U.S. patent application number 10/970280 was filed with the patent office on 2006-01-05 for cloned genes encoding reverse transcriptase lacking rnase h activity.
Invention is credited to Gary Floyd Gerard, Michael Leslie Kotewicz.
Application Number | 20060003341 10/970280 |
Document ID | / |
Family ID | 22503881 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003341 |
Kind Code |
A1 |
Kotewicz; Michael Leslie ;
et al. |
January 5, 2006 |
Cloned genes encoding reverse transcriptase lacking RNase H
activity
Abstract
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 vectors containing
the gene and hosts transformed with the vectors of the invention.
The invention, also relates to a method of producing reverse
transcriptase having DNA polymerase activity and substantially no
RNase H activity by expressing the reverse transcriptase genes of
the present invention in a host. The invention also relates to a
method of producing cDNA from mRNA, using the reverse transcriptase
of the invention. The invention also relates to a kit for the
preparation of cDNA from mRNA comprising the reverse transcriptase
of the invention.
Inventors: |
Kotewicz; Michael Leslie;
(Columbia, MD) ; Gerard; Gary Floyd; (Frederick,
MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Family ID: |
22503881 |
Appl. No.: |
10/970280 |
Filed: |
October 22, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10024131 |
Dec 21, 2001 |
|
|
|
10970280 |
Oct 22, 2004 |
|
|
|
09220329 |
Dec 24, 1998 |
6589768 |
|
|
10024131 |
Dec 21, 2001 |
|
|
|
08798458 |
Feb 10, 1997 |
6063608 |
|
|
09220329 |
Dec 24, 1998 |
|
|
|
08614260 |
Mar 12, 1996 |
5668005 |
|
|
08798458 |
Feb 10, 1997 |
|
|
|
08404907 |
Mar 15, 1995 |
|
|
|
08614260 |
Mar 12, 1996 |
|
|
|
07825260 |
Jan 24, 1992 |
5405776 |
|
|
08404907 |
Mar 15, 1995 |
|
|
|
07671156 |
Mar 18, 1991 |
5244797 |
|
|
07825260 |
Jan 24, 1992 |
|
|
|
07143396 |
Jan 13, 1988 |
|
|
|
07671156 |
Mar 18, 1991 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Y 207/07049 20130101; C12N 9/1276 20130101; Y10S 435/81
20130101; C12Q 1/68 20130101; Y10S 435/975 20130101; C12Q 1/68
20130101; C12Q 2521/107 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A gene which encodes reverse transcriptase having DNA polymerase
activity and substantially no RNase H activity.
2-23. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention is in the field of recombinant genetics.
BACKGROUND OF THE INVENTION
[0002] 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, H. L., et al., Gene
35:249-258 (1985); and Roth, H. 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.
[0003] In the retrovirus life cycle, the RT DNA polymerase activity
is responsible for transcribing viral RNA into double-stranded DNA.
Varmus, H. (1982), in 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.
[0004] 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).
[0005] 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 degrades 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 RHase 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.
[0006] 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. 1, DNA Enzymes, CRC Press,
Inc., Coca 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.
[0007] Computer analysis of the amino acid sequences from the
putative gene products of retroviral pol 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.
[0008] 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., at 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).
[0009] There has been no direct scientific evidence that amino acid
residues involved catalytically or structurally in the RNase H
activity of reverse transcriptase could be altered to eliminate
RNase H activity without affecting the RNA-dependant DNA polymerase
activity of reverse transcriptase. Moreover, there has been no
report of the cloning of RT to give a gene product without RNase
activity.
SUMMARY OF THE INVENTION
[0010] The invention relates to a gene which encodes reverse
transcriptase having DNA polymerase activity and substantially no
RNase H activity.
