to have a DNA polymerase specific activity of 217,600 U/mg protein. The N-terminal amino acid sequence of the recombinant Tfl DNA pol I enzyme was determined and found to be identical to the sequence deduced from the T. flavus DNA Pol I gene sequence.

The foregoing discussion demonstrates that an aspect of the invention is directed to a purified DNA comprising a portion of the insert of plasmid pTFLRT4, the plasmid having ATCC Accession No. 69633, the portion encoding a thermostable polypeptide having DNA polymerase activity. This DNA may be operatively linked to additional DNAs, such as promoter DNAs and/or expression vector DNAs known in the art. A preferred DNA is plasmid pTFLRT4 itself. The present invention is also directed to thermostable polypeptides having DNA polymerase activity. In one aspect, the invention is directed to a Thermus flavus DNA polymerase protein substantially free of other Thermus flavus proteins. Exemplary proteins include a DNA polymerase protein having the amino acid sequence of SEQ ID NO: 2. Similarly, the invention is directed to a thermostable polypeptide having DNA polymerase activity and consisting essentially of the amino acid sequence of SEQ ID NO: 2.

In addition to the cloning and expression of the Tfl DNA pol I holoenzyme, a vector allowing for the expression of a truncated DNA polymerase was generated. As explained in Example 8 and FIGURE IB, a vector lacking the 5 ' one-third of the T. flavus DNA polymerase I gene was constructed. Specifically, the ATG start codon of lacZ was brought in frame with the DNA encoding amino acids 239 to 834 of the Tfl DNA pol I holoenzyme using site-directed mutagenesis, and the resulting plasmid, designated p21EHcMl. l, was expressed in E. coli DH5αF '. The insert of plasmid p21EHcMl. l includes a DNA sequence that corresponds with SEQ ID NO: 3 in the Sequence Listing, and encodes a polypeptide predicted to have the amino acid sequence depicted in SEQ ID NO: 4. The expressed polypeptide product was designated Thermus flavus DNA polymerase I exonuclease-free fragment, or "Tfl exo^' fragment. " An aspect of the invention is directed to a purified DNA comprising a portion of the insert of plasmid p21EHcMl. l, the plasmid having ATCC Accession No. 69632, the portion encoding a thermostable polypeptide having DNA polymerase activity. This DNA may be operatively linked to additional DNAs, such as known promoter DNAs and/or expression vectors. A preferred DNA is plasmid p21EHcMl.1 itself. As detailed in Example 8, the purification protocol for the Tfl exo' fragment expressed in E. coli [p20EHcMl-l] included PEI-precipitation, gel filtration, Procion-Red Sepharose chromatography and immunoaffinity chromatography. The yield using this preparation protocol was approximately 300,000 units of enzyme from 50 g of cells, and the preparation had a DNA polymerase specific activity of 600,000 U/mg protein.

The N-terminal amino acid sequence of the Tfl exo" fragment was determined (Example 8), and interestingly, the purified protein lacked 37 N-terminal amino acids predicted from the DNA encoding the exo^" fragment. The deduced amino acid sequence of the purified Tfl exo^" fragment - based on this amino acid sequence data and the complete DNA sequence ~ is depicted in SEQ ID NO: 5, and corresponds with amino acid 275 to 834 of FIGURE 2. A minor sequence lacking 38 N-terminal amino acids was also detected.

The foregoing demonstrates that another aspect of the invention is directed to a purified DNA encoding a thermostable polypeptide having DNA polymerase activity, the DNA comprising a portion of SEQ ID NO: 3. For example, the invention is directed to a purified DNA comprising nucleotides 112 to 1791 of SEQ ID NO: 3. This DNA also may be operatively linked to other DNAs, such as to nucleotides 1 to 111 of SEQ ID NO: 3, and/or to expression vectors known in the art. In a related aspect, the invention is directed to a vector comprising nucleotides 112 to 1791 of SEQ ID NO: 3, the nucleotides encoding a polypeptide having thermostable DNA polymerase activity. Similarly, the invention is directed to a vector having at least one insert consisting essentially of nucleotides 112 to 1791 of SEQ ID NO: 3, the nucleotides encoding a thermostable polypeptide having DNA polymerase activity.

The recombinant expression and purification of biologically active Tfl exo' fragment demonstrates additional aspects of the present invention. For example the present invention is directed to a purified fragment of Thermus flavus DNA polymerase I protein, the fragment having DNA polymerase activity. Exemplary fragments include a fragment having an amino acid sequence comprising amino acids 2 to 560 of or 1 to 560 of SEQ. ID NO: 5, and a fragment encoded by the insert of plasmid p21EHcMl. l, having ATCC Accession no. 69632. Also, the invention is directed to a polypeptide having DNA polymerase activity and consisting essentially of the amino acid sequence of SEQ ID NO: 5.

The foregoing description of methods and recombinant cells demonstrates that the present invention is directed to more than DNA's and polypetides. Another important aspect of the invention is directed to a host cell transformed with a DNA, vector, or plasmid of the present invention, including those specifically mentioned above. Preferably, the host cell transformed with a DNA is capable of expressing a thermostable polypeptide encoded by the DNA, the polypeptide having DNA polymerase activity. By host cell is meant both prokaryotic host cells, including E. coli cells, and eukaryotic host cells.

In addition to being directed to DNAs, transformed cells, and polypeptides, the present invention is directed to various methods for using DNAs and polypeptides. For example, the invention is directed to a method for purifying a thermostable polypeptide having DNA polymerase activity comprising the steps of: transforming a host cell with a DNA to create a transformed host cell, the DNA selected from the DNA's of the present invention; cultivating the transformed host cell under conditions to promote expression of a thermostable polypeptide encoded by the DNA, the polypeptide having DNA polymerase activity; and purifying the thermostable polypeptide with a monoclonal antibody that is cross-reactive with the thermostable polypeptide. In one preferred method, the cross-reactive monoclonal antibody has specificity for a Thermus aquaticus DNA polymerase and/or for a Thermus flavus DNA polymerase.

In another preferred method, commercially available chromatography columns are used to purify the expressed polypeptide.

The purification protocols for recombinant Tfl DNA polymerase I and Tfl exo^" fragment demonstrate that another aspect of the invention relates to methods of purifying a thermostable polypeptide having DNA polymerase activity. One such method includes the steps of expressing the thermostable polypeptide in a host cell, the polypeptide having an amino acid sequence encoded by a DNA of the present invention; lysing the cell to create a suspension containing the thermostable polypeptide and host cell proteins and cell debris; contacting a soluble portion of the suspension with an antibody that is immunologically cross-reactive with the thermostable polypeptide under conditions wherein the antibody binds to the thermostable polypeptide to form an antibody-polypeptide complex; isolating the antibody-polypeptide complex; and separating the thermostable polypeptide from the isolated antibody- polypeptide complex to provide a purified thermostable polypeptide. Preferably, such a method further includes the steps of heating the suspension to denature the host cell proteins; and centrifuging the suspension to remove the cell debris and denatured host cell proteins. In more preferred methods, the immunologically cross-reactive antibody is a monoclonal antibody, such as a monoclonal antibody that is specific for Thermus aquaticus DNA polymerase I and/or Thermus flavus DNA polymerase I. This preferred method is exemplified herein using a monoclonal antibody purified from a hybridoma designated hybridoma 7B12. This monoclonal antibody is commercially available from Molecular Biology Resources, Inc., Milwaukee, Wisconsin, as Cat. No. 4100-01.

The invention is also directed toward a method of using a DNA encoding a DNA polymerase enzyme to generate active fragments of the DNA polymerase enzyme, comprising the steps of: deleting a portion of the DNA to create a modified DNA, expressing the modified DNA to produce a DNA polymerase enzyme fragment, purifying the DNA polymerase enzyme fragment, assaying the DNA polymerase enzyme fragment for DNA polymerase activity, and selecting a DNA polymerase enzyme fragment having DNA polymerase activity, wherein the DNA is selected from DNAs of the present invention. As detailed in Example 9 and summarized in Table 3 A, a number of experiments were conducted to characterize the exonuclease activities of 7. flavus DNA pol I holoenzyme and exo^" fragment. For both the holoenzyme and the exo^' preparation, each exonuclease and endonuclease activity assayed was either very low or undetectable.

As detailed in Example 10, a number of additional assays were performed to better characterize the recombinant Tfl DNA pol I proteins that had been purified and to compare these proteins to other known thermostable DNA polymerases. For example, the DNA polymerase activity of the Tfl holoenzyme and the exo^' fragment was analyzed at different pH values, and at different MgCl- and MnCl₂ concentrations. FIGURES 5 (pH optima); 6A (MgCl₂ optima); 6B (MnCl₂ optima) and 7 A (temperature optima) summarize the results of some of these assays. The optimal range and the peak values (in parentheses) are summarized in Table IA.

TABLE IA

Holoenzyme Exo Fragment pH 9.5 - 10.5 (10) 7.5 - 10 (8.5)

MgCl₂ [mM] ►50 1.3 - 13 (5)

MnCl₂ [mM] 0.8 - 4 (2) 2.1 - 11 (4)

To assay thermostability the enzymes were incubated for 30 min. at different temperatures to define the temperature optimum. The highest activity (100%) was found at 80°C for the holoenzyme (14% remaining after 30 min. at 90 °C), and 70 to 75 ^°C (8% remaining after 30 min. at 90 °C) for the exo fragment.

The Tfl holoenzyme preparation enzyme was more than 95 % pure as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% gel (FIGURE 9). The apparent molecular weight was 80 kD, which is lower than the calculated molecular weight of approximately 94 kD based on the DNA sequence. The Tfl holoenzyme preparation was found to be free of detectable double-stranded nucleases and of 5 ' - 3 ' exonuclease and endonuclease activities. Low levels of single-stranded nucleases and of 3 ' -» 5 ' exonuclease activity were found. The isoelectric point was determined to be 6.43.

The purified Tfl exo^" fragment was found to possess low 3 ' -* 5 ^' and 5 ' -* 3 ' exonuclease activities. The preparation was more than 95 % pure as judged by SDS-PAGE (FIGURE 9). The apparent molecular weight of 68 kD as judged by SDS-PAGE compares well with the calculated molecular weight of approximately 63 kD. The Tfl exo^" preparation was found to be free of detectable double- and single-stranded nucleases and endonuclease activities. The isoelectric point was determined to be 5.94.

The performances of the Tfl holoenzyme and the exo" fragment were tested in DNA sequencing, PCR and TCL (Examples 11, 12 and 13). Both enzymes were found to be useful in sequencing reactions utilizing labeled primer in conjunction with single-stranded and double-stranded DNA templates, and in a cycle sequencing reaction with a single-stranded template. The enzymes were also useful in sequencing reactions utilizing internal labeling with, for example, [α^SJ-dATP. In all the reactions tested the Tfl exo^' fragment provided DNA sequence information of more than 150 nucleotides, as did recombinant Tfl DNA pol I holoenzyme.

The Tfl DNA pol I holoenzyme and the exo^' fragment were tested in PCR reactions. The recombinant holoenzyme gave similar results to the native enzyme. The Tfl exo" fragment retained 50% of its activity after 16 cycles. The holoenzyme retained 50% of its activity for 20 cycles. The specific amplified products were analyzed at the same time. After 20 cycles, an amplification product was visible on agarose gels. The amount of product increased between 25-50 cycles, but decreased after 100 cycles. The native T. flavus enzyme provided with the ZEPTO™ Labeling kit (CHIMERx, Madison, WI) was replaced by the recombinant holoenzyme or by the (recombinant) exo^' fragment. The efficiency of the labeling of plasmid pUC19 was determined on agarose gels and the efficiency of incorporation was determined in dot blot analysis. A dilution of 1:10^s of labeled probes generated with the holoenzyme was detectable (1: 10^s for probes generated by the exo^" fragment). Both results indicated that the enzymes have the required activity needed for labeling pUC19 DNA in TCL. A protocol is also provided for demonstrating that the present invention is also directed to TCL in which the recombinant Tfl DNA pol I holoenzyme is employed without exogenous primers for enzymatic extension. In this method, referred to as Universal Thermal Cycle Labeling (UTCL), DNA of unknown sequence is combined intact with rTfl DNA Pol I holoenzyme, deoxyribonucleotide triphosphates, and the appropriate buffer. The holoenzyme is then combined with intact template and subjected to repeated cycles of denaturation annealing and extension. Alpha ³²P-dATP, ³²P-dTTP, ³²P-dGTP, ³ P-dCTP, biotin-dUTP, fluorescein-dUTP, or digoxigenin-dUTP is also included in the extension step for subsequent detection purposes. The foregoing results demonstrate further aspects of the invention. For example, the invention is further directed to a method for labeling DNA, comprising the steps of: digesting an aliquot of template DNA with a restriction endonuclease reagent wherein the digestion generates sequence-specific DNA fragments; mixing an aliquot of undigested template DNA with the sequence-specific DNA fragments; denaturing the mixture of template DNA and sequence-specific DNA fragments thereby generating denatured template DNA and oligonucleotide primers; annealing the primers to the denatured undigested template DNA to form a DNA-primer complex; and performing an extension reaction from the primers in the DNA-primer complex using Tfl exo^" fragment in the presence of one or more nucleotide triphosphates, wherein at least one nucleotide triphosphate has a label.

Further, the invention is directed to a method for thermal cycle labeling DNA comprising the steps of: digesting an aliquot of template DNA with a restriction endonuclease reagent wherein the digestion generates sequence-specific DNA fragments; mixing an aliquot of undigested template DNA with the sequence-specific DNA fragments; denaturing the mixture of template DNA and the DNA fragments thereby generating denatured template DNA and oligonucleotide primers; annealing the primers to the denatured undigested template DNA to form a DNA-primer complex; performing an extension reaction from the primers in the DNA-primer complex using Tfl DNA pol I exo^' fragment in the presence of one or more nucleotide triphosphates wherein at least one nucleotide triphosphate has a label; heat- denaturing the labeled extension products; reannealing the excess primers with the template DNA and with the extension products; and performing at least one additional extension reaction from the DNA-primer complex using a Tfl DNA pol I exo^' fragment.

The present invention is further directed to kits for labeling DNA. A kit of the present invention includes, in association: a labeling buffer; a concentrated mixture of 1 or more nucleotide triphosphates; Tfl DNA pol I exo' fragment; and a control DNA, the control DNA being useful for monitoring the efficiency of labeling. Additionally, the kit may include a restriction endonuclease reagent and a restriction endonuclease buffer.

In another aspect, a kit of the present invention for labeling DNA comprises, in association: a Tfl DNA pol I exo^" fragment; and a Tfl

DNA pol I exo' fragment buffer. Preferably, such a kit further comprises a concentrated mixture of 1 or more nucleotide triphosphates and a control

DNA, the control DNA being useful for monitoring the efficiency of labeling.

The following examples are intended to describe various aspects of the invention in greater detail. More particularly, in Example 1 , the purification and amino acid sequencing of native Thermus flavus DNA polymerase I is described. In Example 2, the construction and amplification of a Thermus flavus genomic DNA library is described. In Example 3, the cloning and sequencing of a Thermus flavus DNA polymerase I gene fragment is described. Example 4 details the preparation of gene-specific probes and screening of the Thermus flavus genomic library for clones containing the T. flavus DNA pol I gene. Example 5 details the sequencing of the T.flavus DNA polymerase I gene. In Example 6, the construction and expression of a full-length T. flavus DNA pol I clone and purification of full-length recombinant T. flavus DNA pol I protein are described. In Example 7, the construction and expression of a high-yield, full-length T. flavus DNA pol I clone and purification of full-length recombinant T. flavus DNA pol I is described. Example 8 details the cloning and expression of the exo^" fragment of T. flavus DNA polymerase I. In Example 9, the characterization of recombinant T. flavus DNA polymerase I exonuclease activities is detailed. In Example 10, studies are described comparing the recombinant T. flavus and T. aquaticus DNA polymerases. In Example 11, DNA sequencing with recombinant T. flavus DNA polymerases is detailed. Example 12 demonstrates the utility of recombinant Tfl holoenzyme and the exo fragment in polymerase chain reaction procedures. Example 13 demonstrates the utility of recombinant Tfl DNA pol I holoenzyme and the Tfl exo^' fragment for use in thermal cycle labeling procedures. Example 14 analyzes the utility of T. flavus DNA pol I holoenzyme and exo^" fragment for reverse transcription applications. Example 15 demonstrates the increased processivity of Tfl exo^" fragment as compared to native or recombinant Tfl DNA pol I holoenzyme or Taq holoenzyme. Finally, Example 16 details a large "production scale" purification of recombinant Tfl holoenzyme and exo^" fragment. EXAMPLE 1

Purification and Amino Acid Sequencing of Native Tfl DNA Pol I

Native T. flavus DNA polymerase I was isolated from T. flavus cells and used to generate amino acid sequence information as described below.

Thermus flavus obtained from the American Type Culture

Collection (ATCC 33923, Catalogue of Bacteria and Bacteriophages, 18th

Edition, 1992) was cultured as follows: one ampule of Thermus flavus ATCC 33923 was used to inoculate 100 ml culture medium (0.1 g nitrilotriacetic acid, 3 g NZ Amine A, 3 g yeast extract, 5 g succinic acid [free acid], 0.001 g riboflavin, 0.522 g K₂HPO₄, 0.480 g MgSO₄, 0.020 g NaCl, 2 ml Trace Metal Solution (0.5 ml H₂SO₄, 2.2 g MnSO₄, 0.5 g ZnSO₄, 0.5 g H₃BO₃, 0.016 g CuSO₄, 0.025 g Na₂MoO₄, 0.046 g cobalt nitrate) per liter, adjusted to pH 8.0 with NaOH) and the culture was incubated overnight at 70 °C with shaking. In the morning 10 ml of the overnight culture was used to inoculate 1000 ml of medium. This culture was grown for about 8 hours at 70°C and then used as an inoculum for 170 liters of medium in a New Brunswick 250 liter fermentor equipped with a ML 4100 controller. The settings for a typical fermentation were 3 pounds back pressure, 60 liters/min. (1pm) aeration, 100 rpm agitation, at 70°C. The fermentation was terminated when the cells reached a density of 2 - 3 O.D., as measured at 600nm. The cells were cooled down to room temperature and harvested by centrifugation at 17,000 rpm in a CEP A type 61 continuous flow centrifuge with a flow rate of 2 1pm. The cell paste was stored at -70°C until used.

T. flavus cells (500-1500g) were thawed in 3 volumes of lysis buffer (20mM Tris-HCI . pH 8.0, 0.5mM ethylenediaminetetraacetate (EDTA), 7 mM _/3-mercaptoethanol (/3ME), 10 mM MgCl₂) and homogenized. Phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, was added to a final concentration of 0.3 mM. The suspension was then treated with 0.2 g/1 of lysozyme (predissolved in lysis buffer) at 4°C for 1 hr. Cells were homogenized twice at 9000 psi in a Manton Gaulin homogenizer, with the suspension chilled to approximately 10°C between passes. New PMSF was added to 0.2 g/1 before, between and after passes. NaCl and polyethyleneimine (PEI) (10% w/v, pH 7.0) were added to the crude, homogenized lysate to a final concentration of 0.5 M and to 0.2%, respectively. The sample was mixed well and centrifuged at 13,500 x g for 1 hour.

