Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-12-24
2003-07-08
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S091100, C435S091200, C435S252300, C435S320100, C435S471000
Reexamination Certificate
active
06589768
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of recombinant genetics.
BACKGROUND OF THE INVENTION
Both viral and cloned reverse transcriptase (RT) contain at least two enzymatic activities, DNA polymerase and ribonuclease H (RNase H), that reside on a single polypeptide. Grandgenett, D. P. et al.,
Proc. Natl. Acad. Sci
. (
USA
) 70:230-234 (1973); Moelling, K.,
Virology
62:46-59 (1974); Kotewicz, M. L., et al.,
Gene
35:249-258 (1985); and Roth, M. J., et al.,
J. Biol. Chem.
260:9326-9335 (1985). Little is known about the structure-functional relationship of these two activities, but such knowledge would be important both in understanding retroviral replication and in exploiting the enzyme as a recombinant DNA tool.
In the retrovirus life cycle, the RT DNA polymerase activity is responsible for transcribing viral RNA into double-stranded DNA. Varmus, H. (1982), Weiss, R., et al. (eds.),
RNA Tumor Viruses
, Cold Spring Harbor Laboratory, pp. 410-423. The function of RNase H in replication is less clear, but it is thought to degrade genomic RNA during DNA synthesis to generate oligomeric RNA primers for plus-strand DNA synthesis, and to remove the RNA primers of both minus- and plus-strand DNA. Omer, C. A., et al.,
Cell
30:797-805 (1982); Resnick, R., et al.,
J. Virol.
51:813-821 (1984); Varmus, H. (1985), in Weiss, R., et al. (eds.),
RNA Tumor Viruses
, Cold Spring Harbor Laboratory, pp. 79-80.
The temporal relationship in vivo between DNA polymerization and RNA hydrolysis is not well defined. Furthermore, precisely how the two enzymatic activities are coordinated is not clear. Conditional mutations restricted to either DNA polymerase or RNase H would be invaluable in deciphering the events of retroviral replication. Unfortunately,
conditional viral mutations
in the RT gene
invariably affect both activities
. Lai, M. H. T, et al.,
J. Virol.
27:823-825 (1978); Moelling, K., et al.,
J. Virol.
32:370-378 (1979).
RT is used extensively in recombinant DNA technology to synthesize cDNA from mRNA. One major problem with cDNA synthesis is that the RNase H activity of RT idegrades the mRNA template during first-strand synthesis. The mRNA poly(A)-oligo(dT) hybrid used as a primer for first-strand cDNA synthesis is degraded by RT RNase H. Thus, at the outset of cDNA synthesis, a competition is established between RNase H-mediated deadenylation of mRNA and initiation of DNA synthesis, which reduces the yield of cDNA product. Berger, S. L., et al.,
Biochem.
22:2365-2373 (1983). Furthermore, in some cases, the RNase H causes premature termination of DNA chain growth. Unfortunately, these events eliminate the potential for repeated copying of the RNA template.
Efforts to selectively inactivate RT RNase H with site-specific inhibitors have been unsuccessful (for review, see Gerard, G. F. (1983), in Jacob, S. T., (ed.),
Enzymes of Nucleic Acid Synthesis and Modification
, Vol. I, DNA Enzymes, CRC Press, Inc., Boca Raton, Fla, pp. 1-38). Attempts to physically separate the active centers of RT polymerase and RNase H activity by proteolysis have yielded a proteolytic fragment possessing only RNase-H activity (Lai, M. H. T., et al.,
J. Virol.
25:652-663 (1978); Gerard, G. F.,
J.Virol.
26:16-28 (1978); and Gerard, G. F.,
J. Virol.
37:748-754 (1981)), but no corresponding fragment containing only polymerase activity has been isolated.
Computer analysis of the amino acid sequences from the putative gene products of retroviral vol genes has revealed a 150-residue segment at the carboxyl terminus that is homologous with the ribonuclease H of
E. coli
and a section close to the amino terminus which can be aligned with nonretroviral polymerases. Johnson, M. S., et al.,
Proc. Natl. Acad. Sci
. (
USA
) 83:7648-7652 (1986). Based on these related amino acid sequences, Johnson et al. suggest that ribonuclease H activity should be situated at the carboxyl terminus, and the DNA polymerase activity at the amino terminus.