[0011] The invention also relates to a reverse transcriptase gene
comprising the following DNA sequence: TABLE-US-00001 1078 ATG ACC
CTA AAT ATA GAA GAT GAG CAT CGG CTA CAT GAG ACC TCA AAA GAG CCA GAT
GTT MET Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
Glu Pro Asp Val 1138 TCT CTA GGG TCC ACA TGG CTG TCT GAT TTT CCT
CAG GCC TGG GCG GAA ACC GGG GGC ATG Ser Leu Gly Ser Thr Trp Leu Ser
Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly MET 1198 GGA CTG GCA
GTT CGC CAA GCT CCT CTG ATC ATA CCT CTG AAA GCA ACC TCT ACC CCC GTG
Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser
Thr Pro Val 1258 TCC ATA AAA CAA TAC CCC ATG TCA CAA GAA GCC AGA
CTG GGG ATC AAG CCC CAC ATA CAG Ser Ile Lys Gln Typ Pro MET Ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln 1318 AGA CTG TTG GAC
CAG GGA ATA CTG GTA CCC TGC CAG TCC CCC TGG AAC ACG CCC CTG CTA Arg
Leu Leu Asp Gln Gly Ile Leu Val Pro Cyn Gln Ser Pro Trp Asn Thr Pro
Leu Leu 1378 CCC GTT AAG AAA CCA GGG ACT AAT GAT TAT AGG CCT GTC
CAG GAT CTG AGA GAA GTC AAC Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr
Arg Pro Val Gln Asp Leu Arg Glu Val Asn 1438 AAG CGG GTG GAA GAC
ATC CAC CCC ACC GTG CCC AAC CCT TAC AAC CTC TTG AGC GGG CTC Lys Arg
Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
Leu 1498 CCA CCG TCC CAC CAG TGG TAC ACT GTG CTT GAT TTA AAG GAT
GCC TTT TTC TGC CTG AGA Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg 1558 CTC CAC CCC ACC AGT CAG
CCT CTC TTC GCC TTT GAG TGG AGA GAT CCA GAG ATG GGA ATC Leu His Pro
Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu MET Gly Ile
1618 TCA GGA CAA TTG ACC TGG ACC AGA CTC CCA CAG GGT TTC AAA AAC
AGT CCC ACC CTG TTT Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
Phe Lys Asn Ser Pro Thr Leu Phe 1678 GAT GAG GCA CTG CAC AGA GAC
CTA GCA GAC TTC CGG ATC CAG CAC CCA GAC TTG ATC CTG Asp Glu Ala Leu
His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu
1738 CTA CAG TAC GTG GAT GAC TTA CTG CTG GCC GCC ACT TCT GAG CTA
GAC TGC CAA CAA GGT Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr
Ser Glu Leu Asp Cys Gln Gln Gly 1798 ACT CGG GCC CTG TTA CAA ACC
CTA GGG AAC CTC GGG TAT CGG GCC TCG GCC AAG AAA GCC Thr Arg Ala Leu
Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala
1858 CAA ATT TGC CAG AAA CAG GTC AAG TAT CTG GGG TAT CTT CTA AAA
GAG GGT CAG AGA TGG Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
Leu Leu Lys Glu Gly Gln Arg Trp 1918 CTG ACT GAG GCC AGA AAA GAG
ACT GTG ATG GGG CAG CCT ACT CCG AAG ACC CCT CGA CAA Leu Thr Glu Ala
Arg Lys Glu Thr Val MET Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln
1978 CTA AGG GAG TTC CTA GGG ACG GCA GGC TTC TGT CGC CTC TGG ATC
CCT GGG TTT GCA GAA Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg
Leu Trp Ile Pro Gly Phe Ala Glu 2038 ATG GCA GCC CCC TTG TAC CCT
CTC ACC AAA ACG GGG ACT CTG TTT AAT TGG GGC CCA GAC MET Ala Ala Pro
Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp
2098 CAA CAA AAG GCC TAT CAA GAA ATC AAG CAA GCT CTT CTA ACT GCC
CCA GCC CTG GGG TTG Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
Leu Thr Ala Pro Ala Leu Gly Leu 2158 CCA GAT TTG ACT AAG CCC TTT
GAA CTC TTT GTC GAC GAG AAG CAG GGC TAC GCC AAA GGT Pro Asp Leu Thr
Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly
2218 GTC CTA ACG CAA AAA CTG GGA CCT TGG CGT CGG CCG GTG GCC TAC
CTG TCC AAA AAG CTA Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu 2278 GAC CCA GTA GCA GCT GGG TGG
CCC CCT TGC CTA CGG ATG GTA GCA GCC ATT GCC GTA CTG Asp Pro Val Ala
Ala Gly Trp Pro Pro Cys Leu Arg MET Val Ala Ala Ile Ala Val Leu
2338 ACA AAG GAT GCA GGC AAG CTA ACC ATG GGA CAG CCA CTA GTC ATT
CTG GCC CCC CAT GCA Thr Lys Asp Ala Gly Lys Leu Thr MET Gly Gln Pro
Leu Val Ile Leu Ala Pro His Ala 2398 GTA GAG GCA CTA GTC AAA CAA
CCC CCC GAC CGC TGG CTT TCC AAC GCC CGG ATG ACT CAC Val Glu Ala Leu
Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg MET Thr His
2458 TAT CAG GCC TTG CTT TTG GAC ACG GAC CGG GTC CAG TTC GGA CCG
GTG GTA GCC CTG AAC Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gln
Phe Gly Pro Val Val Ala Leu Asn 2512 CCG GCT ACG CTG CTC CCA CTG
CCT GAG GAA GGG CTG CAA CAC AAC TGC CTT GAT Pro Ala Thr Leu Leu Pro
Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu Asp
or the degenerate variants thereof.
[0012] The invention also relates to a reverse transcriptase gene
comprising the following DNA sequence: TABLE-US-00002 1078 ATG ACC
CTA AAT ATA GAA GAT GAG CAT CGG CTA CAT GAG ACC TCA AAA GAG CCA GAT
GTT MET Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
Glu Pro Asp Val 1138 TCT CTA GGG TCC ACA TGG CTG TCT GAT TTT CCT
CAG GCC TGG GCG GAA ACC GGG GGC ATG Ser Leu Gly Ser Thr Trp Leu Ser
Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly MET 1198 GGA CTG GCA
GTT CGC CAA GCT CCT CTG ATC ATA CCT CTG AAA GCA ACC TCT ACC CCC GTG
Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser
Thr Pro Val 1258 TCC ATA AAA CAA TAC CCC ATG TCA CAA GAA GCC AGA
CTG GGG ATC AAG CCC CAC ATA CAG Ser Ile Lys Gln Tyr Pro MET ser Gln
Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln 1318 AGA CTG TTG GAC
CAG GGA ATA CTG GTA CCC TGC CAG TCC CCC TGG AAC ACG CCC CTG CTA Arg
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro
Leu Leu 1378 CCC GTT AAG AAA CCA GGG ACT AAT GAT TAT AGG CCT GTC
CAG GAT CTG AGA GAA GTC AAC Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr
Arg Pro Val Gln Asp Leu Arg Glu Val Asn 1438 AAG CGG GTG GAA GAC
ATC CAC CCC ACC GTG CCC AAC CCT TAC AAC CTC TTG AGC GGG CTC Lys Arg
Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly
Leu 1498 CCA CCG TCC CAC CAG TGG TAC ACT GTG CTT GAT TTA AAG GAT
GCC TTT TTC TGC CTG AGA Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp
Leu Lys Asp Ala Phe Phe Cys Leu Arg 1558 CTC CAC CCC ACC AGT CAG
CCT CTC TTC GCC TTT GAG TGG AGA GAT CCA GAG ATG GGA ATC Leu His Pro
Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu MET Gly Ile
1618 TCA GGA CAA TTG ACC TGG ACC AGA CTC CCA CAG GGT TTC AAA AAC
AGT CCC ACC CTG TTT Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
Phe Lys Asn Ser Pro Thr Leu Phe 1678 GAT GAG GCA CTG CAC AGA GAC
CTA GCA GAC TTC CGG ATC CAG CAC CCA GAC TTG ATC CTG Asp Glu Ala Leu
His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu
1738 CTA CAG TAC GTG GAT GAC TTA CTG CTG GCC GCC ACT TCT GAG CTA
GAC TGC CAA CAA GGT Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr
Ser Glu Leu Asp Cys Gln Gln Gly 1798 ACT CGG GCC CTG TTA CAA ACC
CTA GGG AAC CTC GGG TAT CGG GCC TCG GCC AAG AAA GCC Thr Arg Ala Leu
Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala
1858 CAA ATT TGC CAG AAA CAG GTC AAG TAT CTG GGG TAT CTT CTA AAA
GAG GGT CAG AGA TGG Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
Leu Leu Lys Glu Gly Gln Arg Trp 1918 CTG ACT GAG GCC AGA AAA GAG
ACT GTG ATG GGG CAG CCT ACT CCG AAG ACC CCT CGA CAA Leu Thr Glu Ala
Arg Lys Glu Thr Val MET Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln
1978 CTA AGG GAG TTC CTA GGG ACG GCA GGC TTC TGT CGC CTC TGG ATC
CCT GGG TTT GCA GAA Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg
Leu Trp Ile Pro Gly Phe Ala Glu 2038 ATG GCA GCC CCC TTG TAC CCT
CTC ACC AAA ACG GGG ACT CTG TTT AAT TGG GGC CCA GAC MET Ala Ala Pro
Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp
2098 CAA CAA AAG GCC TAT CAA GAA ATC AAG CAA GCT CTT CTA ACT GCC
CCA GCC CTG GGG TTG Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
Leu Thr Ala Pro Ala Leu Gly Leu 2158 CCA GAT TTG ACT AAG CCC TTT
GAA CTC TTT GTC GAC GAG AAG CAG GGC TAC GCC AAA GGT Pro Asp Leu Thr
Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly
2218 GTC CTA ACG CAA AAA CTG GGA CCT TGG CGT CGG CGG GTG GCC TAC
CTG TCC AAA AAG CTA Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro
Val Ala Tyr Leu Ser Lys Lys Leu 2278 GAC CCA GTA GCA GCT GGG TGG
CCC CCT TGC CTA CGG ATG GTA GCA GCC ATT GCC GTA CTG Asp Pro Val Ala
Ala Gly Trp Pro Pro Cys Leu Arg MET Val Ala Ala Ile Ala Val Leu
2338 ACA AAG GAT GCA GGC AAG CTA ACC ATG GGA CAG CCA CTA GTC ATT
CTG GCC CCC CAT GCA Thr Lys Asp Ala Gly Lys Leu Thr MET Gly Gln Pro
Leu Val Ile Leu Ala Pro His Ala 2398 GTA GAG GCA CTA GTC AAA CAA
CCC CCC GAC CGC TGG CTT TCC AAC GCC CGG ATG ACT CAC Val Glu Ala Leu
Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg MET Thr His
2458 TAT CAG GCC TTG CTT TTG GAC ACG GAC CGG GTC CAG TTC GGA CCG
GTG GTA GCC CTG AAC Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gln
Phe Gly Pro Val Val Ala Leu Asn 2518 CCG GCT ACG CTG CTC CCA CTG
CCT GAG GAA GGG CTG CAA CAC AAC TGC CTT GAT AAT TCC Pro Ala Thr Leu
Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu Asp Asn Ser
2530 CGC TTA ATT AAT Arg Leu Ile Asn
or the degenerate variants thereof.
[0013] The invention also relates to the vectors containing the
gene of the invention, hosts transformed with the vectors of the
invention, and the reverse transcriptase expressed by the
transformed hosts of the invention.
[0014] The invention also relates to a fusion protein comprising a
polypeptide having RNA-dependent DNA polymerase activity and
substantially no RNase H activity and a second peptide selected
from polypeptide proteins which stabilize the fusion protein and
hydrophobic leader sequences.
[0015] The invention also relates to a method of producing reverse
transcriptase having DNA polymerase activity and substantially no
RNase H activity, comprising culturing transformed hosts of the
invention under conditions which produce reverse transcriptase, and
isolating the reverse transcriptase so produced.
[0016] The invention also relates to a method of preparing cDNA
from mRNA comprising contacting mRNA with a polypeptide having
RNA-dependent DNA polymerase activity and substantially no RNase H
activity, and isolating the cDNA so produced.