The supernatant from the centrifuged lysate was desalted by diluting with 10 liters of DE52 column buffer (20 mM Tris-HCI, pH 8.0, 0.5 mM EDTA, 7 mM /3ME) and concentrated to approximately 4 liters using an Amicon S10Y30 Spiral Ultrafiltration cartridge. The dilution/concentration step was repeated two times, with a final concentrated volume of about 4 liters. The desalted sample was batch contacted with 400 g of equilibrated Whatman DE52 ion exchange resin (Maidstone, England). The suspension was collected on a sintered glass funnel and washed 3 times with 1 volume of DE52 column buffer. The resin was then resuspended in a minimal volume of buffer and poured into a column (4.5 x 50 cm), packed and washed with an additional volume of buffer. The column was eluted with a 0-0.5 M NaCl linear gradient (total gradient volume: 2000 ml). Twenty- five ml fractions were collected at a rate of about 5 ml/min. Peak fractions (fractions containing DNA polymerase activity) were determined by a modified DNA polymerase assay described by Kaledin et al., Biokhimiya 45:644-651 (1980), pooled and dialyzed in approximately twenty-five volumes of Affi-Gel Blue (AGB) column buffer (20 mM Tris-HCI, pH 7.5, 0.5 M EDTA, 10 mM jSME, lOmM MgCl₂, 0.02% Brij 35).

The dialyzed DE52 peak fractions were applied to an AGB column (4.4 x 40 cm, 600 ml packed volume, MBR Blue, Molecular Biology Resources, Milwaukee, WI), which was washed with 2 column volumes of AGB column buffer, and eluted with a 0-1.2 M NaCl linear gradient (total gradient volume: 2000 ml). Twenty-five ml fractions were collected at a rate of 1-5 ml/min. The peak fractions were dialyzed as above in AGB buffer. The dialyzed AGB peak fractions were applied to a heparin agarose column (4.4 x 16.5 cm, 250 ml packed volume (Affigel Heparin, Bio- Rad, Hercules CA; or Heparin Agarose, Molecular Chimerics, Madison, WI)), which was washed with approximately 2 column volumes (until effluent is no longer colored, and column resin is white in appearance), and eluted with a 0.1-1.0 M NaCl linear gradient (total gradient volume: 1500 ml). Twenty-five ml fractions were collected at a rate of 1-5 ml/min. The peak fractions were dialyzed in HP Q Sepharose Column Buffer (20 mM Tris-HCI, pH 7.5, 0.5 M EDTA, 7 mM 0ME, 0.1 % Brij 35).

The dialyzed heparin agarose peak fractions were filtered through a 0.2 μm filter and applied at 4 ml/min. to the HP Q Sepharose column (Pharmacia, Uppsala, Sweden) on FPLC. The column was washed with several column volumes of buffer, and eluted with a 0-0.25 M NaCl linear gradient. Ten ml fractions were collected at 4 ml/minute. The peak fractions were dialyzed in HP S Column Buffer (20 mM Na-Citrate, pH 6.0, 1 mM EDTA, 7 mM ,3ME, 0.1 % Brij 35) or diluted in the same buffer, depending on the volume of the fraction pool.

The dialyzed (or diluted) HP Q peak fractions were filtered through a 0.2 μm filter and the HP S column (Pharmacia) was run as above, washing with HP S Column buffer and eluting with a 0-0.25 M NaCl gradient. Peak fractions were pooled and dialyzed against 4 liters of Final Storage Buffer (50 mM Tris-HCI, pH 7.5, 0.1 mM EDTA, 5mM DTT, 50% glycerol). The final product was diluted to a concentration of 5000 U/ml in the above buffer including 0.5 % Tween 20 (Sigma Chemical Co., St. Louis, MO) and 0.5 % Nonidet P40 (Fluka Biochemika, Buchs, Switzerland) as stabilizers and stored at -20°C. A typical preparation from 1200 g of cells yields approx. 2,000,000 units (1,700 units/g) or about 40 mg of DNA polymerase.

To quantify DNA polymerase activity, a DNA polymerase activity assay was performed using a modification of a protocol described by Kaledin et al., Biokhimiya 45:644-651 (1980). Reactions were performed in a 50 μl reaction mixture of 25 mM Tris-HCI, pH 9.5 at 23 °C; 50mM KC1; 10 mM MgCl₂; 1 mM DTT; 0.2 mM each dCTP, dGTP, dTTP, pH 7.0; 0.2 mM [ ³²P]dATP, pH 7.0, 10 μCi/ml; 50 μg BSA; 15 μg activated DNA (Baril et al. Nucleic Acids Res. 8:2641-2653 (1977)); and 5 μl of diluted enzyme. For control purposes enzymes (in general AmpliTaq DNA polymerase (Perkin Elmer, Cat. No. N801-0060), or Taq DNA polymerase purified according to a procedure described by Kaledin et al., Biokhimiya 45:644-651 (1980)) with known activities are diluted to 20, 40 and 80 units/ml. Two reactions were run without enzyme as negative controls for background subtraction.

A 45 μl reaction mixture, less enzyme, was prepared and the reaction was started by the addition of 5 μl of enzyme. After 10 min. of incubation at 70°C, 40 μl was removed and added to 50 μl of yeast RNA co-precipitant (10 mg/ml in 0.1 M sodium acetate, pH 5.0). One ml of 10% trichloracetic acid (TCA) was added and the samples were placed on ice for at least 10 minutes. The mixture was filtered on a glass fiber filter disc and washed first with 5% TCA/ 2% sodium pyrophosphate, and then with 95 % ethanol. The dried filter disc was counted in 5 ml of scintillation fluid.

One unit of activity is defined as the amount of enzyme required to incorporate 10 nmol of total nucleotide into acid insoluble form in 30 min. at 70 °C in this assay, the standard activity assay.

To estimate protein concentration, an aliquot of a native T. flavus DNA polymerase preparation (1100 U/ml) was separated on a 5 - 25% SDS-polyacrylamide gel, using the Bio-Rad protocols (Hercules, CA) and the Bio-Rad Mini-Protean II electrophoresis unit. The concentration was estimated at 33 μg/ml when compared to co-electrophoresed protein standards. To obtain amino acid sequence information from native T. flavus DNA polymerase, about 53 μg of native polymerase were separated on a preparative 7.5% SDS-polyacrylamide gel, blotted onto PVDF membrane and stained with amido black as described by Matsudaira, J. Biol. Chem. 262: 10035-10038 (1987). The major band at approximately 83 kD was excised and sequenced using an Applied Biosystems (Foster City, CA) 477 A Protein Sequencer. No N-terminal sequence was obtained under these conditions. Due to the apparent block at the N-terminus of the native 7. flavus DNA polymerase I (holoenzyme), another approach was employed to obtain a partial amino acid sequence. Native T. flavus DNA polymerase I was digested with trypsin, and the resulting peptides were separated using reverse phase high-performance liquid chromatography (HPLC). The N-terminal amino acid sequences of four of these peptides (peptides 1-4) were determined. The amino acid sequence of one of the peptides, peptide 1, is LHTRFNQTATATGRLSSSDPNLQNIPVR. This sequence has been determined to map at positions 562 to 589 in the deduced amino acid sequence of the Tfl DNA pol ϊ holoenzyme described herein (FIGURE 2)). As explained in Example 3, knowledge of this amino acid sequence information was used to isolate the T. flavus DNA polymerase I gene.

EXAMPLE 2

Construction and Amplification of a Thermus flavus genomic DNA library A Thermus flavus genomic library was constructed in phage λ

Dash II and amplified in the following manner.

Genomic DNA from the Thermus flavus , cultured overnight as described above, was isolated according to the procedure described by

Ausubel et al. , Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York (1990). In general, yields of genomic DNA between 100 and 900 μg were obtained from the cell pellet of about 1.5 ml of culture.

Twenty-five micrograms of Thermus flavus genomic DNA were partially digested with 0.3 units of Sau3Al in a total reaction volume of 50 μl. At 0, 5, 10, 15, and 30 min. , lOμl samples were removed and the enzyme was inactivated at 65 °C for 15 min. An aliquot from each time point was analyzed on a 1.2% agarose/TBE gel. The 10 min. reaction time produced fragments having the desired size distribution (3 kb to 20 kb). Approximately 2.5 pmoles of 5 '-ends of Sau3Al -digested T. flavus DNA were treated with calf intestinal alkaline phosphatase (CIP) using standard techniques (Ausubel et al. , Current Protocols in Molecular Biology (1990)). One half of the CIP-digested sample was electrophoresed on a 0.7% agarose gel and checked for amount and integrity. The Sau3Al- digested, CIP-treated T. flavus DNA was extracted with phenol/chloroform and chloroform, ethanol precipitated, pelleted, and washed in 70% ethanol. The pellet was stored at -20°C. This DNA is referred to as "CIP TFL DNA. "

The T. flavus library was constructed as described in the manufacturer's instructions using the phage λ DASH II / BamHI Cloning Kit (Stratagene, LaJolla, CA) and the CIP Tfl DNA. The pME/BamHI test insert (0.3 μg) was run in parallel as a control. The ligation mixture was incubated over night at 4°C.

The T. flavus DNA ligated to λ DASH II arms was packaged in vitro using the Gigapack II Gold Packaging Extract from Stratagene, according to the manufacturer's instructions. Control DNA provided by the manufacturer was also packaged.

Following the protocol provided by Stratagene with the λ DASH II / BamHI Vector Kit, host bacteria were prepared: Escherichia coli VCS 257 (Stratagene) for wild type phage; E. coli SRB and SRB(P2) (Stratagene) for the T. flavus library and the control. VCS 257 was grown in NZY+ maltose medium; SRB and SRB(P2) were grown in NZY+ maltose medium with 50 μg/ml kanamycin at 37°C for 6 hours. After centrifugation of the cells at 2800 x g for 10 min. , the cells were resuspended in sterile 10 mM MgSO₄ to give an A^ (optical density at 600nM) of 0.5.

Two 1: 10 serial dilutions were prepared from the control phage and the CIP Tfl DNA library. Ten microliters of undiluted, 1: 10, and 1: 100 dilutions of phage were added to 200 μl of SRB cells. The cells were incubated with light shaking at 37^"C for 15 minutes and after the addition of top agar, the mixture was poured onto LB/M/M plates. The plates were incubated overnight at 37°C.

The T. flavus library was amplified using techniques described by Ausubel et al. , Current Protocols in Molecular Biology (1990), and the primary and amplified libraries were titered on SRB cells and the titers are shown in Table IB. The amplified library was stored at 4^°C.

TABLE IB

Titer (plaque forming units/ml)

Construct Primary Library Amplified Library

CIP TFL DNA 4.4 x 10^s 9.6 x 10⁷ pME/BamHI 3 x 10⁶ 1.5 x 10⁹ λ Control DNA 1.1 x 10⁹ Not determined

EXAMPLE 3 Cloning and Sequencing a Tfl DNA Pol I Gene Fragment

The amino acid sequence information derived from four Tfl DNA pol I peptides (Example 1) was used to design the synthesis of two primers for the amplification of a T. flavus DNA polymerase gene fragment: primer FTFL2 (primer "2"; 21mer) (SEQ ID NO: 8) and primer RTF- (primer "4"; 25mer) (SEQ ID NO: 9) (synthesized by Synthetic Genetics, San Diego, CA). The sequence of the two primers was also compared to the T. aquaticus DNA polymerase sequence (Lawyer et al., J. Biol. Chem 264: 6427-6437 (1989)); the primer nucleotide sequences, with cross-references to the Sequence Listing and Sequence ID Nos. are shown in Table 2. Primer FTFL2 was chosen because the amino acid sequence obtained from peptide 1 (Example 1) was identical to a sequence in the Taq DNA polymerase I protein. Primer FTFL2 corresponds to nucleotides 1719-1740 of the T. aquaticus DNA polymerase coding sequence, top strand (i.e., to a portion of the sequence that encodes a portion of the Taq DNA pol I protein that is homologous to Peptide 1). Primer RTFL4 hybridizes to the 3 '-end of the Taq DNA pol I gene at position 2476 - 2500 and has sequence identical to the bottom strand (Lawyer et al. , J. Biol Chem 264: 6427-6437 (1989)).

A typical amplification reaction (100 μl) contained 0.2 mM deoxynucleotide triphosphates (dNTPs), 1 x Taq Polymerase Reaction Buffer (10 x buffer is 100 mM Tris-HCI, pH 8.4, 500 mM KC1, 15 mM MgClJ, 0.5 μM of each primer FTFL2 and RTFL4 (primer set 2-4), 50 μl mineral oil and 15 ng T. flavus genomic DNA. After the initial denaturation step (Step 1), 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer No. N801-0060, Foster City, CA) were added. Negative control reactions containing either no enzyme or no template were performed. The amplification program was carried out in a thermocycler as follows: Step 1: 95° C for 5 min.; Step 2: hold at 72 °C (for the time required to add the enzyme); Step 3: 55 °C for 45 sec.; Step 4: 72 °C for 5 min.; Step 5: 95 °C for 15 sec.; Step 6: repeat Steps 3-5 34 times; Step 7: 55 °C for 45 sec. ; Step 8: 72°C for 20 min.; Step 9: hold at 4°C until processing the product. Under these conditions primer set 2-4 gave a single amplification product from T. flavus genomic DNA. The observed mobility of the amplification product ("the 2-4 fragment") in 1 % and 1.2% agarose gels was in agreement with the 782 bp predicted from the T. aquaticus coding sequence.

The 2-4 fragment was cloned, sequenced, and compared to a previously published DNA sequence for a purported T.flavus DNA polymerase I as follows.

First, to improve cloning efficiency, the 2-4 fragment was fractionated, blunt ended, and phosphorylated as follows. Approximately 20 μl of the 2-4 fragment was loaded onto a Sephacryl S-500 (400 μl in a spin filter, preswollen, pre-equilibrated and stored in 100 mM Tris-HCI, pH 8.0, 1 mM EDTA) column and centrifuged at 2,000 x g for 5 min. to trap the unused primers from the PCR reaction. DNA that passed through the column was ethanol-precipitated and resuspended in double-distilled water (ddH₂O). The 2-4 fragment was blunt-ended using mung bean nuclease (MBN) (Molecular Biology Resources, Inc., Cat. No. 1190-01.), and phosphorylated with T4 polynucleotide kinase (Molecular Biology Resources, Inc., Cat. No. 1260-01) prior to ligation to a vector by procedures well known in the art.

M13mpl 8 RF DNA (Life Technologies, Grand Island, NY) was restriction-digested with Hinc II and Eel 136 II to create blunt ends for ligation to the above 2-4 fragment. The vector ends were dephosphorylated with CIP to reduce the probability of self-ligation. The digested and dephosphorylated M13mpl 8 vector and the 2-4 fragment (fractionated/MBN/kinased) were ligated for 2 hr. at room temperature using procedures that are well known in the art.

Using standard techniques, 5 μl of the ligation reaction were added to 50 μl of DH5αF ' E. coli (Life Technologies, Grand Island NY) cells, which had first been made competent, and the cells were transformed (see Ausubel et al. , Current Protocols in Molecular Biology (1990)). Different numbers of cells were spread on 2XTY plates and grown until plaques appeared. Several plaques were picked, and DNA was prepared using the Minute Miniprep ssDNA Purification Kit (CHIMERx, Madison WI). It was determined that plaques designated M13-TFL 4.21 and 4.22 contained the 2-4 fragment in opposite orientations.

The DNAs M13-TFL 4.21 and 4.22 were sequenced by Sanger's dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. 74:5463 (1977)) using the SEQUAL™ Sequencing Kit from CHIMERx. The forward sequencing primers (FSP, Table 2, SEQ ID NO: 6) used in sequencing M13-TFL 4.21 and M13-TFL 4.22 were end-labeled using [y³²P]-ATP and T4 polynucleotide kinase. The extension/termination reactions were performed according to the protocol provided with the SEQUAL™ Sequencing Kit (CHIMERx). One microtiter of each extension/termination reaction was loaded onto a 6% sequencing gel, which was electrophoresed at 3000 volts for 3 hours. The bands were detected by autoradiography and the sequence was determined.

When the nucleotide sequence from both ends of the 2-4 DNA was aligned, 771 bp of the approximately 780 bp DNA could be determined. This 2-4 DNA sequence was compared with the purported T. flavus DNA polymerase sequence reported by Akhmetzjanov and Vakhitov, Nucleic Acids Res. 20:5839 (1992), which theoretically should have been amplified by this primer set. Eighty-four percent maximum matching, as calculated by the MacDNAsis software program (Hitachi Software Engineering America, San Bruno, CA), was found. This degree of homology, compared with a reported DNA polymerase gene, suggested that the 2-4 DNA was indeed part of the T. flavus (ATCC 33923) gene and could serve as a useful probe for screening the T. flavus genomic library. The homology of 84% also suggested that either (1) the purported T.flavus strain studied by Akhmetzjanov and Vakhitov and the T. flavus strain (ATCC 33923) do not have identical DNA polymerase I genes; or (2) the two strains have more than one gene or gene-like sequences having homology to DNA polymerase I genes. EXAMPLE 4

Preparation of Gene-Specific Probes and Screening of the Thermus flavus Genomic Library for Clones

Containing the T. flavus DNA Pol I Gene

The 2-4 fragment described in Example 3 was used to isolate the Thermus flavus DNA pol I gene from the 7. flavus genomic library Example 2). Using M13-TFL 4.21 as template and primers FTFL2 and RTFL4, the 2-4 fragment was amplified by PCR as described above to obtain larger quantities of the fragment for use in preparing probes to screen the T. flavus genomic library. The amplified 2-4 fragment, migrating at about 780 bp, was cut out of preparative 0.7% agarose gel, eluted, phenol-chloroform extracted and ethanol-precipitated. Approximately 1 μg of the 2-4 fragment was digested with CviYi (CHIMERx) to generate sequence specific primers for labeling. A variety of thermal cycle labeling (TCL) probes were prepared with the 2-4 intact fragment (i.e., Biotin-11-dUTP, fluorescein and [α³³P]dCTP probes) in the manner described below. Each set of duplicate plaque lifts or targets was screened using two different types of labeled probes. Digestion with CviJI*, as well as this method of labeling, is described in a co-owned, copending U.S. Patent Application Ser. No. 08/217,459, filed March 24, 1994, entitled "Methods and Materials for Restriction Endonuclease Applications," incorporated herein by reference in its entirety. The PCT counteipart of this application, filed March 24, 1994, is PCT App. No. US94/03246.

The 2-4 intact fragment was labeled with Biotin-11-dUTP as described in the manual for the ZEPTO™ Labeling Kit (CHIMERx). To determine the relative efficiency of the amplification reaction, 5μl of the amplified 2-4 TCL probe was electrophoresed on a 0.7% agarose gel along side a 1 kb molecular size ladder. The amplified probe was evident as a smear from 0.1-5 kb, which is an indication of a successful TCL reaction.

To determine the efficiency of incorporation of the biotin-11-dUTP, a dot blot assay was performed as follows: A serial dilution of the probe from 1: 10 to 1: 10⁸ was made in TE (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) and lμl of each dilution was spotted on a Hybond-N membrane (Amersham, Arlington Heights, IL), UV-cross-linked for 3 min., followed by colorimetric detection of the incorporated biotin-11-dUTP using streptavidin-alkaline phosphatase as described in the ZEPTO™ labeling manual. The probe was detected at 10⁶ dilution suggesting that the biotin- labeled 2-4 fragment was efficiently labeled and is highly sensitive for the screening of the Tfl genomic library.