There have been a number of reports concerning the cloning of genes which encode RT and their expression in hosts. Weiss et al., U.S. Pat. No. 4,663,290 (1987); Gerard, G. F.,
DNA
5:271-279 (1986); Kotewicz, M. L., et al.,
Gene
35:249-258 (1985); Tanese, N., et al.,
Proc. Natl. Acad. Sci
. (
USA
) 82:4944-4948 (1985); and Roth, M. J., et al.,
J. Biol. Chem.
260:9326-9335 (1985).
SUMMARY OF THE INVENTION
The invention relates to a gene which encodes reverse transcriptase having DNA polymerase activity and substantially no RNase H activity.
The invention also relates to a reverse transcriptase gene comprising the following DNA sequence:
. . . . .
ATG ACC CTA AAT ATA GAA GAT GAG CAT CGG CTA CAT GAG ACC TCA AAA GAG CCA GAT GTT
1078
MET Thr Leu Asn Ile Glu Asp Glu his Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val
TCT CTA GGG TCC ACA TGG CTG TCT GAT TTT CCT CAG GCC TGG CGC GAA ACC GGG GGC ATG
1138
Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly MET
GGA CTG GCA GTT CGC CAA GCT CCT CTG ATC ATA CCT CTG AAA GCA ACC TCT ACC CCC GTG
1198
Gly Leu Ala Val Arg Gln Ala Pro Leu ile Ile Pro Leu Gly Ile Lys Pro His Ile Gln
TCC ATA AAA CAA TAC CCC ATG TCA CAA GAA GCC AGA CTG GGG ATC AAG CCC CAC ATA CAG
1258
Ser Ile Lys Gln Tyr Pro MET ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
AGA CTG TTG GAC CAG GGA ATA CTG GTA CCC TGC CAG TCC CCC TGG AAC ACG CCC CTG CTA
1318
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu
CCC GIT AAG AAA CCA GGG ACT AAT GAT TAT AGG CCT GTC CAG GAT CTG AGA GAA GTC AAC
1378
Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn
AAG CGG GTG GAA GAC ATC CAC CCC ACC GTG CCC AAC CCT TAC AAC CTC TTG AGC GGG CTC
1438
Lys Arg val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu
CCA CCG TCC CAC CAG TGG TAC ACT GTG CTT GAT TTA AAG GAT GCC TTT TTC TGC CTG AGA
1498
Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
CTC CAC CCC ACC AGT CAG CCT CTC TTC GCC TTT GAG TGG AGA GAT CCA GAG ATG GGA ATC
1558
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu MET Glu Ile
TCA GGA CAA TTG ACC TGG ACC AGA CTC CCA CAG GGT TTC AAA AAC AGT CCC ACC CTG TTT
1618
Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe
GAT GAG GCA CTG CAC AGA GAC CTA GCA GAC TTC CGG ATC CAG CAC CCA GAC TTG ATC CTG
1678
Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu
CTA CAG TAC GTG GAT GAC TTA CTG CTG GCC GCC ACT TCT GAG CTA GAC TGC CAA CAA GGT
1738
Leu Gln Tyr Val Asp Asp Leu leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
ACT CGG GCC CTG TTA CAA ACC CTA GGG AAC CTC GGG TAT CGG GCC TCG GCC AAC AAA GCC
1798
Thr Arg Ala leu leu Gln Thr Leu Gly Asn Lru Gly Tyr Arg Ala Ser Ala Lys Lys Ala
CAA ATT TGC CAG AAA CAG GTC AAG TAT CTG GGG TAT CTT CTA AAA GAG GGT CAG AGA TGG
1858
Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp
CTG ACT GAG GCC AGA AAA GAG ACT GTG ATG GGG CAG CCT ACT CCG AAG ACC CCT CGA CAA
1918
Leu Thr Glu Ala Arg Lys Glu thr Val MET Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln
CTA AGG GAG TTC CTA GGG ACG GCA GGC TTC TGT CGC CTC TGG ATC CCT GGG TTT GCA GAA
1978
Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
ATG GCA GCC CCC TTG TAC CCT CTC ACC AAA ACG GGG ACT CTG TTT AAT TGG GGC CCA GAC
2038
MET Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Tro Gly Pro Asp
CAA CAA AAG GCC TAT CAA GAA ATC AAG CAA GCT CTT CTA ACT GCC CCA GCC CTG GGG TTG
2098
Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu
CCA GAT TTG ACT AAG CCC TTT GAA CTC TTT GTC GAC GAG AAG CAG GGC TAC GCC AAA GGT
2158
Pro Asp
Gerard Gary Floyd
Kotewicz Michael Leslie
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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