[0017] The invention also relates to a kit for the preparation of
cDNA from mRNA comprising a carrier being compartmentalized to
receive in close confinement therein one or more containers,
wherein [0018] (a) a first container contains reverse transcriptase
having DNA polymerase activity and substantially no RNase H
activity; [0019] (b) a second container contains a buffer and the
nucleoside triphosphates; [0020] (c) a third container contains
oligo(dT)primer; and [0021] (d) a fourth container contains control
RNA.
[0022] The invention is related to the discovery that portions of
the RT gene can be deleted to give a deletion mutant having DNA
polymerase activity but no detectable. RNase H activity. This
purified mutant RT lacking RNase H activity can be used to
effectively synthesize cDNA from mRNA.
DESCRIPTION OF THE FIGURES
[0023] FIG 1. This figure depicts the restriction map of plasmid
pRT601. The M-MLV RT gene extends from position 1,019 to 3,070.
[0024] FIG. 2. This figure depicts schematic representation of
pRT601 and related plasmids, and the enzymatic activities and
predicted structure of the M-MLV RT protein coded by each
plasmid.
[0025] FIG. 3. This figure depicts an SDS-polyacrylamide gel of
M-MLV RT. pRTdEcoRV-C RT (A) and pRT601 RT (B) (3 .mu.g of each)
were run on an SDS 10% polyacrylamide gel (Laemmli, U. K., Nature
227:680-685 (1970)). The gel was stained with Coomassie blue. Lane
M contained Kr standards.
[0026] FIG. 4. This figure depicts an autoradiogram of
.sup.32P-labeled cDNA synthesized from 6.2 kb RNA (Materials and
Methods) by pRTdEcoRV-C RT (A) or pRT601 RT (B). A 1 kb ladder was
used as a standard (C). Electrophoresis was performed on an
alkaline 1.4% agarose gel (McDonnel, M. W., et al., J. Mol. Biol.
110:119-146 (1977)).
[0027] FIG. 5. This figure depicts an autoradiogram of
.sup.32P-labeled 2.3 kb poly(A)-tailed RNA after oligo(dT)-primed
cDNA synthesis catalyzed by pRTdEcoRV-C-RT or pRT601 RT. Aliquots
were removed from reaction mixtures containing no enzyme (-E) or
200 units of RT at the times indicated (in min). The minus enzyme
control was incubated for 60 min. Samples were electrophoresed as
described in Materials and Methods. A 1 kb ladder was used as
marker (M).
[0028] FIG. 6. This figure depicts the DNA sequence which encodes
reverse transcriptase having DNA polymerase activity and
substantially no RNase H activity. Also shown is the corresponding
amino acid sequence.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] The invention relates to the production of reverse
transcriptase having DNA polymerase activity and substantially no
RNase H activity, using recombinant DNA techniques.
[0030] Recombinant plasmids constructed as described herein provide
reverse transcriptase for use in recombinant. DNA technology to
synthesize cDNA from mRNA without the problem associated with RNase
H activity which degrades mRNA template during first-strand
synthesis.
[0031] By the terms "substantially no RNase H activity" is intended
reverse transcriptase purified to near homogeneity and having an
RNase H activity of less than 0.001 pmoles [.sup.3H](A).sub.n
solubilized per .mu.g protein with a [.sup.3H](A).sub.n+(dT).sub.n
substrate in which the [.sup.3H](A).sub.n has a specific
radioactivity of 2,200 cpm/pmole. RNase H activities of this
specific activity or less allows the preparation of cDNA without
significant degradation of the mRNA template during first-strand
synthesis.
[0032] By the terms "degenerate variants" is intended cloned genes
having variations of DNA sequence, but which encode the same amino
acid sequence.