The fluorescein labeled 2-4 fragment was prepared and analyzed as above except fluorescein- 12-dUTP was used instead of biotin-11-dUTP. The fluorescein- 12-dUTP incorporation was detected using alkaline phosphatase conjugated anti-fluorescein antibody (Boehringer- Mannheim, Indianapolis, IN) instead of streptavidin-alkaline phosphatase. These probes were detected at a 1:10⁶ dilution by the colorimetric assay as described in the ZEPTO™ labeling manual or by chemiluminescence. Both the biotin and flurescein non-radioactive probes were aliquoted and used throughout the entire screening process;.

The preferred detection method for both the biotin- 11-dUTP probes and the fluorescein- 12-dUTP probes was chemiluminescence. For this method of detection the filters with hybridized probes were incubated either with streptavidin-alkaline phosphatase or alkaline phosphatase conjugated to anti-fluorescein antibody for 30 min. at room temperature. They were then rinsed three times with wash buffer (1 x phosphate buffered saline (PBS), 0.3% Tween 20 (Sigma Chemical Co. , St. Louis, MO) 0.02% Na-azide) for 15 min. each and finall) in assay buffer (0.1 M diethanolamine, 1 mM MgCl₂ and 0.02% Na-azide, pH 10) for 5 minutes. They were finally incubated in assay buffer containing CSPD™ (Tropix, Bedford, MA) a chemiluminescence substrate, for 15 min. in the dark followed by exposure to X-ray films. The normal exposure times for the biotin- 11-dUTP probes were 5-30 min. and for the fluorescein- 12-dUTP probes were 2-6 hours. The 2-4 intact fragment was labeled with [α³³P]dCTP as described in the ZEPTO™ labeling manual; a total of 6 x 10⁷ cpm of [α³³P]dCTP at 1 x 10⁹ cpm/μg was incorporated. For probes, 1-5 x 10⁶ cpm of radio-labeled DNA was added to each plaque lift.

The sensitivity and specificity of the labeled probes was demonstrated by screening blots of digested T. flavus genomic DNA. Specifically, T. flavus genomic DNA was restricted with different restriction enzymes, such as BamHI, Bgll, Dral, EcoRI, EcoRV and Pad. 250 ng/lane of restricted DNA, along with 500 ng of IL-3A viral DNA as negative control (Xia, Y., et al., Nucleic Acids Research 15: 6075-6090 (1987)), were electrophoresed on a 0.7% agarose gel. A Southern transfer of this gel onto Hybond-N was prepared. The denatured DNA on the Southern blots was UV- cross-linked to the filter for 3 minutes. Duplicate blots were prehybridized in 2 ml of hybridization buffer (50% deionized formamide, 7% SDS, 120 mM Na phosphate, pH 7.2, 250 mM NaCl, 1 M EDTA and 1 mM cetyldimethylethylammonium bromide and 20μl of denatured salmon sperm DNA at 10 mg/ml) in a heat-sealed plastic bag at 52°C for 1 hour. Seven μl of either the biotin- 11-dUTP 2-4 TCL probe or the fluorescein- 12-dUTP 2-4 probe was added to one set of the Southern blots and 1-5 x 10⁶ cpm of the [α³³P]dCTP 2-4 TCL probe was added to the duplicate blot. The filters were hybridized by incubation overnight at 52°C.

The filters with the radioactive probe were incubated with low stringency buffer (1 x SSC, 1 % SDS) for 1 hr. at 52°C, washed with high stringency buffer (0.1 x SSC, 1 % SDS) for 1 hr. at 50°C, dried, and then exposed to X-ray film for 3 hours. The detection of non-radioactive probes was accomplished as described above. Both the biotin- 11-dUTP and the [α³³P]dCTP 2-4 TCL probes recognized a large molecular weight band at about 20 kb in all the lanes containing digested T.flavus genomic DNA, although the mobility of the bands varied somewhat in the lanes containing different digests. The probes did not bind to the control IL-3A DNA, suggesting that the probes were specific for the target and could be used to screen the T. flavus genomic library.

To screen the amplified T. flavus genomic library (Example 2), the phage library was plated on two plates each at 10⁵ plaque-forming units (pfu)/100 mm 2XTY pxates. Duplicate plaque lifts on Hybond N from each plate were obtained and prepared for hybridization by methods well known in the art (Sambrook, Fritsch, and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989)). The DNA on the plaque lifts was UV-cross-linked to the Hybond N for 3 minutes and each plaque lift was placed in a heat-sealed plastic bag and prehybridized as described above. Seven μl of either the 2-4 biotin- 11-dUTP TCL probe or the 2-4 fluorescein- 12-dUTP probe were added to one set of the plaque lifts and 1-5 x 10⁶ cpm of the [α³³P]dCTP 2-4 TCL probe were added to the duplicate filters. The filters were incubated overnight at 52 °C and washed the next day with low and high stringency buffers as described above. The filters with non-radioactive probes were incubated with 10 ml of conjugation buffer (0.5% casein, 1 x PBS and 0.02% Na-azide) for 30 min. at room temperature. Hybridization conditions, washes and detection were as described above. Approximately 25 positive plaques (hybridizing with the labeled probes) out of 10⁵ pfu from the amplified CIP Tfl DNA library were detected on the duplicate plaque lifts.

Ten positive plaques were selected (λ21 , λ31, λ-51 , λ61, λ71, λ81 , λ91 , λlOl, λl l l and X121) and were purified by two rounds of dilution - 41

and screening with the labeled 2-4 probes, until well-isolated, single, positive plaques were obtained.

The four stocks of phage λ21 , λ 1 , λ71 , and λ91 were grown at 5 x 10^s pfu/2XTY plate, 5 plates per stock. The phages were eluted from the plates by a standard protocol (Sambrook et al. (1989)). The eluant was treated with 20μg/ml DNase and 50μg/ml RNAse A for 1 hr. at 37 °C and extracted with both phenol-chloroform and chloroform. The DNA was ethanol-precipitated, pelleted, rinsed with ethanol, resuspended in 1 ml of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and purified using the Lambda prep kit from CHIMERx.

The phage DNA was restriction-digested with EcoRI and BamHI and electrophoresed on a 0.7% agarose gel, transferred to Hybond N and probed with the 2-4 TCL probes. Based on agarose gel band distribution and Southern blot detection by the 2-4 probes, the four phages were grouped into two classes. Clones λ21 and λ91 belong to one class and the λ51 and λ71 belong to a second class. Clones λ21 and λ51 were chosen for further analyses.

Clone λ21 was digested with BamHI and the T. flavus insert was subcloned into pTZ18U (Mead et al., Protein Engineering 1: 67-74 (1986)). Eight of these clones were sequenced using the SEQUAL™ Sequencing kit from CHIMERx. One of these clones, designated p21BG, hybridized to the 2-4 TCL probes and yielded sequences identical to the sequence of the 2-4 fragment between the BamHI and Eco47III sites (these sites begin at positions 2084 and 2387 in Fig. 4, respectively). This sequence information confirmed that the clones contained authentic T. flavus DNA polymerase gene sequence, and confirmed the orientation of this gene sequence in the clones.

Based or. agarose gel analysis neither λ21 nor λ51 had any internal Eco Rl sites, hence λ21 and λ51 were restriction-digested with Eco Rl and the insert was cloned into pTZ18U for ease in further analyses. The resulting recombinants were designated p21E10 and p51E9, respectively (FIGURE IA). Each clone had an insert of about 14-16 kb.

EXAMPLE 5 Sequencing the T. flavus DNA Polymerase I Gene Three main strategies were adopted for sequencing the Tfl DNA pol I gene.

In a first strategy, several primers were designed based on the purported T. flavus DNA pol I sequence published by Akhmetzjanov and Vakhitov (1992) and were synthesized by Synthetic Genetics, San Diego, CA (e.g., primers FTFL10, FTFL11, RTFL12, RTFL13, FTFL15, RTFL16, FTFL17 and RTFL18 (Table 2)). Dideoxy- sequencing of the λ21 and λ51 clones was attempted using these primers, but only primers FTFL17 and RTFL 18 yielded good sequence data and only very faint bands were obtained with primers FTFL11 and RTFL13, suggesting only partial homology to this purported DNA pol I sequence.

In a second sequencing strategy, deletion vectors of p21E10 were obtained by restriction digestion of the plasmid with Hindi, Hindlll, Sphl, Kpnl and BamHI. These restriction enzymes cut once in the multiple cloning site and once or twice in the insert. The digests were diluted to allow self-ligation and transformed into E. coli strain DH5αF' by standard methods. The clones that ligated back to the vector were selected on ampicillin- containing plates and picked for further sequence analysis.

All of these clones had deletions of different lengths at the 3' end. The size of the insert in the Hindi deletion vector (p21EHc) was approximately 4.6 - 4.7 kb; in the Kpnl deletion vector (p21EK) about 7 kb; in the Hindlll deletion vector (p21EHd) about 1.4 kb; in the Sphl deletion vector (p21ES) about 1.6 kb; and in the BamHI deletion vector (p21EB) about 1.2 kb. The plasmids p21EHd, p21EB and p21ES were sequenced with [7-³²P] end-labeled FSP by dideoxy sequencing. The sequences obtained were within the region of the 2-4 fragment, further confirming the orientation of the insert and the presence of the 3' region of the T. flavus DNA polymerase I in the p21E10 parent clone. Clone p21EHc is a deletion derivative containing the entire portion of the Tfl DNA polymerase I gene DNA present in p21E10 and about 3 kb DNA downstream from the stop codon of the Tfl DNA pol I gene, but lacking about 9.0 kb of unwanted 3' end sequence (FIGURE IA). In addition, the DNA sequence obtained from p21E10 and p21EHc using [7-³²P] end-labeled reverse sequencing primer (RSP, Table 2) suggested that p21E10 contained only about 2/3 of the DNA polymerase gene and lacked the 5 ' one- third of the gene. In contrast, sequence obtained from p51E9 suggested that this clone contains a 5' portion of the Tfl DNA pol I gene that overlaps the coding sequence contained in p21E10, as well as significant Tfl DNA upstream of the gene.

A primer walking sequencing strategy was employed to obtain the remainder of the sequence of the Tfl DNA pol I gene. This strategy is described as "Directed Sequencing with Progressive Oligonucleotides" in Sambrook et al. , Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press (1989). To obtain additional sequence information from the clones by primer walking, primers homologous or complimentary to the ends of previously determined sequences obtained from p21E10, from the deletion vectors, and from primers FTFL17 and RTFL18 were synthesized as described above and used in additional sequencing reactions. By repeating this process the entire length of the gene was sequentially sequenced.

Specifically, TFL primers FTFL17A, RTFL18A, TFLEF1 and TFLER1 (Table 2 and FIGURE 4) were synthesized by Synthetic Genetics for primer walking based on the sequence information obtained from primers FTFL17, RTFL18 and RSP on the p21E10 template DNA (Table 2 and FIGURE 4). Primer TFLSF1 was designed as a forward primer for walking into the 3' end of the DNA pol I gene, by utilizing sequence information from p21ES. As more information became available, additional TFL primers RTFLA - FTFLZ (Table 2; FIGURE 4) were designed for sequencing both strands. The DNA sequence of the T. flavus DNA polymerase I gene and flanking sequences are given in FIGURE 2 (SEQ. ID NO. 1), along with the deduced amino acid sequence (SEQ. ID NO. 2). The sequence of 3326 b.p. has been determined. FIGURE 2 depicts 3048 bases of this sequence, of which 2505 bases are deduced to encode a polypeptide of 834 amino acids (plus stop codon). The coding sequence was determined to be 86% homologous to the Taq polymerase gene and 83% homologous to the purported Tfl polymerase gene published by Akhmetzjanov and Vakhitov.

The deduced amino acid sequence of the T. flavus DNA polymerase I gene was aligned and compared to the deduced amino acid sequences of the purported Tfl DNA pol I published by Akhmetzjanov and Vakhitov (85 % homology) and to the deduced amino acid sequence of the Taq pol I gene (87% homology). As shown in FIGURE 3, the amino acid alignment chosen to maximize homology reveals two single amino acid insertions in the T. flavus DNA pol I reported here, relative to the other two reported sequences. The single amino acid inserts are depicted by dashes (-) in the sequences for Taq pol I and for Akhmetzjanov and Vakhitov' s purported Tfl DNA pol I.

TABLE2

Primer name: Primer Seσuence Seq Id.

No;

FSP CGC CAG GGT TTT CCC AGT CAC GAC 6

RSP AGC GGA TAA CAA TTT CAC ACA GGA 7

FTFL2 CTA AGT AGC TCC GAT CCC AAC 8

RTFL4 ATC ACT CCT TGG CGG AGA GCC AGT C 9

FTFL10 ATT TAG CAC ATA TGG CGA TGC TTC CC 10

FTFL11 CTT TCC AGC TCC GAC CCC AAC 11

RTFL12 CCT ACT CCT TGG CGG AGA GCC AGT C 12

RTFL13 TGG ATG TCC CTC CCC TCC TGA AAG A 13

FTFL15 ccc TTT CCC GGA AGC TTT CCC AGG TGC A 14

RTF 16 TGC ACC TGG GAA AGC TTC CGG GAA AGG G 15

FTF 17 CCT GCA GTA CCG GGA GCT CAC CAA GCT CAA 16

RTFL18 TTG AGC TTG GTG AGC TCC CGG TAC TGC AGG 17

FTFL17A TGG ACT ATA GCC AGA TAG AGC T 18

RTFL18A AAG CGA AGA CCT CCT CCT CGA 19

TFLEF1 AGT TCG GCA GCC TCC TCC ACG A 20

TFLER1 TCC AAG GAA AGC CTG AGG TCT T 21

TFLSF1 AAG CTC GCC ATG GTG AAG CTC TT 22

RTFLA TCG GAG ACG AGT TGG TAG AGG T 23

FTFLB ACC TCT ACC AAC TCG TCT CCG A 24

RTFLC AGA GGA CGA AGC CCA CGA A 25

RTFLD AGG AGG TAG GCG AGG AGC AT 26

FTFLE ATG CTC CTC GCC TAC CTC CT 27

FTFLF TCG AGG AGG AGG TCT TCG CTT 28

RTFLG AGC TCT ATC TGG CTA TAG TCC A 29

FTFLH ATA GGC TCT CCC AGG AGC TT 30

RTFLI AAG AGC TTC ACC ATG GCG AGC TT 31

RTFLJ TTC CCC TGG AGG CGT TTC TGA 32

RTFLK AAA GAC CAC GAA GAC GGC CTT 33

FTFLL AAG GCC GTC TTC GTG GTC TTT 34

FTFLM AAG GAG TGG GGA AGC CTG GAA 35

RTFLN TTC CAG GCT TCC CCA CTC CTT 36

RTFLO TTC TTC CGA AGA GGG TTT CCA 37

RTFLP GCG TCC AGG AGC GCC CTG AGG A 38

FTFLQ CCT CAG GGC GCT CCT GGA CGC CA 39

FTFLR TTC GTC CTC TCC CGC CCC GA 40

FTFLS CCA ACC TGC AGA ACA TCC CCG T 41

RTFLT GGT GTG GAT GTC CTT CCC CT 42

FTFLU CCC TGC CGT TTA GAG GAA GTT CAA G 43

RTFLV CTT GAA CTT CCT CTA AAC GGC AGG G 44

RTFLW ACC CGG CCT TTG GGT TCA AAG A 45

FTFLX TCT TTG AAC CCA AAG GCC GGG T 46

RTFLY TTC CCG TGC TCC TTC CGC TC 47

FTFLZ CTC GCC TTC CTC GTG CCC TT 48

5'lac PCR GCT TCC GGC TCG TAT GTT GTG TG 49

TFL-SDM-1 GGA AAG CCT GAG GTC TTC CAT AGC TGT TTC CTG50

TGT GAA ATT GTT ATC CGC TCA CAA TTC CAC ACA

ACA T

TFL-SDM-3 ACC CGG CCT TTG GGT TCA AAG AGC GGA ACG ATC51

GCC TCC ATA GCT GTT TCC TGT GTG AAA TTG TTA

TCC GCT CAC AAT TCC EXAMPLE 6

Construction and Expression of a

Full-Length T. flavus DNA Pol I Clone and Purification of Full-length Recombinant T. flavus DNA Pol I An expression vector containing the full-length T. flavus DNA polymerase I gene was constructed as described below, utilizing plasmid p51E9, which contains the 5' portion of the gene, and plasmid p21E10, which contains a 3' portion of the gene that overlaps the 5' portion contained in p51E9. FIGURES IA and IB are provided to illustrate steps in the construction of expression vectors of this invention, and are not intended to be a scale representation of clone inserts, or to contain a complete restriction map of clones depicted therein for enzymes shown.

Referring to FIGURE IA, clone p51E9 which carries the 5' portion of the Tfl DNA pol I gene, was digested with BamHI and a 3.7 kb digestion product was subcloned into the BamHI cloning site of pTZlδU to produce recombinant plasmid, p51B4, which was characterized as containing about 1.5 kb of DNA upstream of the DNA pol I start codon contiguous with the 5' region of the Tfl DNA pol I gene extending to the BamHI site in the 2-4 fragment. Plasmid p51B4 was then digested with Xbal, and a 2.5 kb digestion product was subcloned into the Xbal site of pTZ18U to create plasmid p51X16, which contained only approximately 0.3 kb of DNA upstream of the DNA pol I start codon.

Next, plasmid p21EHc (a subclone of p21E10 described above) was digested with BamHI and Sail. The 3.7 kb fragment containing the 3' region of the Tfl DNA pol I gene, beginning with the BamHI site in the 2-4 fragment, was isolated and subcloned into pTZ18U that had been digested with BamHI and Sail to create clone p21BHc. Clone p21BHc was digested with EcoRV and BamHI and the 1.3 kb fragment containing the 3' region of Tfl DNA pol I was ligated into pTZ18U that had been digested with BamHI and Hindi, yielding p21BRV2. -

47

Plasmid p51X16 was digested with BamHI and the 2.5 kb BamHI insert was isolated. Plasmid p21BRV2 was linearized with BamHI and ligated to the BamHI fragment of p51X16. The resulting clones were designated pTFL 1.3 and pTFL 1.4. The integrity of the Tfl DNA pol I gene in clone pTFL 1.4 was verified by DNA sequence analysis using the primer RTFLG (Table 2 and FIGURE 4).

Competent £. coli DH5αF' were transformed with plasmid pTFL 1.4 (the 1st generation expression clone), from which a Tfl DNA pol I protein was isolated and purified as follows. E. coli DH5αF'[pTFL-1.4] were grown in a 50 liter fermentor (10 pounds back pressure, 30 1pm aeration, 200 rpm agitation, at 37°C) in TB medium (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (19δ9): 12 g Bactotryptone, 24 g yeast extract, 4 ml glycerol, 0.1 g MgSO₄, 2.31 g KH₂PO₄, 12.54 g K₂HPO₄ per liter), supplemented with 50 μg/ml ampicillin with vigorous aeration at 37°C. At 0.0.₆₀₀= 1.0, IPTG was added to a final concentration of 0.5 mM and the cells were cultured for an additional 2 hrs. The culture was cooled down to 20°C and 100 ml of 100 mM phenylmethyl sulfonyl fluoride (PMSF) in isopropanol was added. After brief mixing, the culture was spun down in a Sharpies centrifuge and the pellet (or paste) was stored frozen at -70°C. Fifty grams of cells were thawed in 250 ml of lysis buffer A (20 mM Tris-HCI pH 7.4, 0.5 mM EDTA, 100 mM NaCl, 5% glycerol, 5 mM 3-mercaptoethanol, 0.5% Nonidet P40, 0.5% Tween 20, 50 μg/ml PMSF, 0.5 μg/ml pepstatin A, 0.5 μg/ml leupeptin). The cell suspension was homogenized twice in a Manton-Gaulin press. Because PMSF is unstable in aqueous solutions, new PMSF was added again to a final concentration of 50 μg/ml after the first and second homogenizations.

The suspension of broken cells was divided into 100 ml aliquots and heated to 65 °C for 1 hr. to denature the bulk of E. coli proteins, including nucleases, proteases and E. coli polymerases. Cell debris and denatured proteins were centrifuged at 6,δ00 x g for 30 min. and the NaCl - 4δ

concentration of the supernatant was adjusted to 400 mM. The presence of DNA polymerase activity in the supernatant was confirmed using the standard activity assay described above. Then 10% polyethyleneimine (PEI, pH 7.5) was slowly added to a final concentration of 0.2%. After 30 min. of stirring at 4°C, the suspension was centrifuged (1 hr., 6,δ00 x g) and the resulting supernatant was diluted with 6 volumes of buffer C (20 mM Tris-HCI, pH 7.4, 0.5 mM EDTA, 5% glycerol, 5 mM /3-mercaptoethanol, 0.5% Nonidet P40, 0.5% Tween 20, 50 μg/ml PMSF, 0.5 μg/ml pepstatin A, 0.5 μg/ml leupeptin). Ammonium sulphate was added to 0.55 g/ml and the mixture was stirred slowly overnight at 4°C.

After centrifugation for 2 hrs. at 6,δ00 x g the supernatant was carefully removed and tested for DNA polymerase activity. The polymerase- containing pellet was dissolved in a total of δO ml of Buffer A (10 mM KPO₄ pH 7.0, 0.5 mM EDTA, 100 mM NaCl, 5 % glycerol, 5 mM β- mercaptoethanol, 0.5% Nonidet P40, 0.5% Tween 20, 50 μg/ml PMSF). Insoluble material was removed by centrifugation at 6,δ00 x g for 20 min. The supernatant obtained from this centrifugation (which contains the polymerase activity) was loaded onto a 5 x 50 cm Sephadex G-25 column equilibrated in buffer A to desalt the solution and to remove traces of PEI. The flow rate used on this column was about 200 l/hr. Fractions of 25 ml were collected and assayed for DNA polymerase activity. The flow-through fractions contained the activity. It was essential to remove all the PEI for efficient adsorption to the next column.

The crude Tfl DNA polymerase described above was applied to a Bio-Rex 70 column (5 x 10 cm) (Bio-Rad) equilibrated in Buffer A. The column was washed with 1.5 1 of buffer A and the DNA polymerase was eluted with 4 1 of a 0 - 1 M NaCl gradient in buffer A. Fractions of 25 ml were collected and assayed for DNA polymerase activity. Fractions containing DNA polymerase activity (as assayed below) were pooled, concentrated in an Amicon concentrator with a YM30 membrane to about 40 ml and dialyzed against two changes (1 liter each) of antibody column high salt buffer B (20 mM Tris-HCI, pH 7.5, 0.5 mM EDTA, 0.5 M NaCl, 0.05% Brij-35) and applied to an immunoaffinity column (1.5 x δ cm).

The immunoaffinity column was prepared using techniques well-known in the art. First, a mouse is injected with purified DNA polymerase I to provide an immune response; preferred DNA polymerases for generating antibodies are thermophilic DNA polymerases, including those isolated from Thermus flavus and Thermus aquaticus. A ten week old female BALB/c mouse (Harlan Sprague Dawley, Madison, WI) was immunized by intraperitoneal injection with Taq polymerase (purified from Thermus aquaticus ATCC #25104). To prepare the Taq polymerase for injection, Taq storage buffer was removed with a Centricon 30 protein concentrator (Amicon Corp.), and the concentrated protein was diluted with phosphate-buffered saline. For the initial immunization, 40 μg of Taq emulsified with complete Freund's adjuvant was injected. Five booster injections of 40μg Taq polymerase mixed with equal volumes of the Ribi Adjuvant System (Ribi Immunochem Research, Inc., Hamilton, MT) were administered over a 6 month period, with successive intervals between injections of approximately five weeks, 4 weeks, 4 weeks, 12 weeks, and 4 weeks. Five days after the final booster injection, the mouse was sacrificed and spleen cells were isolated and fused with myeloma cells (myeloma P3X63-AGδ.653 (ATCC CRL 15δ0)) to generate hybridomas, using techniques well-known in the art. See E. Harlow and D. Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988). In particular, fusions were performed in 50% polyethylene glycol and selected in HAT medium. All hybridomas were screened as described below.

The fifth fusion experiment yielded a useful hybridoma, as selected in the following manner. The hybridomas were distributed into 96- well plates. Of 1176 wells filled, approximately 913 showed growth. To initially screen these wells, an ELISA assay was employed. First, polystyrene ELISA plates were coated with Thermus flavus (ATCC #33923) DNA polymerase (1 μg/ml Tfl DNA pol I (MBR lot 20229) in 100 mM Tris-HCI, pH 8.5/0.05% NaN₃). Five microliter samples of supernatant from each culture were diluted into 95 μl of Tris-buffered saline, pH 8.5/0.05 % Triton X-100 (TBST) and then incubated in the coated ELISA plates for 2 hours at room temperature. The plates were then washed with TBST. To detect positive anti-Tf DNA polymerase cross-reactivity, a commercially available, peroxidase-conjugated goat anti-mouse IgG, chain specific (Jackson ImmunoResearch, West Grove, PA), was diluted 5000-fold in TBST, added to the ELISA plates, and incubated for 1 hour at room temperature. Positive cross-reactivity was detected colorimetrically with 3-methyl-2- benzothiazolinonehydrazone/3-dimethylaminobenzoicacid/hydrogenperoxide.

Supematants from wells that tested even weakly positive by ELISA were further screened by immunoprecipitation of both Tfl and Taq DNA polymerases using techniques well known in the art. See Harlow and Lane, supra. The immunoprecipitation assay employed relies on the presence of protein A (which binds IgG) on the surface of Staphylococcus aureus (SAC, Sigma Chemical Co., St. Louis, MO). Since protein A does not bind strongly to a common subclass of mouse IgG, IgG], but does bind rabbit IgG strongly, a pellet of centrifuged SAC cells was first treated with rabbit anti- mouse IgG antibodies. The pellet from 10 μl of a 10% suspension of these cells was then incubated with 20 μl of hybridoma culture supernatant for one hour at room temperature. The resultant SAC cells were centrifuged, washed, and resuspended in diluted Taq or Tfl polymerase. The polymerase enzyme- cell suspensions were incubated overnight at 4°C and centrifuged. The resultant supernatant was removed and tested for depletion of polymerase activity using a standard radiochemical assay.

One hybridoma, designated hybridoma 7B12, tested strongly in the ELISA assay and immunoprecipitated both Taq and Tfl DNA polymerases (70-99% depletion in polymerase activity). More particularly, in a series of immunoprecipitations in which the polymerase concentration (Taq or Tfl polymerase) was varied, the results shown in Table 2B were obtained.

TABLE 2B IMMUNOPRECIPITATION RESULTS

Trial Polymerase Source of Monoclonal antibody Depletion of polymerase activity

2* 0.04 units Tfl hybridoma 7B12 supernatant, >99% polymerase 20 μl

3 0.09 units Tfl hybridoma 7B12 supernatant, 80% polymerase 20 μl

3 0.27 units Taq hybridoma 7B12 supernatant, 79% polymerase 20 μl

4 0.30 units Tfl 1.375 μg purified IgG from 99% polymerase hybridoma 7B12

Trial 1 was unsuccessful due to an excess of polymerase enzyme relative to the amount of antibody.

In control immunoprecipitation experiments in which six anti-£. coli DNA polymerase I monoclonal antibodies were employed (25 μl of hybridoma culture supernatant), 91 to 112% of the original Tfl polymerase activity was still detectable in solution (i.e., at most a 9% depletion of Tfl polymerase activity). The monoclonal antibody from hybridoma 7B12 was further characterized and found to neutralize Taq and Tfl polymerase activity at lower temperatures (41 °C). This activity assay was performed at 41 °C, rather than at higher temperatures (70°C) where the enzymes are more active, because the antibody itself denatures at higher temperatures.

Cells from hybridoma 7B12 were cloned three times by limiting dilution until all wells with growth tested positive in the ELISA assay (66/66 wells). The monoclonal antibody of hybridoma 7B12, a mouse IgG (γl, K) antibody, is commercially available from Molecular Biology Resources, Inc., Milwaukee, Wisconsin as Cat. No. 4100-01.

Subsequent experiments produced two additional anti-Taq/anti- Tfl monoclonal antibodies that may be (but have not been) used to affinity- purify DNA polymerase enzymes. In particular, two hybridomas producing anti-Tfl DNA polymerase monoclonal antibodies, formed using spleen cells from a mouse immunized with Tfl DNA polymerase, were identified using the sceening procedures outlined above. In the immunoprecipitation assay, 25 μl of supernatant from these two hybridoma cultures, designated hybridomas 10F10 and 11G4, depleted 92% and 95% , respectively, of the DNA polymerase activity from a solution containing 0.30 units of Tfl DNA polymerase.

The monoclonal antibodies from hybridoma 7B12 were coupled with Emphaze™ resin (3M, St. Paul, MN) as follows. Twenty-five milliliters of antibody solution (2 mg/ml in 0.6 M sodium citrate, 0.05 M sodium chloride, 0.05 M HEPES pH 8.6) was added to 1.25 g of Emphaze™ resin and allowed to react for 2 hrs at room temperature. Ethanolamine (1 ml of a 3 M solution, pH 9.0) was then added to quench unreacted azlactone functional groups and incubated for 1 hr. at room temperature, then overnight at 4°C. The resin was washed with and stored in PBS with 0.05% sodium azide.

The immunoaffinity column used to purify the DNA polymerase was prepared with about 10 ml dead volume of the resin washed with 300 ml of antibody column high salt buffer B (20mM Tris-HCI pH 7.5, 0.5 mM EDTA, 0.5 M NaCl, 0.05% Brij - 35). The enzyme was eluted with 10 mM triethylamine (pH 11.6). Fractions (5 ml each) were collected into tubes with 0.01 volumes of 1 M HEPES. Those fractions containing the DNA pol I enzyme were identified by activity assay, pooled, and dialyzed against storage buffer S (50% glycerol, 50 mM Tris-HCI, pH 7.5 at 23 °C, 5 mM DTT, 0.1 mM EDTA, 0.5% Tween 20, 0.5% Nonidet P40). The final product was stored at -20°C. The above purification procedure yielded about 60,000 units of purified T. flavus DNA polymerase I from 50 g of E. coli [pTFL-1.4] cells, which is equivalent to 1 ,200 units/g of cells.

The protein concentration was determined by the method of Lowry using a modification of the Sigma (St. Louis, MO) Protein Assay Kit (Cat. No. P5656) with Bovine Serum Albumin as a standard. Both standard and sample were precipitated with TCA prior to the protein analysis. Using the standard activity assay, the DNA polymerase specific activity was calculated to be 79,500 U/mg protein for the recombinant Tfl holoenzyme purified as described.

EXAMPLE 7

Construction and Expression of a High- Yield,

Full-Length T. flavus DNA Pol I Clone and Purification of Recombinant T. flavus DNA Pol I Holoenzyme To increase expression of the DNA Tfl pol I gene and to increase the yield and DNA polymerase specific activity of recombinant Tfl DNA pol I, the lacZ promoter was fused directly to the ATG start codon of the Tfl DNA pol I gene using site-directed mutagenesis, the resultant improved expression plasmid was expressed, and the recombinant DNA pol I was purified using a modified procedure.

Site-directed mutagenesis of single-stranded uracil-(U-) containing DNA from p51X16 was performed using the oligonucleotide TFL-SDM-3 (Table 2). Single-stranded U-containing DNA was prepared according to the protocol provided by Bio-Rad (Hercules, CA) in their Mutagenesis Kit. The new clone, p51X16M1, had the lacZ promoter fused to about 2 kb of the 5' portion of the Tfl DNA pol I gene (FIGURE IA). Plasmid p51X16Ml was digested with BamHI and Hindi and ligated to the 1.3 kb BamHI/EcoRV fragment isolated from p21BHc, which provided the 3' region of the Tfl DNA pol I gene. The resulting plasmid, pTFLRT4 (ATCC - 54

Accession No. 69633), was used to transform E. coli DH5αF'IQ (Life Technologies, Grand Island, NY), generating the 2nd generation expressing clone (FIGURE IA). The presence and integrity of the Tfl DNA pol I gene in the insert of pTFLRT4 was confirmed by DNA sequence analysis using primers RTFLJ, RTFLG, FTFLB, and FTFLE as set out in Table 2.

E. coli DH5αF'IQ transformed with pTFLRT4 were grown in a 250 liter fermentor in TB medium supplemented with 50 μg/ml ampicillin. At O.D._JOO = 0.7, expression of the plasmid was induced by the addition of IPTG to a final concentration of 0.5mM and the cells were cooled down to 20 °C, harvested three hours later, and stored at -70 °C until use.

Five hundred grams of induced cells were thawed and suspended in 2500 ml of lysis buffer A (20 mM Tris-HCI, pH 7.4, 0.5 mM EDTA, 100 mM NaCl, 5% glycerol, 5 mM jS-mercaptoethanol, 0.5% Nonidet P40, 0.5% Tween 20, 50 μg/ml PMSF, 0.5 μg/ml pepstatin A, 0.5 μg/ml leupeptin).

The cell suspension, to which lysozyme was added to a concentration of 0.5 mg/ml, was homogenized in a Manton-Gaulin press. Fresh PMSF again was added to the lysed cells to a final concentration of 50 μg/ml. The suspension of lysed cells was divided into 300 ml portions, heated to 65 °C for 1.5 hrs., and centrifuged for 30 min. at 6,δ00 x g.

Following this centrifugation, the resulting supernatant was adjusted to an additional NaCl concentration of 400 mM and 10% PEI, pH 7.5, was added to a final concentration of 0.2%. After 1 hr. of stirring at 4°C, the suspension was centrifuged (1 hr., 6,δ00 x g) and the resultant supernatant was precipitated with ammonium sulphate. After centrifugation for 2 hours at 6,800 x g the resultant pellet was resuspended with 200 ml Buffer A and applied to a Bio-Rex 70 column (5 x 10 cm) (Bio-Rad). The column was washed with 1.5 1 of buffer A and the DNA polymerase protein was eluted with 4 1 of a 0-1 M NaCl gradient in buffer A. Fractions of 25 ml were collected, and the peak fractions were pooled and dialyzed against two changes (2.5 1 each) of antibody column high salt buffer B and applied to an immunoaffinity column (1.5 x 8 cm) prepared as described above. After washing the immunoaffinity column with 250 ml antibody column high salt buffer B, the enzyme was eluted with 10 mM triethylamine (pH 11.6). Fractions of 5 ml were collected and the peak fractions were dialyzed against storage buffer S. This procedure yielded about 2,000,000 units of purified T. flavus DNA polymerase from 500 g of E. coli [pTFLRT4] cells, or about 4,000 units/g of cells as measured using the standard assay described above. The calculated DNA polymerase specific activity was 217,600 U/mg for this preparation of Tfl holoenzyme.

The N-terminal amino acid sequence from recombinant DNA pol I holoenzyme isolated from E. coli [pTFLRT4] was determined as Met- Glu-Ala-Ile-Val-Pro-Leu-Phe-Glu-Pro. This sequence matches the amino terminal sequence deduced by translation of the T flavus DNA pol I gene sequence (FIGURE 2), indicating that the translation starts at the predicted position. Unlike the native holoenzyme studied, no blockage of the terminal methionine in the cloned holoenzyme was observed.

EXAMPLE 8

Cloning and Expression of the Exo^" Fragment of T. flavus DNA Polymerase I

Expression studies using plasmids p21E10 and p21EHc were performed because these plasmids contain the 3' two-thirds of the DNA polymerase I gene fused to the lacZ operator/promoter. As deduced from the

DNA sequence, the first amino acid encoded by the insert of plasmid p21E10 corresponds to Glu₂₃₉ in FIGURE 2 (circled). It was hypothesized that the insert in p21E10 would encode a fragment of DNA polymerase I lacking the exonuclease domain (the exo^" fragment) due to the absence of the 5' one-third portion of this gene. From translation of sequence information obtained from the 5'-end of p21E10 using primer RSP (Table 2), it was concluded that the insert encoding the 3' two-thirds of the Tfl DNA pol I gene was out-of- frame. It was assumed that the same out-of-frame fusion was present in p21EHc. However, in spite of frame shift some heat-stable DNA polymerase activity was obtained from the clone harboring p21E10.

The ATG start codon of lacZ was brought in frame with the TFL DNA polymerase exo^' fragment in p21EHc using site-directed oligonucleotide mutagenesis (FIGURE IB). A mutagenic oligonucleotide TFL-SDM-1 was designed (Table 2), part of TFL-SDM-1 having homology to nucleotides 1015-1032 in FIGURE 4, the other part having homology to the vector. Single-stranded U-containing DNA was prepared by standard procedures and the chemically synthesized oligonucleotide TFL-SDM-1 was used to obtain site-directed changes in the newly synthesized DNA. This DNA was used to tiansform competent E. coli DH5αF'. Several transformants were selected and grown up for plasmid analysis. Of these, sequence analysis was performed on four clones using the [ ²P] end-labeled primer "5 'lac PCR" (Table 2) (Synthetic Genetics). The clones with the DNA polymerase gene fragment in the proper reading frame (which includes the ATG from the lacZ coding sequence, followed by "GAA GAC..." derived from the Tfl DNA pol I gene - see FIGURE 2, nucleotides 1015-1020 et seq.) were included in expression studies. Overexpressed recombinant protein was then isolated and purified from E. coli transformed with one of the clones, p21EHcMl. l (ATCC Accession No. 69632) by following the procedures outlined below.

E. coli DH5c-F' [p21EHcMl . l] was grown in a 50 liter fermentor in TB medium (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (1989)) supplemented with 50 μg/ml ampicillin with vigorous aeration at 37°C. At O-O.^ = 1.0, IPTG was added to final 0.5 mM concentration and cells were cultured for an additional 2 hours. The culture was cooled down to 20°C and 100 ml of 100 mM PMSF in isopropanol was added. After brief mixing, the culture was spun down in a Sharpies centrifuge and stored frozen at -70°C.