[0033] The reverse transcriptase gene (or the genetic information
contained therein) can be obtained from a number of different
sources. For instance, the gene may be obtained from eukaryotic
cells which are infected with retrovirus, or from a number of
plasmids which contain either a portion of or the entire retrovirus
genome. In, addition, messenger RNA-like RNA which contains the RT
gene can be obtained from retroviruses. Examples of sources for RT
include, but are not limited to, Moloney murine leukemia virus
(M-MLV); human T-cell leukemia virus type I (HTLV-I); bovine
leukemia virus (BLV); Rous Sarcoma virus (RSV); human
immunodeficiency virus (HIV); yeast, including Saccharomyces,
Neurospora, Drosophila; primates; and rodents. See, for example,
Weiss et al., U.S. Pat. No. 4,663,290 (1987); Gerard, G. R., DNA
5:271-279 (1986); Kotewicz, M. L., et al., Gene 35:249-258 (198051
Tanese, N., et al., Proc. Natl. Acad. Sci. (USA) 82:4944-4948
(1985) Rothe M. J., et al. J. Biol. Chem. 260:9326-9335 (1985).
Michel, F., et al., Nature 316:641-643 (1985); Akins, R. A., et
al., Cell 47: 505-516 (1986), EMBO J. 4:1267-1275 (1985); and
Fawcett, D. F., Cell 47:1007-1015 (1986).
[0034] RT proviral DNA can be isolated using standard isolation
techniques. The DNA is cleaved into linear fragments, any one of
which may contain the genes which encode RT. Such fragmentation can
be achieved using enzymes which digest or cleave DNA, such as
restriction enzymes which cleave DNA as specific base sequences.
After the linear DNA fragments are generated, they are separated
according to size by standard techniques. Such recombinant DNA
techniques may be performed as described by Maniatis, T., et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1982).
[0035] Identification of the DNA fragment which contains the gene
may be accomplished in a number of ways. For example, it is
possible to sequence the DNA fragments (Maxam and Gilbert, Methods
in Enzymology 64:499. (1980); Messing, J., Meth. in Enz. 101C:20
(1983)) to identify which fragment contains the reverse
transcriptase gene. Alternatively, hybridization techniques
(Southern, J. Mol. Biol. 98:503 (1975)) using a labeled (e.g.,
radioactively labeled) DNA probe may be employed.
[0036] The fractions containing the desired DNA are pooled, ligated
into a suitable vector, and used to transform a host. Screening for
transformed hosts containing the RT gene may be accomplished by,
for example, the method disclosed by Gerard et al., Biochem.
13:1632-1641 (1974) or by Gerard et al., J. Virol. 15:785-797
(1975). Alternatively, clones containing reverse transcriptase may
be identified by hybridization with complementary labeled DNA.
[0037] An alternative to isolating the reverse transcriptase gene
from a retroviral proviral DNA is to make cDNA to the mRNA-like RNA
which codes for reverse transcriptase. To this end, mRNA-like RNA
coding for reverse transcriptase is isolated from retrovirus. By
standard techniques, the isolated mRNA is then converted into cDNA
using reverse transcriptase. The cDNA can then be inserted into a
plasmid vector in a conventional manner.
[0038] The choice of a suitable vector depends on a number of
considerations known to one of ordinary skill in the art, such as
the size of the fragment, nature of the host, number and position
of restriction sites desired, and the selection marker and markers
desired. Such vectors may include replicon and control sequences
from species compatible with a host cell (see Maniatis et al.,
supra). Expression of the RT genes may also be placed under control
of other regulatory sequences homologous or heterologous to the
host organism in its untransformed state. For example,
lactose-dependent E. coli chromosomal DNA comprises a lactose or
lac operon which mediates lactose utilization by elaborating the
enzyme .beta.-galactosidase. The lac control elements may be
obtained from bacteriophage lambda plac 5, which is infectious for
E. coli. The lac promoter-operator system can be induced by
IPTG.