Fifty grams of E. coli [p21EHcMl. l] were thawed in 250 ml of lysis buffer A (20 mM Tris-HCI pH 7.4, 0.5 mM EDTA, 100 mM KC1, 10 mM MgCl₂, 5% glycerol, 5 mM /5-mercaptoethanol, 0.5% Nonidet P40, 0.5% Tween 20, 50 μg/ml PMSF, 0.5 μg/ml pepstatin A, 0.5 μg/ml leupeptin). The cell suspension was homogenized twice in a Manton-Gaulin press. After the first and second passes, fresh PMSF was added again to a new, final concentration of 50 μg/ml. The suspension of broken cells was divided into 100 ml portions and heated to 65 °C for 1 hr. Cell debris and denatured proteins were centrifuged at 6,800 x g for 30 min. and the supernatant was adjusted to an additional NaCl concentration of 400 mM. Then 10% PEI, pH 7.5, was slowly added to a final concentration of 0.2%. After 30 min. of stirring at 4°C, the suspension was centrifuged (1 hr., 6,800 x g) and the supernatant was concentrated on a YM30 membrane to 100-120 ml. The concentrate was run through a 5 x 50 cm Sephadex G-25 column equilibrated in buffer A, as described in Example 6. The crude Tfl exo fragment was applied to a Procion-Red Sepharose column (5 x 10 cm). The column was washed with 1.5 liters of buffer A and the DNA polymerase fragment was eluted with 4 liters of a 0-1.5 M NaCl gradient in buffer A. Fractions of 25 ml were collected and the fractions with DNA polymerase activity were dialyzed against two changes (1 liter each) of antibody column high salt buffer B (20 mM Tris-HCI, pH 7.5, 0.5 mM EDTA, 0.5 M NaCl, 0.05% Brij-35) and applied to an immunoaffinity column (1.5 x 8 cm). After washing the column with 250 ml of the same buffer, the enzyme was eluted with 10 mM triethylamine (pH 11.6) and treated as described above. In general, about 300,000 units of purified T.flavus exo^' fragment were obtained from 50 g of E. coli [p21EHcMl. l] cells (6,000 units/g). The protein concentration was determined as described above. The calculated DNA polymerase specific activity for the Tfl exo^" fragment was 600,000 U/mg.

Once the Tfl exo^" fragment was cloned and expressed the N-terminal amino acid sequence was determined. About 50 μg of the purified enzyme was separated using SDS-PAGE and blotted onto PVDF membrane as described for the holoenzymes. The major band was excised and subjected to sequence analysis. The chromatogram of the sequencer indicated the presence of a major and a minor sequence. The minor sequence represents the major sequence shifted by one amino acid. The major sequence reads: Leu-Glu-Arg-Leu-Glu-Phe-Gly-Ser-Leu-Leu-His-Glu-Phe-X-Leu-Leu-X-Ala- Pro-Ala (where X represents an amino acid whose identity was uncertain from the chromatogram). The minor sequence has the amino acid sequence: Glu- Arg-Leu-Glu-Phe-Gly-Ser-Leu-X-His-Glu-Phe-Gly-X-X-Pro-X-X-Ala-Pro. The major sequence is identical to the amino acid sequence deduced from the recombinant Tfl exo' fragment DNA sequence, except for the lack of 37 N-terminal amino acids, including the N-terminal methionine. SEQ ID NO: 3 and 4 contain the DNA sequence and the deduced amino acid sequence of the Tfl exo^' fragment, as expected from construct p21EHcMl.1. The loss of the 37 N-terminal amino acids may be due to processing of the exo^" fragment in the E. coli host. SEQ ID NO: 5 contains the amino acid sequence of the major band exo^' fragment, as deduced from the N-terminal amino acid sequence of the purified exo^" fragment and from the DNA sequence of plasmid p2 lEHcMl .1. The minor sequence presented here is the Tfl exo' fragment lacking both the N-terminal methionine and the next 37 amino acids. Although the amount sequenced in the minor species was small there was good correlation with the deduced amino acid sequence, except for the proline at position 16, that was expected to be glutamic acid. EXAMPLE 9

Characterization of 7- flavus DNA Polymerase I Exonuclease Activities

The purity and molecular weight of the T. flavus DNA polymerase and the Tfl exo^" fragment were estimated by SDS-polyacrylamide gel electrophoresis using the Pharmacia PhastSystem (Piscataway, NJ).

FIGURE 9 shows the purity of the holoenzyme and the Tfl exo^" fragment, which were separated on a 12.5% SDS-PAGE gel and stained with silver.

Assays were performed to determine intrinsic/extrinsic exonuclease, endonuclease, and DNase activities of the DNA polymerase enzyme preparations purified as described above and for T. aquaticus DNA pol I holoenzyme (Taq holo) and Stoffel fragment (Stoffel, Perkin Elmer, Foster City, CA, Cat. No. Nδ0δ-003δ), and for T. thermophilus holoenzyme (Tth holo) (Molecular Biology Resources, Inc., Cat. No. 1115-01 , Milwaukee, WI). The protocols are described below and results summarized in Table 3A.

A 3'-> 5' exonuclease activity assay was performed in a final volume of 10 μl containing 50 mM Tris-HCI, pH 7.6, 10 mM MgCl₂, 1 mM DTT, 0.15 μg of [3'-³H dCTP and dGTP labeled]λ DNA/Taq I fragments and 5, 10 and 20 units of enzyme. Each sample was overlaid with 10 μl of light mineral oil and incubated at 70°C for 1 hour. The reaction was terminated by the addition of 50 μl yeast RNA and 200μl of 10% TCA. After incubation for 10 min. on ice, the samples were centrifuged for 7 min. in a microcentrifuge. 200μl of supernatant was added to 6 ml of scintillation fluid and counted in a scintillation counter. The results are presented in Table 3A as the slope % -label released per unit of enzyme.

A 5' -_* 3' assay was performed in a manner identical to the 3' → 5' exonuclease assay, except for the use of [5'-³²P] λ DNA/ Haelll fragments instead of the 3'-labeled substrate. Double-stranded and single stranded DNase assays were performed using the protocol for the 3' → 5' exonuclease assay, except for the use of [³²P] λ DNA instead of the 3'-labeled substrate. The DNA was treated for 3 min. at 100°C and immediately chilled on ice prior to assaying for single stranded DNase activity.

An assay for endonuclease activity was performed as follows. The reagents (final concentrations of 50 mM Tris-HCI, pH 7.6, 10 mM MgCl₂, 1 mM 0-mercaptoethanol), 0.5 μg pBR322, less enzyme/H₂O, were mixed and kept on ice. The required amount of H₂O and 10 μl mineral oil were added to each tube. The reaction was started with the addition of 5, 10 or 20 units of enzyme; the final reaction volume was 10 μl. The samples were incubated at 70 °C for 1 hour. Two μl of a solution containing 0.25% bromophenol blue, 1 mM EDTA, and 40% sucrose was added to the reaction, and after a short centrifugation, 6 μl of the bottom layer was removed and electrophoresed on 1.5% agarose gels in 1 x TBE. The mobility change from the supercoiled to the linear form of pBR322 was recorded.

TABLE 3A

Enzyme 3'->5' exo¬ 5'->3' exo¬ ss ds Endo¬ nuclease nuclease DNase DNase nucle¬ ase

Tfl holo (r) 0.19 0 0.66 0 0

Tfl holo (n) 0.04 0.01 0 0 0

Tfl exo^" 0.03 0.002 0 0 0

Taq holo 0.031 0.09 0 0 0

Stoffel 0 0.01 0.28 0.1 0

Tth holo 0.07 0.04 0.2 0 0 The values for 3'-* 5' exonuclease activity and for 5'→ 3' exonuclease activity are low for all DNA polymerases tested. The differences in exonuclease and DNase activities between naturally occurring and recombinant Tfl holoenzyme are not believed to be statistically significant.

EXAMPLE 10

Comparison of the T. flavus and T. aquaticus DNA Polymerases

Biological properties of native T. flavus DNA pol I (nTfl Holo, lot #30419; Molecular Biology Resources, Inc. , Cat. No. 1112-01 , Milwaukee, WI); recombinant T. flavus DNA pol I holoenzyme (rTfl Holo) purified from E. coli [pTFLRT4]; T.flavus DNA pol I exo' fragment (Tfl exo ) purified from E. coli [p21EHcMl.1]; T. aquaticus DNA polymerase I (native Taq or recombinant AmpliTaq) holoenzyme; and the -AmpliTaq DNA polymerase Stoffel fragment were compared using a number of protocols described below.

The molecular weights and purities of the preparations of the various enzymes were estimated by acrylamide gel electrophoresis utilizing the Pharmacia PhastSystem (Piscataway, NJ) for electrophoresis and silver staining. A comparison of the apparent molecular weights estimated from 7.5% and 12.5% acrylamide gels and the calculated molecular weights derived from available sequence data is given in Table 3B. The apparent molecular weight of the holoenzymes using either acrylamide concentration was less than the calculated molecular weights. A purity of greater than 95 % was estimated for all DNA polymerases analyzed: i.e. Tfl and Taq holo enzymes, Tfl exo^' fragment and Stoffel fragment. TABLE 3B

Apparent Mol. Weight

Enzyme 7.5% gel 12.5% gel Calculated Mol. Weight

Tfl holoenzyme* 80,000 δ4,000 93,969

Recombinant Tfl exo^' 59,000 59,000 62,979 fragment (recomb.)

Taq holoenzyme 82,000 δ5,000 93,904

Stoffel 60,000 61,000 61,000

* native and recombinant

Using the Pharmacia PhastSystem, the polymerases and standards were subjected to isoelectric focusing. The experimentally derived pi values of the samples, including samples of E. coli DNA pol I holoenzyme (E. coli pol I) and Klenow fragment, were compared to values calculated from derived amino acid sequence information. The results are given in Table 4.

TABLE 4 pi Values

Enzyme Calculated pi Measured pi nTfl Holo 6.23 6.25 rTfl Holo 6.23 6.43

A&V Tfl^* 5.73 (not available)

Tfl exo^' 6.37 5.94

Taq Holo 6.00 6.14

Taq Stoffel 5.93 5.83

E. coli pol I 5.29 5.12

Klenow 5.60 5.75

'Purported T. flavus DNA pol I protein sequence published by Akhmetzjanov and Vakhitov. The relative DNA polymerase activities of the enzymes were assayed at 70 °C at different pH values. The pH of selected buffers were adjusted at 23°C, to permit direct comparison to published results. Table 5A shows the measured pH values at 70 °C for lx buffers which first had been titrated at 23 °C. Unless otherwise indicated, pH values reported herein were adjusted at about 23 °C.

The activity assays were performed in a 100 μl (final volume) reaction mixture, containing 0.1 mM dCTP, dTTP, dGTP, [α³³P]dATP, 0.3 mg/ml activated calf thymus DNA and 0.5 mg/ml BSA in a set of buffers containing: 50 mM KC1, 1 mM DTT, 10 mM MgCl₂ and 50 mM of one of three buffering compounds: PIPES, Tris or Triethylamine. Three dilutions (20, 40 or 80 U/μl) of each polymerase enzyme were prepared, and 5 μl of a dilution was added to the reaction mixture, followed by incubation at 70°C for 30 min. The experiment was performed twice, each time using duplicate samples. FIGURE 5 graphically depicts the relative activities of the enzymes studied, calculated as the ratio of counts per minute (corrected for background and enzyme dilution) at a given pH to counts per minute at the maximum value for that enzyme. The optimal ranges (> 80% activity) for the five enzymes tested are provided in Table 5B.

TABLE 5B

Optimal pH ranges (as titered at 23 °C)

Enzyme pH

Native Tfl holoenzyme 9.5-10.5

Recombinant Tfl holoenzyme 9.5-10.5

Tfl exo^' fragment 7.5-9.8

Stoffel fragment 7.5-9.8

Ampli Taq 7.5-9.3

These values are about 1 pH unit higher than for buffers measured at 70 °C (see Table 5A).

The pH protocol described above was modified to determine the influence of MgCl₂ concentration on the activities of the DNA polymerases. The reaction buffers included 50 mM Tris-HCI pH 8.3 (23 °C) and MgCl₂ concentrations from 0.36 to 50 mM. Three independent experiments were performed and curves were constructed (FIGURE 6A) showing the relative activity of Tfl exo" fragment, Tfl holoenzyme (native and recombinant), and Taq Stoffel fragment. The higher limit for the Stoffel fragment was extrapolated. The optimal ranges (> 80% activity) are 1.3-13 mM MgCl₂ for the Tfl exo' fragment, and 2.3-33 mM MgCl₂ for the Stoffel fragment. The recombinant and the native Tfl holoenzyme showed greatest activity at 50 mM MgCl₂.

The above protocol was modified to determine the influence of MnCl₂ concentration on the activities of the DNA polymerases (in the absence of magnesium ions). The reaction buffers included MnCl₂ concentrations from 0.1 to 20 mM. Due to the precipitation of oxidation products (MnO^) of MnCl₂, the reaction buffers were prepared just prior to the assay. The pH of the buffer was adjusted to pH δ.7 before the addition of a 1 M stock solution of MnCl₂. The pH was finally adjusted to δ.3 at 23°C. Three independent experiments were performed and a curve was constructed (FIGURE 6B) showing relative activity of the enzymes. The optimal ranges for the four enzymes tested are 2.1-11 mM MnCl₂ for the Tfl exo^" fragment, 4-20 M MnCl₂ for the Stoffel fragment and O.δ-4 mM MnCl₂ for the recombinant and native Tfl holoenzymes. The thermostability and temperature optimum of the polymerase enzymes were determined by incubating 10 units of enzyme for 30 min. at 23, 37, 60, 65, 70, 75, δO, and 90°C, in 100 μl of buffer used for the determination of polymerase activity (including 50 mM Tris-HCI pH δ.3 (23 ^CC) and 1.5 mM MgCl₂) in a DNA polymerase activity assay as described above. The polymerase activity was then determined by acid precipitation of the polymerization product as described above. FIGURE 7 A depicts the percent relative activity, calculated as described above. The temperature optima were 70-75 °C for the Stoffel and Tfl exo^' fragments and δ0°C for the native and recombinant Tfl holoenzymes. At 90 °C there was 14%, 6% and δ% of the activity left in the Tfl holoenzymes, the Stoffel fragment, and the Tfl exo" fragment, respectively.

The PCR half lives of the enzymes were determined in 100 μl PCR reactions, performed in duplicate, substituting the appropriate buffer in the PCR cocktail prepared for each individual enzyme. The cocktail for the Tfl exo' fragment contains 1 x Tfl pol buffer (50 mM Tris-HCI, pH 9.0 at 23°C, 20 mM (NH₄)₂SO₄, 1.5 mM MgCl₂), 200 μM of each dNTP, 0.5 μM of primer FTFL2 and primer RTFL4, and 15 ng of T. flavus genomic DNA. The buffers for other enzymes tested were as follows: Taq pol I (1 x Taq pol buffer: 10 mM Tris-HCI pH δ.4, 50 mM KC1, 1.5 mM MgCl₂); Tfl DNA pol I holoenzyme (1 x Tfl pol buffer); and Stoffel fragment (1 x Stoffel buffer: 10 mM Tris-HCI, pH δ.3, 10 mM KC1 and 2.5 mM MgCl₂). Duplicate samples were denatured at 95 °C for 5 min. and held at 72 °C until 10 units of enzyme were added, and the samples were then cycled 0, 20, 25, 30, 35, 50 and 100 times as described in Example 3. The samples were analyzed on 1.2% agarose gels using ethidium bromide to visualize the presence of specific PCR product. The expected length of PCR product was about δOO bp. Reactions containing Taq DNA pol I, Stoffel and Tfl exo^" fragments had visible product after 20 cycles, whereas reactions with Tfl holoenzyme showed product only after 30 cycles. The Tfl exo^' fragment synthesized more product than the Stoffel fragment (FIGURE 7B). In general, there was some background, very likely because of the large amount of enzyme in the reaction. This background was not observed when 1 to 5 units of Tfl exo' fragment were used in a 35 cycle regimen. (FIGURE 7B, Right lane.) The polymerase activity in each tube was also determined as described above following completion of the PCR cycling, and the result plotted in a enzyme cycling stability curve (FIGURE δ). The half life was estimated to be: 25 cycles for both the Taq holoenzyme and Stoffel fragment, 20 cycles for the Tfl holoenzyme, and 16 cycles for the Tfl exo^' fragment.

EXAMPLE 11 DNA Sequencing with T. flavus DNA Polymerases

A. Native and recombinant Tfl holoenzyme, Tfl exo^' fragment, AmpliTaq, and Stoffel fragment were employed in the SEQUAL-¹' DNA Polymerase Sequencing System (CHIMERx) to test their performance in DNA sequencing using ssDNA template and labeled primer.

The primer FSP (Table 2) was end-labeled with T4 kinase and [7³²P]ATP according to the CHIMERx protocol. A 10 μl labeling reaction was prepared containing 0.5 μl primer (10 pmol/μl), 1.0 μl T4 Kinase 10X buffer (500 mM Tris-HCI pH 7.5, 100 mM MgCl₂, 50 M DTT, 1 mM spermidine), 3.0 μl [7-³²P] ATP (6000 Ci/mmol, 10μCi/μl), 0.5 μl T4 kinase (15 U/μl), and 5.0 μl H₂O. The labeling reaction was incubated at 37°C for 10 min., and the kinase was inactivated by incubation at 65 °C for 10 min. The sequencing reactions for native and recombinant Tfl holoenzyme and the exo^" fragment were set up using 2-5 units of enzyme, according to CHIMERx conditions. Briefly, a reaction cocktail was prepared containing 16.0 μl ssM13mplδ DNA (approx. 1 μg), 5.0 μl Sequal 5x buffer (250mM Tris-HCI, pH 9.5, 12.5 mM MgCl₂), 1.0 μl labeled primer (0.5 pmol/μl), and 2-5 units of enzyme. Four d/ddNTP mixtures were also prepared (A mix: 20 μM dATP, 60 μM dCTP, 60 μM dGTP, 60 μM dTTP, 300 μM ddATP; C mix: 60 μM dATP, 20 μM dCTP, 60 μM dGTP, 60 μM dTTP, 150 μM ddCTP; G mix: 60 μM dATP, 60 μM dCTP, 20 μM dGTP, 60 μM dTTP, 30 μM ddGTP; T mix: 60 μM dATP, 60 μM dCTP, 60 μM dGTP, 20 μM dTTP, 400 μM ddTTP). The sequencing reactions were performed by mixing 5 μl of reaction cocktail with 1 μl of the appropriate d/ddNTP mixture and heating the reaction tube at 65 °C for 10 min. After this incubation 3 μl of stop solution (EDTA/DTT/Bromophenol blue/xylene cyanol) were added and the reactions were placed on ice.

AmpliTaq reaction cocktail was prepared by using the lOx Reaction Buffer provided with the Cycle Sequencing Kit, which contains 2 units of enzyme in a final volume of 20 μl. Stoffel fragment reaction cocktail (20 μl) contained 4 μl of 5x Stoffel fragment reaction buffer, 2 μl of 25 mM MgCl₂, both provided with the enzyme (Perkin Elmer), and 2 units of enzyme. Both cocktails included lμg of ssDNA template and 1 μl of labeled primer FSP. For both enzymes, sequencing reactions were performed by mixing 5μl reaction cocktail with lμl d/ddNTP mixtures and incubating for 10 min. at 65 °C. Three microliters of stop solution were then added to the reactions, and the reactions were placed on ice.

The reactions were heated at 90°C for 5 min. just before loading onto a 6% sequencing gel. One microtiter of each sample was loaded 6δ -

and electrophoresed at 3000 volts for 1.5 hours. The gel was autoradiographed and analyzed. FIGURE 10A photographically depicts a portion of a sequencing gel showing the same DNA sequence for all enzymes used, except for the native T. flavus DNA pol I holoenzyme control. Very little background was observed when the Tfl exo^" fragment, Tfl holoenzyme and AmpliTaq were used. The Stoffel fragment had more ghost bands than the other enzymes. However, no attempt had been made to optimize the reaction conditions for the Stoffel fragment.

B. To demonstrate the utility of the recombinant Tfl holoenzyme and the Tfl exo^" fragment in cycle sequencing with single- stranded DNA template, these enzymes were substituted into the SEQUAL™ DNA Polymerase and Cycle- SEQUAL™ Sequencing System (CHIMERx) and the protocols provided were followed. The labeling protocol described above was repeated to create end-labeled primer. A 22 μl reaction cocktail was then prepared containing approx. 20 ng ssM13mplδ DNA, 5.0 μl 5X Sequal sequencing buffer, 1.0 μl labeled primer (0.5 pmol/μl), balance H₂O. Native or recombinant Tfl holoenzyme or Tfl exo' fragment was then added to the cocktail (0.5 units) and gently mixed. For comparison purposes, two units of AmpliTaq or Stoffel fragment were added to the ssM13mplδ DNA template (20 ng); the manufacturer's reaction conditions for the Perkin Elmer Cycle Sequencing Kit were followed for AmpliTaq, and, for the Stoffel fragment, 4 μl of the Stoffel buffer and 2 μl of the MgCl₂ solution provided with the enzyme were used. The sequencing reactions were performed by mixing 5 μl of reaction cocktail with 1 μl of each d/ddNTP mixture (in separate tubes), adding a drop ( - 10μl) of mineral oil to each tube, and placing the tubes into a preheated (94 °C) thermal cycler programmed to run the following cycle twenty times: 94°C for 15 seconds (denaturation), 70°C for 60 seconds (extension). The reactions were cooled to 4°C after 20 cycles until 4μl stop solution were added, and then the reactions were set on ice.

Immediately after heating the reaction mixtures at 90 °C for 5 min., one microtiter of each reaction mixture was loaded onto a 6% sequencing gel. FIGURE 10B shows that the Tfl exo^" fragment and recombinant Tfl holoenzyme yield clean sequence data, whereas in the AmpliTaq lanes some ghost bands were observed. The Stoffel fragment, under the conditions used here, did not produce comparable sequencing data.

C. The utility of recombinant Tfl holoenzyme and Tfl exo^' fragment for sequencing with internal labeling using double-stranded DNA template was demonstrated in a sequencing reaction in which a [α³⁵S]-dATP labeling protocol and double stranded pUC19 template were used. The experiment was performed as outlined in the SEQUAL™ DNA Polymerase Sequencing System (CHIMERx) with 2 μg of pUC19 dsDNA and 2.5 units of the enzymes.

To promote efficient priming, the pUC 19 double-stranded DNA template was denatured by adding deionized H₂O to lδ μl, adding 2 μl of 2M

NaOH, and incubating for 5 min. at room temperature. The reaction was neutralized by adding 2 μl of 2M ammonium acetate, pH 4.6, ethanol precipitated, air-dried, and resuspended in 10 μl deionized water.

For each enzyme, a 22.75 μl extension/labeling cocktail was prepared with the 2 μg denatured pUC19 dsDNA, 5.0 μl 5X Sequal buffer,

1.0 μl primer (0.5 pmol/μl), 1.0 μl alpha labeling mix (- 45 μM each of dCTP, dGTP, dTTP), 0.25 μl [α-³⁵S] dATP (1000 Ci/mmol), 2.5 units enzyme, balance H₂O. This cocktail was incubated at 65° for 10 min.

Extension/termination reactions were performed by adding 5 μl of extension/labeling cocktail to tubes containing 1 μl of the appropriate d/ddNTP mix, and mixing gently. The reaction tubes were immediately placed at 65 °C for 4 min., stopped by addition of 4 μl step solution, and set on ice.

Each reaction was heated at 90° for 5 min. immediately before loading 1-2 μl onto a sequencing gel. Results are depicted in FIGURE IOC. Native Tfl holoenzyme (not shown) was compared to recombinant holoenzyme and to the Tfl exo^' fragment. The bands were comparable for the holoenzymes. The quality of the sequence data is comparable although the signal was weaker when the Tfl exo^" fragment was used.

D. The utility of recombinant Tfl holoenzyme and the exo^' fragment for double-stranded sequencing using a labeled sequencing primer was demonstrated by substitution of these enzymes into the SEQUAL™ System which uses 2 μg pUC19 dsDNA and [τ³²P]-labeled primer FSP. The reactions were performed according to CHIMERx 's protocol. More particularly, the double-stranded template was first denatured as described above, and then sequencing reactions were performed essentially as decribed in part A (substituting the pUC19 denatured dsDNA for ssM13mplδ template). As can be seen in FIGURE 10D, both the Tfl holoenzyme and the Tfl exo" fragment produced good sequence data.

EXAMPLE 12

Polymerase Chain Reaction

The utility of recombinant Tfl holoenzyme and the exo fragment in PCR was demonstrated as follows. In a 0.5 ml reaction tube 85 μl water, 2 μl 10 mM dNTPs, 10 ml 10 x Tfl Polymerase Reaction Buffer (10 x buffer is 500 mM Tris-HCI, pH 9.0, 200 mM (NH^SO,, 15 mM MgCl₂), 1 μl each of 50 μM primers FTFL11 and RTFL12 (primer set 11-12), 50 μl mineral oil and 1 μl of 15 μg/ml T.flavus genomic DNA were combined. After the initial denaturation step (Step 1), 5.5 and 11 units of Tfl exo^' fragment, or 5 units of Tfl holoenzyme were added. As a control Taq pol 1 in 1 x Taq Polymerase Reaction Buffer (Example 10) was used to amplify the genomic DNA. Amplifications were performed in a MJ Research PTC-100 Cycler with external sensor control. The amplification program was: Step 1: 95°C for 5 min. ; Step 2: hold at 72°C; Step 3: 55°C for 45 sec.; Step 4: 72°C for 5 min.; Step 5: 95°C for 15 sec.; Step 6: repeat steps 3-5 thirty-four times; Step 7: 55°C for 45 sec.; Step 8: 72°C for 20 min.; Step 9: hold at 4°C.

The amplification products were separated on 1.2% agarose gels. Primer set 11-12 gave a single amplification product from T. flavus genomic DNA. Five units of the Tfl exo^' fragment produced a single product: the yield was slightly less than that obtained with Taq polymerase and better than the yield from Tfl holoenzyme.

EXAMPLE 13 Thermal Cycle Labeling With Tfl DNA Pol I The protocol described in Example 4 was used to demonstrate the utility of recombinant Tfl DNA pol I holoenzyme and the Tfl exo^" fragment for thermal cycle labeling (TCL). See co-owned, co-pending U.S. Patent Application Serial No. 08/217,459, filed March 24, 1994, entitled "Materials and Methods for Restriction Endonuclease Applications." PCT Application No. US94/03246, filed March 24, 1994.

Thermal Cycle Labeling (TCL) is a procedure for labeling double-stranded DNA while simultaneously amplifying large amounts of the labeled probe. TCL of DNA requires two general steps: 1) generation of the sequence-specific oligonucleotides by CV/^'JI^* (Molecular Biology Resources, Milwaukee, WI) restriction of the template DNA; and 2) repeated cycles of denaturation, annealing, and extension in the presence of a thermostable DNA polymerase or a functional fragment thereof which maintains polymerase activity. Optimal results are obtained after 20 such cycles, which is best performed in an automated thermal cycling instrument such as a Perkin-Elmer Model 480 thermocycler. In conjunction with such an instrument, about 1.5 hr. is required to complete this protocol. If a thermal cycler is not available these reactions may be performed using heat blocks. As few as 5 cycles may yield probes with acceptable detection sensitivities. The generation of sequence specific oligonucleotides for use in this method may also be accomplished using the restriction endonuclease reagent CGase I (Molecular Biology Resources) or the restriction endonuclease Aci I which has as a recognition sequence CCGC.

Non-radioactive labeling of DNA using TCL is accomplished by mixing: 10 pg - 100 ng linearized template, 50 ng CViJI'-digested primers, 1.5 μl 10X labeling buffer, 2.5 - 5 units thermostable DNA polymerase, 1 μl of ImM Biotin- 11-dUTP (Enzo Diagnostics, New York, New York), 1.5 μl each of dATP, dCTP, and dGTP (2 mM), and 1.0 μl 2mM dTTP. The reaction mixture is brought to a volume of 15 μl with deionized H₂O, overlaid with mineral oil and cycled through 20 rounds of denaturation, annealing and extension. A typical cycling regimen employs 20 cycles of denaturation at 91 °C for 5 sec, annealing at 50 °C for 5 sec and extension at 72 °C for 30 sec. The reaction is then terminated by adding 1 μl of 0.5M EDTA, pH δ.0. The amplified, labeled probe is a very heterogeneous mixture of fragments, which appears as a smear when analyzed by agarose gel electrophoresis.

The performance of recombinant Tfl DNA pol I holoenzyme, Tfl exo" fragment, Taq holoenzyme, and the Stoffel fragment (control) was assayed by substitution of these enzymes for the enzyme provided with the CHIMERx TCL kit (ZEPTO™ Labeling Kit). Five units of each enzyme and biotin- 11-dUTP as the label were used. The substrate was pUC 19 DNA.

After cycling of the samples the relative efficiency of the labeling reaction was determined by electrophoresis on a 0.7% agarose gel. The ethidium bromide gel staining of amplified DNA shows the characteristic smear for all enzymes used. The efficiency of incorporation was then determined by dot blot analysis. The hybridized and developed filter showed that the holoenzymes (native and recombinant Tfl, and Taq) can be diluted 1: 10⁶ and still generate a visible dot. The samples which were labeled with Tfl exo^" or the Stoffel fragment can clearly be seen after a 1: 10* dilution. The 1: 10^s dilution gave a weak signal when the exo^" fragment was used for the TCL reaction.

Another aspect of the present invention involves a variation of TCL called Universal Thermal Cycle Labeling (UTCL) in which the extension primers are not supplied by CvLFI* restriction. Without intending to be limited to a particular theory, one explanation for the mechanism of UTCL is that the Tfl DNA pol I holoenzyme itself may supply endogenous "random" primers for enzymatic extension in a TCL-type reaction. Alternatively, some other explanation accounts for the mechanism of UTCL.

In a UTCL reaction, recombinant Tfl DNA pol I holoenzyme is combined with intact DNA template and is subjected to repeated cycles of denaturation, annealing, and extension. A radioactive- or non-isotopically- labeled deoxynucleotide triphosphate is incorporated during the extension step for subsequent detection purposes. The amplified, labeled probe represents a very heterogenous mixture of fragments, which appears as a large molecular weight smear when analyzed by agarose gel electrophoresis. The utility of recombinant Tfl DNA pol I for Universal Thermal Cycle Labeling is demonstrated by substituting this enzyme in the UTCL protocol described in co-owned, copending U.S. Patent App. Ser. No. 0δ/217,459, filed March 24, 1994 (Example 12), incorporated herein by reference.

EXAMPLE 14

Reverse Transcription with T. flavus DNA Pol I Holoenzyme and exo^" Fragment

RNA-dependent DNA polymerase activity of the Tfl DNA polymerases was analyzed using the following procedure: In a 0.5 ml reaction tube, 2.5 μl 1 M Tris-HCI, pH δ.3, 5μl of 0.6 M KC1, 5 μl of 0.04 M MgCl₂, 17.5 μl of water, 10μl of 2 mM poly rA:dT (the substrate) and 5 μl 5 mM [α-³²P]TTP at 10 μCi/ml were combined. After incubation at 55 °C for 5 min. , the reaction was started by the addition of 5 μl of enzyme (Five DNA- dependent DNA polymerase units per μl). The reaction was allowed to proceed at 55 °C for 30 min., and terminated by taking a 40 μl aliquot and adding it to 50 μl of 10% tRNA, 2% sodium pyrophosphate. The samples were precipitated with TCA and the enzyme activity was determined as described above. The RNA-dependent polymerase activity of the native and the recombinant Tfl DNA pol I was determined to be about 6% of the DNA-dependent polymerase activity. When 10 (RNA-dependent DNA polymerase) units of AMV-RT (Molecular Biology Resources, Inc., Cat. No. 1372-01) were compared to 10 (DNA-dependent DNA polymerase) units of Tfl DNA pol I it was found that nTfl DNA pol I possess 2.4% and rTfl DNA pol I 1.6% of the RNA-dependent DNA polymerase activity of AMV-RT.

Titration of the MgCl₂ and the MnCl concentration revealed that the native and the recombinant holoenzymes prefer MgCl₂ over MnCl₂ for RT activity.

The Tfl exo^" fragment has a lower RT activity than the holoenzyme, but has a broader temperature range for activity. First strand cDNA synthesis with the holoenzymes apparently yields a product of die same length as that obtained by using AMV-RT. The recombinant T. flavus DNA polymerase I and the exo^' fragment both exhibit reverse transcriptase function which can be used in applications such as RT-PCR or cDNA preparation at elevated temperatures.

EXAMPLE 15

Comparison of the Processivity of DNA Polymerase Enzymes

Using a modification of a procedure described by Tabor et al. ,

J. Biol. Chem. 262: 16212-16223 (1987), the processivity of native and recombinant Tfl DNA pol I holoenzyme, Tfl exo^" fragment, and Taq DNA pol I holoenzyme were compared. The "processivity" of a DNA polymerase enzyme is a measure of the rate at which the enzyme moves forward along a template while catalyzing DNA synthesis, i.e., a measure of the speed at which DNA polymerization takes place in the presence of the enzyme.

To prepare the assay, a 60 μl reaction cocktail was prepared with 3μg M13 mplδ ssDNA, 12 μl ddATP mix (20 μM dATP; 60 μM each of dCTP, dGTP, and dTTP; 300 μM ddATP), 3.0 μl α-³³P labeled forward sequencing primer (3 μg/μl), 12μl 5x reaction buffer (250 mM Tris-HCI, pH 9.5; 12.5 mM MgClj), balance H₂O. Additionally, dilutions of native and recombinant Tfl holoenzyme, Tfl exo^' fragment, and Taq holoenzyme were prepared with appropriate storage buffer to create enzyme solutions of 0.125 and 0.0125 units/μl for the holoenzymes and 0.25 and 0.025 units/ml for Tfl exo^" fragment. To perform the assay, 7.0 μl of the reaction cocktail were mixed with 2.0 μl of diluted DNA polymerase enzyme. By using 0.25 and 0.025 units of Taq, nTfl, and rTfl holoenzyme and 0.5 and 0.05 units of exo' fragment per reaction, reactions containing approximately 1: 100 and 1: 1000 enzyme molecule: template molecule are obtained. The use of such low polymerase concentrations minimizes the "bumping off" from template by competing polymerase molecules. Reaction mixtures were incubated at 65 °C and 3μl samples were removed at 1.0, 2.5 and 6.0 minute time points. Reactions were stopped by adding 1.0 μl stop buffer (EDTA/DTT/BromoPhenol Blue/xylene cyanol), were heated at 90°C for 3 min., and were loaded onto 7.5% polyacrylamide sequencing gels. The gels were electrophoresed until the bromophenol blue dye was about 3/4 down the gel, and an autoradiograph of the gel was taken overnight at -70°C.

With this assay, a highly processive enzyme produces stong, slow-mobility (larger) labeled bands on an autoradiograph, whereas a less processive DNA polymerase produces higher-mobility (smaller) fragments and/or bands with less intensity. Autoradiographs of the 6 min. incubation/ 1:100 enzyme: template reactions revealed die exo' fragment produced bands witi the most intensity, followed by the rTfl and nTfl holoenzyme, then the Taq holoenzyme. The length of fragments obtained by the four enzymes was very comparable. Autoradiographs from the 1: 1000 enzyme:template reaction indicate that processivity (from best to least) is Tfl exo^" fragment > Taq holoenzyme > nTfl holoenzyme and rTfl holoenzyme. These results indicate that Tfl exo" fragment has greater processivity than either Tfl holoenzyme (native or recombinant) or Taq holoenzyme.

EXAMPLE 16

Large-Scale Purification of Recombinant Tfl DNA Polymerase I Holoenzyme and Exo^" Fragment

Both the recombinant Tfl holoenzyme and Tfl exo^" fragment were purified on a large "production" scale by modifying the procedure described above for purifying native Tfl holoenzyme.

Four hundred sixty grams of induced £. coli DH5αF'IQ cells transformed with pTFLRT4 (cultured and frozen as described above) were thawed and suspended in 2500 ml of lysis buffer A (20 mM Tris-HCI, pH 8; 0.5 mM EDTA; 7 mM /-■-mercaptoethanol; 10 mM MgCl₂). For Tfl exo fragment, 787 grams of £. coli transformed with p21EHcMl.1 (cultured and frozen as described above) were used. Phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 0.3 mM.

The suspension was then treated with 0.2 g/1 of lysozyme (predissolved in lysis buffer) at 4 °C for 1 hr. Cells were homogenized twice at 9000 psi in a Manton Gaulin homogenizer, with the suspension chilled to approximately 10°C between passes. New PMSF was added to 0.2 g/1 before, between and after passes. The suspension of lysed cells was divided into 300 ml portions, heated to 65 °C for 1 hr., cooled down to 4°C, and centrifuged for 30 min. at 13,500 x g. Following the centrifugation, NaCl and polyethyleneimine (PEI) (10% w/v, pH 7.0) were added to the heat-treated supernatant to a final concentration of 0.5 M and to 0.1 %, respectively. The sample was mixed well and centrifuged at 13,500 x g for 1 hour. The supernatant from the twice-centrifuged, heat-treated lysate was desalted by diluting with 10 liters of DE52 column buffer (20 mM Tris- HCI, pH 8.0; 0.5 mM EDTA; 7 mM 0ME) and concentrated to approximately 4 liters using an Amicon S10Y30 Spiral Ultrafiltration cartridge. The dilution/concentration step was repeated two times, with a final concentrated volume of about 4 liters.

The desalted sample was batch contacted with 400 g of equilibrated Whatman DE52 ion exchange resin (Maidstone, England). The suspension was collected on a sintered glass funnel and washed 3 times with 1 volume of DE52 column buffer. The resin was then resuspended in a minimal volume of buffer and poured into a column (4.5 x 50 cm), packed and washed with an additional volume of buffer. The column was eluted with a 0-0.5 M NaCl linear gradient (total gradient volume: 2000 ml). Twenty- five ml fractions were collected at a rate of about 5 ml/min. Peak fractions (fractions containing DNA polymerase activity) were determined by a modified DNA polymerase assay described by Kaledin et al., Biokhimiya 45:644-651 (1980), pooled and dialyzed in approximately twenty-five volumes of Affi-Gel Blue (AGB) column buffer (20 mM Tris-HCI, pH 7.5; 0.5 mM EDTA; 10 mM 0ME; 10 mM MgCl₂; 0.02% Brij 35).

The dialyzed DE52 peak fractions were applied to an AGB column (4.4 x 40 cm, 600 ml packed volume, MBR Blue, Molecular Biology Resources, Milwaukee WI), which was washed with 2 column volumes of AGB column buffer, and eluted with a 0-1.2 M NaCl linear gradient (total gradient volume: 2000 ml). To elute the exo^" fragment, a 0-1.5 M NaCl linear gradient was employed. Twenty-five ml fractions were collected at a rate of 1-5 ml/min. The peak fractions were dialyzed as above in AGB buffer.

The dialyzed AGB peak fractions were applied to a Heparin Agarose column (4.4 x 16.5 cm, 250 ml packed volume (Bio-Rad Affigel Heparin or Heparin Agarose from Molecular Chimerics, Madison, WI)), which was washed with approximately 2 column volumes (until effluent is no longer colored, and column resin is white in appearance), and eluted with a 0.1-1.0 M NaCl linear gradient (total gradient volume: 1500 ml). To elute the exo^' fragment, a 0.15-1.0 M NaCl linear gradient was employed. Twenty- five ml fractions were collected at a rate of 1-5 ml/min. The peak fractions were dialyzed in HP Q Sepharose Column Buffer (20 mM Tris-HCI, pH 7.5; 0.5 mM EDTA; 7 mM 0ME; 0.1% Brij 35).

The dialyzed heparin agarose peak fractions were filtered through a 0.2 μm filter and applied at 4 ml/min. to the HP Q Sepharose column (Pharmacia, Uppsala, Sweden) on FPLC. The column was washed with several column volumes of buffer, and eluted with a 0-0.25 M NaCl linear gradient. Ten ml fractions were collected at 4 ml/minute. The peak fractions were dialyzed in HP S Column Buffer (20 mM Na-Citrate, pH 6.0; 1 mM EDTA; 7 mM 3ME; 0.1 % Brij 35) or diluted in the same buffer, depending on the volume of the fraction pool.

The dialyzed (or diluted) HP Q peak fractions were filtered through a 0.2 μm filter and the HP S column (Pharmacia) was run as above, washing with HP S Column buffer and eluting with a 0-0.25 M NaCl gradient. Peak fractions were pooled and dialyzed against 4 liters of Final Storage Buffer (50 mM Tris-HCI, pH 7.5, 0.1 mM EDTA, 5 mM DTT, 50% glycerol). The final product was diluted to a concentration of 5000 U/ml in the above buffer including 0.5 % Tween 20 (Sigma Chemical Co., St. Louis, MO) and 0.5 % Nonidet P40 (Fluka Biochemika, Buchs, Switzerland) as stabilizers and stored at -20°C. To purify the recombinant holoenzyme, the HP S column purification was unnecessary, and therefore was omitted. Samples from the above-described preparations were electrophoresed using SDS-Page and visualized with silver staining. The rTfl holoenzyme and exo^" fragments appeared as single bands having apparent molecular weights of 8δ,000 and 63,000 kDa, respectively, each being greater than 95 % pure. A quantitative analysis of the enzymes prepared using the above-described purification procedure is as shown in Table 6:

TABLE 6

Enzvme Quantity of Specific Activity Yield Cells (Units/mg protein) (Units/g cells) nTfl Holo 1200 g 50,000 U/mg 1,700 U/g (Example 1) rTfl Holo 460 g 70,000 U/mg 4,300 U/g rTfl exo 7δ7 g 192,000 U/mg 5,600 U/g

The biological activities of the recombinant enzymes purified by the above-described protocol were analyzed using the assays described in preceding Examples. In the endonuclease activity assay described in Example 9, five, ten, and twenty unit challenges resulted in less than 5% conversion of supercoiled pBR322 to the linear form. The results of other assays described in Example 9 are summarized in Table 7:

TABLE 7

ASSAY rTfl holo Tfl Exo^' ds DNase 0% slope/unit 0% slope/unit ss DNase 0% slope/unit 0% slope/unit

3' Exonuclease 0% slope/unit 0.06% slope/unit

5' Exonuclease 0.4δ% slope/unit 0% slope/unit - δO -

Deposit of Biological Materials: The following plasmids have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Dr. , Rockville MD 20δ52 (USA) pursuant to the provisions of the Budapest Treaty:

Designation Deposit Date ATCC No. Host Strain pTFLRT4 May 26, 1994 69633 DH5αF'IQ

P21EHcMl. l May 26, 1994 69632 DH5αF'

Availability of the deposited materials is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The present invention has been described with reference to specific examples and embodiments. However, this application is intended to cover those changes and substitutions which are apparent and may be made by those skilled in the art without departing from the spirit and scope of the claims.

δi

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(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Gass, David A.

(B) REGISTRATION NUMBER: 38,153

(C) REFERENCE/DOCKET NUMBER: 28003/32330

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312/474-6300

(B) TELEFAX: 312/474-0448

(C) TELEX: 25-3856

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3048 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

( ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 301..2805

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TACTTCGGCG GGGTGAAGCT CGGGGCCGGG GGGCTTGTGC GGGCCTACGG GGGGGTGGCG 60

GCGGAGGCCT TAAGCGGGCG CCCAAGGTCC CCTTGGTGGA GCGGGTGGGG CTCGCCTTCC 120

TCGTGCCCTT CGCCGAGGTG GGCCGGGTCT ACGCCCTCCT GGAGGCCCGC GCCCTGAAGG 180

CCGAGGAGAC CTACACCCCG GAGGGCGTGC GCTTCGCCCT CCTCCTCCCC AAGCCCGAGC 240

GGGAAGGTTT CCTCAGGGCG CTCCTGGACG CCACCCGGGG ACAGGTGGCC CTGGAGTAGC 300

ATG GAG GCG ATC GTT CCG CTC TTT GAA CCC AAA GGC CGG GTC CTC CTG 348 Met Glu Ala He Val Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15

GTG GAC GGC CAC CAC CTG GCC TAC CGC ACC TTC TTC GCC CTG AAG GGC 396 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20 25 30

CTC ACC ACG AGC CGG GGC GAA CCG GTG CAG GCG GTC TAC GGC TTC GCC 444 Leu Thr Thr Ser Arg Gly Glu Pro Val Gin Ala Val Tyr Gly Phe Ala 35 40 45

AAG AGC CTC CTC AAG GCC CTG AAG GAG GAC GGG TAC AAG GCC GTC TTC 492 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe 50 55 60

GTG GTC TTT GAC GCC AAG GCC CCC TCC TTC CGC CAC GAG GCC TAC GAG 540 Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu 65 70 75 80

GCC TAC AAG GCG GGG AGG GCC CCG ACC CCC GAG GAC TTC CCC CGG CAG 588 Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gin 85 90 95

CTC GCC CTC ATC AAG GAG CTG GTG GAC CTC CTG GGG TTT ACC CGC CTC 636 Leu Ala Leu He Lye Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu 100 105 110

GAG GTC CCC GGC TAC GAG GCG GAC GAC GTC CTC GCC ACC CTG GCC AAG 684 Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys 115 120 125

AAG GCG GAA AAG GAG GGG TAC GAG GTG CGC ATC CTC ACC GCC GAC CGC 732 Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg He Leu Thr Ala Asp Arg 130 135 140

GAC CTC TAC CAA CTC GTC TCC GAC CGC GTC GTC GTC CTC CAC CCC GAG 780 Asp Leu Tyr Gin Leu Val Ser Asp Arg Val Val Val Leu His Pro Glu 145 150 155 160 GGC CAC CTC ATC ACC CCG GAG TGG CTT TGG GAG AAG TAC GGC CTC AAG 828 Gly His Leu He Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys 165 170 175

CCG GAG CAG TGG GTG GAC TTC CGC GCC CTC GTG GGG GAC CCC TCC GAC 876 Pro Glu Gin Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180 185 190

AAC CTC CCC GGG GTC AAG GGC ATC GGG GAG AAG ACC GCC CTC AAG CTC 924 Asn Leu Pro Gly Val Lys Gly He Gly Glu Lys Thr Ala Leu Lys Leu 195 200 205

CTC AAG GAG TGG GGA AGC CTG GAA AAC CTC CTC AAG AAC CTG GAC CGG 972 Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg 210 215 220

GTA AAG CCA GAA AAC GTC CGG GAG AAG ATC AAG GCC CAC CTG GAA GAC 1020 Val Lys Pro Glu Asn Val Arg Glu Lys He Lys Ala His Leu Glu Asp 225 230 235 240

CTC AGG CTT TCC TTG GAG CTC TCC CGG GTG CGC ACC GAC CTC CCC CTG 1068 Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu 245 250 255

GAG GTG GAC CTC GCC CAG GGG CGG GAG CCC GAC CGG GAG GGG CTT AGG 1116 Glu Val Asp Leu Ala Gin Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg 260 265 270

GCC TTC CTG GAG AGG CTG GAG TTC GGC AGC CTC CTC CAC GAG TTC GGC 1164 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 275 280 285

CTC CTG GAG GCC CCC GCC CCC CTG GAG GAG GCC CCC TGG CCC CCG CCG 1212 Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290 295 300

GAA GGG GCC TTC GTG GGC TTC GTC CTC TCC CGC CCC GAG CCC ATG TGG 1260 Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305 310 315 320

GCG GAG CTT AAA GCC CTG GCC GCC TGC AGG GAC GGC CGG GTG CAC CGG 1308 Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg 325 330 335

GCA GCA GAC CCC TTG GCG GGG CTA AAG GAC CTC AAG GAG GTC CGG GGT 1356 Ala Ala Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly 340 345 350

CTC CTC GCC AAG GAC CTC GCC GTC TTG GCC TCG AGG GAG GGG CTA GAC 1404 Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp 355 360 365

CTC GTG CCC GGG GAC GAC CCC ATG CTC CTC GCC TAC CTC CTG GAC CCC 1452 Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375 380

TCC AAC ACC ACC CCC GAG GGG GTG GCG CGG CGC TAC GGG GGG GAG TGG 1500 Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 385 390 395 400 ACG GAG GAC GCC GCC CAC CGG GCC CTC CTC TCG GAG AGG CTC CAT CGG 1548 Thr Glu Aβp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg 405 410 415

AAC CTC CTT AAG CGC CTC GAG GGG GAG GAG AAG CTC CTT TGG CTC TAC 1596 Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lye Leu Leu Trp Leu Tyr 420 425 430

CAC GAG GTG GAA AAG CCC CTC TCC CGG GTC CTG GCC CAC ATG GAG GCC 1644 His Glu Val Glu Lye Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435 440 445

ACC GGG GTA CGG CTG GAC GTG GCC TAC CTG CAG GCC CTT TCC CTG GAG 1692 Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Glu 450 455 460

CTT GCG GAG GAG ATC CGC CGC CTC GAG GAG GAG GTC TTC CGC TTG GCG 1740 Leu Ala Glu Glu He Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465 470 475 480

GGC CAC CCC TTC AAC CTC AAC TCC CGG GAC CAG CTG GAA AGG GTG CTC 1788 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gin Leu Glu Arg Val Leu 485 490 495

TTT GAC GAG CTT AGG CTT CCC GCC TTG GGG AAG ACG CAA AAG ACG GGC 1836 Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gin Lys Thr Gly 500 505 510

AAG CGC TCC ACC AGC GCC GCG GTG CTG GAG GCC CTA CGG GAG GCC CAC 1884 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His 515 520 525

CCC ATC GTG GAG AAG ATC CTC CAG CAC CGG GAG CTC ACC AAG CTC AAG 1932 Pro He Val Glu Lys He Leu Gin His Arg Glu Leu Thr Lys Leu Lys 530 535 540

AAC ACC TAC GTG GAC CCC CTC CCA AGC CTC GTC CAC CCG AGG ACG GGC 1980 Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly 545 550 555 560

CGC CTC CAC ACC CGC TTC AAC CAG ACG GCC ACG GCC ACG GGG AGG CTT 2028 Arg Leu His Thr Arg Phe Asn Gin Thr Ala Thr Ala Thr Gly Arg Leu 565 570 575

AGT AGC TCC GAC CCC AAC CTG CAG AAC ATC CCC GTC CGC ACC CCC TTG 2076 Ser Ser Ser Asp Pro Asn Leu Gin Asn He Pro Val Arg Thr Pro Leu 580 585 590

GGC CAG AGG ATC CGC CGG GCC TTC GTG GCC GAG GCG GGA TGG GCG TTG 2124 Gly Gin Arg He Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605

GTG GCC CTG GAC TAT AGC CAG ATA GAG CTC CGC GTC CTC GCC CAC CTC 2172 Val Ala Leu Asp Tyr Ser Gin He Glu Leu Arg Val Leu Ala His Leu 610 615 620

TCC GGG GAC GAG AAC CTG ATC AGG GTC TTC CAG GAG GGG AAG GAC ATC 2220 Ser Gly Asp Glu Asn Leu He Arg Val Phe Gin Glu Gly Lys Asp He 625 630 635 640 δ5 -

CAC ACC CAG ACC GCA AGC TGG ATG TTC GGC GTC CCC CCG GAG GCC GTG 2268 His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val 645 650 655

GAC CCC CTG ATG CGC CGG GCG GCC AAG ACG GTG AAC TTC GGC GTC CTC 2316 Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670

TAC GGC ATG TCC GCC CAT AGG CTC TCC CAG GAG CTT GCC ATC CCC TAC 2364 Tyr Gly Met Ser Ala Hie Arg Leu Ser Gin Glu Leu Ala He Pro Tyr 675 680 685

GAG GAG GCG GTG GCC TTT ATA GAG CGC TAC TTC CAA AGC TTC CCC AAG 2412 Glu Glu Ala Val Ala Phe He Glu Arg Tyr Phe Gin Ser Phe Pro Lye 690 695 700

GTG CGG GCC TGG ATA GAA AAG ACC CTG GAG GAG GGG AGG AAG CGG GGC 2460 Val Arg Ala Trp He Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly 705 710 715 720

TAC GTG GAA ACC CTC TTC GGA AGA AGG CGC TAC GTG CCC GAC CTC AAC 2508 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Aβp Leu Aβn 725 730 735

GCC CGG GTG AAG AGC GTC AGG GAG GCC GCG GAG CGC ATG GCC TTC AAC 2556 Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Aβn 740 745 750

ATG CCC GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG CTC GCC ATG GTG 2604 Met Pro Val Gin Gly Thr Ala Ala Aβp Leu Met Lys Leu Ala Met Val 755 760 765

AAG CTC TTC CCC CGC CTC CGG GAG ATG GGG GCC CGC ATG CTC CTC CAG 2652 Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gin 770 775 780

GTC CAC GAC GAG CTC CTC CTG GAG GCC CCC CAA GCG CGG GCC GAG GAG 2700 Val His Asp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg Ala Glu Glu 785 790 795 800

GTG GCG GCT TTG GCC AAG GAG GCC ATG GAG AAG GCC TAT CCC CTC GCC 2748 Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala 805 810 815

GTG CCC CTG GAG GTG GAG GTG GGG ATG GGG GAG GAC TGG CTT TCC GCC 2796 Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Aβp Trp Leu Ser Ala 820 825 830

AAG GGT TAGGGGGGCC CTGCCGTTTA GAGGAAGTTC AAGGGGTTGT CCCTCAGAAA 2852 Lys Gly

CGCCTCCAGG GGAACGCCCT CTGCGGCTAC CAGGAGGCCT TTAGCCCCAA AGGTGCGGGT 2912

GAAGGCTTCC AGGCCCTGGG TTCTTTTAAA GGGGGCGCTT TTGACCTCGA GGGCCAGGAG 2972

GCGCTTTCCC TTTTGAAGGA CAAAGTCACT TCCTGGTCCC TTTCCCGCCA GTAGTACACC 3032

TCAAACCCCC CCTGGT 3048

(2) INFORMATION FOR SEQ ID NO:2: - δ6 -

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 834 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Ala He Val Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

1 5 10 15

Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20 25 30

Leu Thr Thr Ser Arg Gly Glu Pro Val Gin Ala Val Tyr Gly Phe Ala 35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe 50 55 60

Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu 65 70 75 80

Ala Tyr Lye Ala Gly Arg Ala Pro Thr Pro Glu Aβp Phe Pro Arg Gin 85 90 95

Leu Ala Leu He Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu 100 105 110

Glu Val Pro Gly Tyr Glu Ala Aβp Aβp Val Leu Ala Thr Leu Ala Lye 115 120 125

Lys Ala Glu Lye Glu Gly Tyr Glu Val Arg He Leu Thr Ala Aβp Arg 130 135 140

Asp Leu Tyr Gin Leu Val Ser Aβp Arg Val Val Val Leu His Pro Glu 145 150 155 160

Gly His Leu He Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Lys 165 170 175

Pro Glu Gin Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180 185 190

Asn Leu Pro Gly Val Lys Gly He Gly Glu Lys Thr Ala Leu Lys Leu 195 200 205

Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg 210 215 220

Val Lys Pro Glu Asn Val Arg Glu Lys He Lys Ala His Leu Glu Asp 225 230 235 240

Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu 245 250 255

Glu Val Asp Leu Ala Gin Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg 260 265 270

Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 275 280 285 Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro 290 295 300

Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305 310 315 320

Ala Glu Leu Lys Ala Leu Ala Ala Cyβ Arg Aβp Gly Arg Val Hie Arg 325 330 335

Ala Ala Aβp Pro Leu Ala Gly Leu Lye Aβp Leu Lye Glu Val Arg Gly 340 345 350

Leu Leu Ala Lys Aβp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Aβp 355 360 365

Leu Val Pro Gly Aβp Aβp Pro Met Leu Leu Ala Tyr Leu Leu Aβp Pro 370 375 380

Ser Aβn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 385 390 395 400

Thr Glu Aβp Ala Ala Hie Arg Ala Leu Leu Ser Glu Arg Leu Hie Arg 405 410 415

Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lye Leu Leu Trp Leu Tyr 420 425 430

His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435 440 445

Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Glu 450 455 460

Leu Ala Glu Glu He Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala 465 470 475 480

Gly His Pro Phe Asn Leu Asn Ser Arg Aβp Gin Leu Glu Arg Val Leu 485 490 495

Phe Aβp Glu Leu Arg Leu Pro Ala Leu Gly Lye Thr Gin Lys Thr Gly 500 505 510

Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His 515 520 525

Pro He Val Glu Lys He Leu Gin His Arg Glu Leu Thr Lys Leu Lys 530 535 540

Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly 545 550 555 560

Arg Leu His Thr Arg Phe Asn Gin Thr Ala Thr Ala Thr Gly Arg Leu 565 570 575

Ser Ser Ser Asp Pro Asn Leu Gin Asn He Pro Val Arg Thr Pro Leu 580 585 590

Gly Gin Arg He Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605

Val Ala Leu Asp Tyr Ser Gin He Glu Leu Arg Val Leu Ala His Leu 610 615 620 Ser Gly Asp Glu Asn Leu He Arg Val Phe Gin Glu Gly Lye Aβp He 625 630 635 640

His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val 645 650 655

Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670

Tyr Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala He Pro Tyr 675 680 685

Glu Glu Ala Val Ala Phe He Glu Arg Tyr Phe Gin Ser Phe Pro Lys 690 695 700

Val Arg Ala Trp He Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly 705 710 715 720

Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn 725 730 735

Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn 740 745 750

Met Pro Val Gin Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val 755 760 765

Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gin 770 775 780

Val His Aβp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg Ala Glu Glu 785 790 795 800

Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lye Ala Tyr Pro Leu Ala 805 810 815

Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala 820 825 830

Lys Gly

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1794 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1794

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG GAA GAC CTC AGG CTT TCC TTG GAG CTC TCC CGG GTG CGC ACC GAC 48 Met Glu Asp Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Aβp 1 5 10 15

CTC CCC CTG GAG GTG GAC CTC GCC CAG GGG CGG GAG CCC GAC CGG GAG 96 Leu Pro Leu Glu Val Asp Leu Ala Gin Gly Arg Glu Pro Asp Arg Glu 20 25 30

GGG CTT AGG GCC TTC CTG GAG AGG CTG GAG TTC GGC AGC CTC CTC CAC 144 Gly Leu Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His 35 40 45

GAG TTC GGC CTC CTG GAG GCC CCC GCC CCC CTG GAG GAG GCC CCC TGG 192 Glu Phe Gly Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp 50 55 60

CCC CCG CCG GAA GGG GCC TTC GTG GGC TTC GTC CTC TCC CGC CCC GAG 240 Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu 65 70 75 80

CCC ATG TGG GCG GAG CTT AAA GCC CTG GCC GCC TGC AGG GAC GGC CGG 288 Pro Met Trp Ala Glu Leu Lye Ala Leu Ala Ala Cys Arg Asp Gly Arg 85 90 95

GTG CAC CGG GCA GCA GAC CCC TTG GCG GGG CTA AAG GAC CTC AAG GAG 336 Val His Arg Ala Ala Asp Pro Leu Ala Gly Leu Lye Aβp Leu Lye Glu 100 105 110

GTC CGG GGT CTC CTC GCC AAG GAC CTC GCC GTC TTG GCC TCG AGG GAG 384 Val Arg Gly Leu Leu Ala Lye Aβp Leu Ala Val Leu Ala Ser Arg Glu 115 120 125

GGG CTA GAC CTC GTG CCC GGG GAC GAC CCC ATG CTC CTC GCC TAC CTC 432 Gly Leu Asp Leu Val Pro Gly Asp Aβp Pro Met Leu Leu Ala Tyr Leu 130 135 140

CTG GAC CCC TCC AAC ACC ACC CCC GAG GGG GTG GCG CGG CGC TAC GGG 480 Leu Asp Pro Ser Aβn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly 145 150 155 160

GGG GAG TGG ACG GAG GAC GCC GCC CAC CGG GCC CTC CTC TCG GAG AGG 528 Gly Glu Trp Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg 165 170 175

CTC CAT CGG AAC CTC CTT AAG CGC CTC GAG GGG GAG GAG AAG CTC CTT 576

Leu His Arg Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu 180 185 190

TGG CTC TAC CAC GAG GTG GAA AAG CCC CTC TCC CGG GTC CTG GCC CAC 624 Trp Leu Tyr Hie Glu Val Glu Lye Pro Leu Ser Arg Val Leu Ala Hie 195 200 205

ATG GAG GCC ACC GGG GTA CGG CTG GAC GTG GCC TAC CTG CAG GCC CTT 672 Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu 210 215 220

TCC CTG GAG CTT GCG GAG GAG ATC CGC CGC CTC GAG GAG GAG GTC TTC 720 Ser Leu Glu Leu Ala Glu Glu He Arg Arg Leu Glu Glu Glu Val Phe 225 230 235 240 CGC TTG GCG GGC CAC CCC TTC AAC CTC AAC TCC CGG GAC CAG CTG GAA 768

Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gin Leu Glu 245 250 255

AGG GTG CTC TTT GAC GAG CTT AGG CTT CCC GCC TTG GGG AAG ACG CAA 816

Arg Val Leu Phe Aβp Glu Leu Arg Leu Pro Ala Leu Gly Lye Thr Gin 260 265 270

AAG ACG GGC AAG CGC TCC ACC AGC GCC GCG GTG CTG GAG GCC CTA CGG 864

Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg

275 280 285

GAG GCC CAC CCC ATC GTG GAG AAG ATC CTC CAG CAC CGG GAG CTC ACC 912

Glu Ala His Pro He Val Glu Lys He Leu Gin His Arg Glu Leu Thr 290 295 300

AAG CTC AAG AAC ACC TAC GTG GAC CCC CTC CCA AGC CTC GTC CAC CCG 960

Lys Leu Lys Aβn Thr Tyr Val Aβp Pro Leu Pro Ser Leu Val Hie Pro

305 310 315 320

AGG ACG GGC CGC CTC CAC ACC CGC TTC AAC CAG ACG GCC ACG GCC ACG 1008

Arg Thr Gly Arg Leu Hie Thr Arg Phe Aβn Gin Thr Ala Thr Ala Thr 325 330 335

GGG AGG CTT AGT AGC TCC GAC CCC AAC CTG CAG AAC ATC CCC GTC CGC 1056

Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gin Asn He Pro Val Arg 340 345 350

ACC CCC TTG GGC CAG AGG ATC CGC CGG GCC TTC GTG GCC GAG GCG GGA 1104

Thr Pro Leu Gly Gin Arg He Arg Arg Ala Phe Val Ala Glu Ala Gly

355 360 365

TGG GCG TTG GTG GCC CTG GAC TAT AGC CAG ATA GAG CTC CGC GTC CTC 1152

Trp Ala Leu Val Ala Leu Asp Tyr Ser Gin He Glu Leu Arg Val Leu 370 375 380

GCC CAC CTC TCC GGG GAC GAG AAC CTG ATC AGG GTC TTC CAG GAG GGG 1200

Ala His Leu Ser Gly Asp Glu Asn Leu He Arg Val Phe Gin Glu Gly

385 390 395 400

AAG GAC ATC CAC ACC CAG ACC GCA AGC TGG ATG TTC GGC GTC CCC CCG 1248

Lys Asp He His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro 405 410 415

GAG GCC GTG GAC CCC CTG ATG CGC CGG GCG GCC AAG ACG GTG AAC TTC 1296

Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lye Thr Val Asn Phe 420 425 430

GGC GTC CTC TAC GGC ATG TCC GCC CAT AGG CTC TCC CAG GAG CTT GCC 1344

Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala

435 440 445

ATC CCC TAC GAG GAG GCG GTG GCC TTT ATA GAG CGC TAC TTC CAA AGC 1392

He Pro Tyr Glu Glu Ala Val Ala Phe He Glu Arg Tyr Phe Gin Ser 450 455 460

TTC CCC AAG GTG CGG GCC TGG ATA GAA AAG ACC CTG GAG GAG GGG AGG 1440

Phe Pro Lys Val Arg Ala Trp He Glu Lys Thr Leu Glu Glu Gly Arg

465 470 475 480 AAG CGG GGC TAC GTG GAA ACC CTC TTC GGA AGA AGG CGC TAC GTG CCC 1488 Lys Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro 485 490 495

GAC CTC AAC GCC CGG GTG AAG AGC GTC AGG GAG GCC GCG GAG CGC ATG 1536 Asp Leu Asn Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met 500 505 510

GCC TTC AAC ATG CCC GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG CTC 1584 Ala Phe Asn Met Pro Val Gin Gly Thr Ala Ala Asp Leu Met Lye Leu 515 520 525

GCC ATG GTG AAG CTC TTC CCC CGC CTC CGG GAG ATG GGG GCC CGC ATG 1632 Ala Met Val Lye Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met 530 535 540

CTC CTC CAG GTC CAC GAC GAG CTC CTC CTG GAG GCC CCC CAA GCG CGG 1680 Leu Leu Gin Val Hie Aβp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg 545 550 555 560

GCC GAG GAG GTG GCG GCT TTG GCC AAG GAG GCC ATG GAG AAG GCC TAT 1728 Ala Glu Glu Val Ala Ala Leu Ala Lye Glu Ala Met Glu Lys Ala Tyr 565 570 575

CCC CTC GCC GTG CCC CTG GAG GTG GAG GTG GGG ATG GGG GAG GAC TGG 1776 Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Aβp Trp 580 585 590

CTT TCC GCC AAG GGT TAG 1794

Leu Ser Ala Lye Gly 595

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 597 amino acidβ

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Glu Asp Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Aβp

1 5 10 15

Leu Pro Leu Glu Val Asp Leu Ala Gin Gly Arg Glu Pro Asp Arg Glu 20 25 30

Gly Leu Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His 35 40 45

Glu Phe Gly Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp 50 55 60

Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu 65 70 75 80

Pro Met Trp Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg 85 90 95 Val His Arg Ala Ala Asp Pro Leu Ala Gly Leu Lys Aep Leu Lye Glu 100 105 110

Val Arg Gly Leu Leu Ala Lys Aβp Leu Ala Val Leu Ala Ser Arg Glu 115 120 125

Gly Leu Asp Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu 130 135 140

Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly 145 150 155 160

Gly Glu Trp Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg 165 170 175

Leu His Arg Asn Leu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu 180 185 190

Trp Leu Tyr His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His 195 200 205

Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu 210 215 220

Ser Leu Glu Leu Ala Glu Glu He Arg Arg Leu Glu Glu Glu Val Phe 225 230 235 240

Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gin Leu Glu 245 250 255

Arg Val Leu Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gin 260 265 270

Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg 275 280 285

Glu Ala His Pro He Val Glu Lys He Leu Gin His Arg Glu Leu Thr 290 295 300

Lys Leu Lys Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro

305 310 315 320

Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gin Thr Ala Thr Ala Thr 325 330 335

Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gin Asn He Pro Val Arg 340 345 350

Thr Pro Leu Gly Gin Arg He Arg Arg Ala Phe Val Ala Glu Ala Gly 355 360 365

Trp Ala Leu Val Ala Leu Asp Tyr Ser Gin He Glu Leu Arg Val Leu 370 375 380

Ala His Leu Ser Gly Asp Glu Asn Leu He Arg Val Phe Gin Glu Gly 385 390 395 400

Lys Asp He His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro 405 410 415

Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe 420 425 430 Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala 435 440 445

He Pro Tyr Glu Glu Ala Val Ala Phe He Glu Arg Tyr Phe Gin Ser 450 455 460

Phe Pro Lye Val Arg Ala Trp He Glu Lys Thr Leu Glu Glu Gly Arg 465 470 475 480

Lys Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro 485 490 495

Asp Leu Aβn Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met 500 505 510

Ala Phe Asn Met Pro Val Gin Gly Thr Ala Ala Aβp Leu Met Lye Leu 515 520 525

Ala Met Val Lye Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met

530 535 540

Leu Leu Gin Val His Asp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg 545 550 555 560

Ala Glu Glu Val Ala Ala Leu Ala Lye Glu Ala Met Glu Lys Ala Tyr 565 570 575

Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Aβp Trp 580 585 590

Leu Ser Ala Lye Gly 595

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 560 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 1 5 10 15

Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 20 25 30

Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu 35 40 45

Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg Ala Ala 50 55 60

Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly Leu Leu 65 70 75 80 Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp Leu Val 85 90 95

Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 100 105 110

Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 115 120 125

Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu

130 135 140

Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu 145 150 155 160

Val Glu Lys Pro Leu Ser Arg Val Leu Ala Hie Met Glu Ala Thr Gly 165 170 175

Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Glu Leu Ala 180 185 190

Glu Glu He Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala Gly Hie 195 200 205

Pro Phe Asn Leu Asn Ser Arg Aβp Gin Leu Glu Arg Val Leu Phe Aβp 210 215 220

Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gin Lys Thr Gly Lys Arg 225 230 235 240

Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro He 245 250 255

Val Glu Lys He Leu Gin His Arg Glu Leu Thr Lys Leu Lys Asn Thr 260 265 270

Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg Leu 275 280 285

His Thr Arg Phe Asn Gin Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 290 295 300

Ser Asp Pro Asn Leu Gin Asn He Pro Val Arg Thr Pro Leu Gly Gin 305 310 315 320

Arg He Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val Ala 325 330 335

Leu Asp Tyr Ser Gin He Glu Leu Arg Val Leu Ala His Leu Ser Gly 340 345 350

Asp Glu Asn Leu He Arg Val Phe Gin Glu Gly Lys Asp He His Thr 355 360 365

Gin Thr Ala Ser Trp Met Phe Gly val Pro Pro Glu Ala Val Asp Pro

370 375 380

Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly 385 390 395 400

Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala He Pro Tyr Glu Glu 405 410 415 Ala Val Ala Phe He Glu Arg Tyr Phe Gin Ser Phe Pro Lys Val Arg 420 425 430

Ala Trp He Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly Tyr Val 435 440 445

Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala Arg 450 455 460

Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 465 470 475 480

Val Gin Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 485 490 495

Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gin Val His 500 505 510

Asp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg Ala Glu Glu Val Ala 515 520 525

Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala Val Pro 530 535 540

Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala Lys Gly 545 550 555 560

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGCCAGGGTT TTCCCAGTCA CGAC 24

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGCGGATAAC AATTTCACAC AGGA 24

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTAAGTAGCT CCGATCCCAA C 21

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATCACTCCTT GGCGGAGAGC CAGTC 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATTTAGCACA TATGGCGATG CTTCCC 26

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CTTTCCAGCT CCGACCCCAA C 21 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCTACTCCTT GGCGGAGAGC CAGTC 25

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TGGATGTCCC TCCCCTCCTG AAAGA 25

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCCTTTCCCG GAAGCTTTCC CAGGTGCA 28

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TGCACCTGGG AAAGCTTCCG GGAAAGGG 28 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCTGCAGTAC CGGGAGCTCA CCAAGCTCAA 30

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TTGAGCTTGG TGAGCTCCCG GTACTGCAGG 30

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TGGACTATAG CCAGATAGAG CT 22

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: AAGCGAAGAC CTCCTCCTCG A 21

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: AGTTCGGCAG CCTCCTCCAC GA 22

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TCCAAGGAAA GCCTGAGGTC TT 22

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: AAGCTCGCCA TGGTGAAGCT CTT 23

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TCGGAGACGA GTTGGTAGAG GT 22

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ACCTCTACCA ACTCGTCTCC GA 22

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: AGAGGACGAA GCCCACGAA 19

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AGGAGGTAGG CGAGGAGCAT 20

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATGCTCCTCG CCTACCTCCT 20

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

TCGAGGAGGA GGTCTTCGCT T 21

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 baee pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AGCTCTATCT GGCTATAGTC CA 22

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: ATAGGCTCTC CCAGGAGCTT 20

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AAGAGCTTCA CCATGGCGAG CTT 23

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

TTCCCCTGGA GGCGTTTCTG A 21

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AAAGACCACG AAGACGGCCT T 21

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AAGGCCGTCT TCGTGGTCTT T 21

(2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: AAGGAGTGGG GAAGCCTGGA A 21

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

TTCCAGGCTT CCCCACTCCT T 21

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: TTCTTCCGAA GAGGGTTTCC A 21

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GCGTCCAGGA GCGCCCTGAG GA 22 (2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CCTCAGGGCG CTCCTGGACG CCA 23

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

TTCGTCCTCT CCCGCCCCGA 20

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CCAACCTGCA GAACATCCCC GT 22

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GGTGTGGATG TCCTTCCCCT 20

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CCCTGCCGTT TAGAGGAAGT TCAAG 25

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CTTGAACTTC CTCTAAACGG CAGGG 25

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ACCCGGCCTT TGGGTTCAAA GA 22

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TCTTTGAACC CAAAGGCCGG GT 22

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: TTCCCGTGCT CCTTCCGCTC 20

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CTCGCCTTCC TCGTGCCCTT 20

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GCTTCCGGCT CGTATGTTGT GTG 23

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GGAAAGCCTG AGGTCTTCCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC 60 CACACAACAT 70

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 81 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: ACCCGGCCTT TGGGTTCAAA GAGCGGAACG ATCGCCTCCA TAGCTGTTTC CTGTGTGAAA 60 TTGTTATCCG CTCACAATTC C 81

Claims

What is Claimed is:

1. A purified DNA encoding a thermostable polypeptide having DNA polymerase activity, said DNA comprising nucleotides 301 to 2802 of SEQ ID NO: 1.

2. The DNA of claim 1 in an expression vector.

3. Plasmid pTFLRT4, having ATCC Accession No. 69633.

4. A host cell transformed with a DNA selected from the group consisting of the DNAs of claims 1 and 2.

5. The host cell of claim 4, wherein said host cell is capable of expressing a thermostable polypeptide encoded by said DNA, said polypeptide having DNA polymerase activity.

6. The host cell of claim 5, wherein said host cell is a prokaryotic cell.

7. The host cell of claim 6, wherein said host cell is an £. coli cell.

8. A host cell transformed with plasmid pTFLRT4 having ATCC Accession No. 69633.

9. A vector wherein nucleotides 301 to 2δ02 of SEQ ID NO: 1 are operably attached to a promoter, said nucleotides encoding a polypeptide having thermostable DNA polymerase activity.

10. The vector of claim 9 having at least one insert consisting essentially of nucleotides 301 to 2802 of SEQ ID NO: 1.

11. A method for producing a thermostable polypeptide having DNA polymerase activity comprising the steps of: transforming a host cell with a DNA to create a transformed host cell, said DNA selected from the group consisting of the DNAs of claims 1 or 2; cultivating said transformed host cell under conditions to promote expression of a thermostable polypeptide encoded by said DNA, said polypeptide having DNA polymerase activity; and purifying said thermostable polypeptide.

12. The method of claim 11 wherein an antibody is used to purify said thermostable polypeptide.

13. A method for purifying a recombinant Thermus flavus thermostable polypeptide having DNA polymerase activity comprising the steps of: transforming a host cell with a plasmid pTFLRT4, having ATCC Accession No. 69633, to create a transformed host cell; cultivating said transformed host cell under conditions to promote expression of a thermostable polypeptide encoded by said plasmid, said polypeptide having DNA polymerase activity; and purifying said thermostable polypeptide with a monclonal antibody that is immunologically cross-reactive with said Thermus flavus thermostable polypeptide.

14. The method of claim 13 wherein said immunologically cross-reactive monoclonal antibody has specificity for a Thermus aquaticus DNA polymerase.

15. A method of purifying a thermostable polypeptide having DNA polymerase activity comprising the steps of: a) expressing said thermostable polypeptide in a host cell, said polypeptide having an amino acid sequence encoded by a DNA selected from the group consisting of the DNAs of claims 1 and 2; b) lysing the cell to create a suspension containing said thermostable polypeptide and host cell proteins and cell debris; c) contacting a soluble portion of said suspension with an antibody that is immunologically cross-reactive with said thermostable polypeptide and under conditions wherein the antibody binds to said thermostable polypeptide to form an antibody-polypeptide complex; d) isolating the antibody-polypeptide complex; and e) separating said thermostable polypeptide from said isolated antibody-polypeptide complex to provide a purified thermostable polypeptide.

16. The method of claim 15 further comprising between steps (b) and (c) the steps of: heating said suspension to denature host cell proteins; and centrifuging said suspension to remove said cell debris and denatured host cell proteins.

17. The method of claim 15 or 16 wherein said immunologically cross-reactive antibody is a monoclonal antibody. - I l l -

18. The method of claim 17 wherein said immunologically cross-reactive monoclonal antibody is specific for Thermus aquaticus DNA polymerase I.

19. The method of claim 17 wherein the purified thermostable polypeptide has a DNA polymerase activity between 79,500 U/mg protein and

217,600 U/mg protein.

20. A method of purifying a recombinant Thermus flavus thermostable polypeptide having DNA polymerase activity comprising the steps of: a) expressing said thermostable polypeptide in a host cell, said polypeptide having an amino acid sequence encoded by plasmid pTFLRT4, having ATCC Accession No. 69633; b) lysing the cell to create a suspension containing said thermostable polypeptide and host cell proteins and cell debris; c) contacting a soluble portion of said suspension with an antibody that is immunologically cross-reactive with said thermostable polypeptide and under conditions wherein the antibody binds to said thermostable polypeptide to form an antibody-polypeptide complex; d) isolating the antibody-polypeptide complex; and e) separating said thermostable polypeptide from said isolated antibody-polypeptide complex to provide a purified thermostable polypeptide.

21. The method of claim 20 further comprising between steps (b) and (c) the steps of: heating said suspension to denature host cell proteins; and centrifuging said suspension to remove said cell debris and denatured host cell proteins.

22. The method of claim 20 or 21 wherein said immunologically cross-reactive antibody is a monoclonal antibody.

23. The method of claim 22 wherein said immunologically cross-reactive monoclonal antibody is specific for Thermus aquaticus DNA polymerase.

24. The method of claim 22 wherein the purified thermostable polypeptide has a DNA polymerase activity between 79,500 U/mg protein and 217,600 U/mg protein.