[0039] Other promoter-operator systems or portions thereof can be
employed as well. For example, galactose, alkaline phosphatase,
tryptophan, xylose, tac, lambda pL, lambda pR and the like can be
used. Once the vector or DNA sequence containing the constructs has
been prepared, the vectors may be introduced into an appropriate
host. Various techniques may be employed such as protoplast fusion,
CaCl.sub.2, calcium phosphate precipitation, electroporation, or
other conventional DNA transfer techniques. The vectors may then be
introduced into a second host by similar transfer methods, and also
by cell to cell transfer methods such it as conjugation. This
cell-to-cell transfer may be accomplished using known techniques
which depend upon the nature of the transferer bacterium, the
recipient bacterium, and the cloning vector used to propagate the
RT DNA. The transfer may require the use of a helper plasmid. See,
for example, Ditta, G., et al., Proc. Natl. Acad. Sci. (USA)
77:7347-7351 (1980).
[0040] RT genes having DNA polymerase activity and substantially no
RNase H activity may be obtained by deletion of
deoxyribonucleotides at the 3' end of the gene which encode the
portion of the polypeptide having RNase H activity. Deletions of
the RT gene may be accomplished by cutting the plasmid at selected
restriction sites within the RT gene and discarding the excised
fragment. Further deletion of consecutive deoxyribonucleotides may
be accomplished by treating the fragment with an exonuclease. The
DNA ends may then be joined in such a way that the translation
reading frame of the gene is maintained. The plasmid thus obtained
may then be used to transform hosts which may then be screened for
altered RT activity. RT RNase H activity may be assayed according
to Gerard et al., J. Virol. 15:785-79.7 (1975). DNA polymerase
activity may be assayed according to Gerard et al,, Biochem.
13:1632-1641 (1974). Clones having DNA polymerase activity and
substantially no RNase H activity may be used to prepare RT with
altered activity.
[0041] According to these methods, the portion of the RT gene
derived from M-MLV which encodes DNA polymerase was localized to
about 1495 base pairs (about 1018 to about 2512) as shown in FIG.
6. The protein expressed by this gene has about 503 amino acids
(FIG. 6). This protein has DNA polymerase activity and
substantially no RNase H activity.
[0042] The invention also relates to fusion proteins which comprise
the reverse transcriptase of the invention. Such fusion proteins
may comprise, for example, a carrier protein which has a leader
sequence of hydrophobic amino acids at the amino terminus of the
reverse transcriptase. This carrier protein is normally excreted
through the membrane of the cell within which it is made. By
cleavage of the hydrophobic leader sequence during excretion, a
means is provided for producing reverse transcriptase which can be
recovered either from the periplasmic space or the medium in which
the bacterium is grown. The use of such a carrier protein allows
isolation of reverse transcriptase without contamination by other
proteins within the bacterium, and achieves production of a form of
reverse transcriptase having greater stability by avoiding the
enzymes within the bacterial cell which degrade foreign proteins.
The DNA and amino acid sequences for such hydrophobic leader
sequences, as well as methods of preparing such fusion proteins are
taught, for example, by Gilbert et al., U.S. Pat. No. 4,411,994
(1983).
[0043] It is also possible to prepare fusion proteins comprising
the reverse transcriptase of the invention which is substituted at
the amino or carboxy termini with polypeptides which stabilize or
change the solubility of the reverse transcriptase. An
amino-terminal gene fusion which encodes reverse transcriptase,
having both DNA polymerase and RNase activity, and IME taught, for
example, by Tanese, N. et al., Proc. Nat'l. Acad. Sci. 82:4944-4948
(1985). A carboxy-terminal gene fusion which encodes reverse
transcriptase and part of the plasmid pBR322 tet gene is taught,
for example, by Kotewicz, M., et al., Gene 35:249-258 (19851 and
Gerard, G., DNA 5:271-279 (1986).
[0044] The transformed hosts of the invention may be cultured under
protein producing conditions according to any of the methods which
are known to those skilled in the art.
[0045] The reverse transcriptase having DNA-polymerase activity and
substantially no RNase activity may be isolated according to
conventional methods known to those skilled in the art. For
example, the cells may be collected by centrifugation, washed with
suitable buffers, lysed, and the reverse transcriptase isolated by
column chromatography, for example, on DEAE-cellulose,
phosphbcellulose (see Kotewicz et. al., Gene 35:249-258. (198.5) or
other standard isolation and identification techniques using, for
example, polyribocytidylic acid-agarose, or hydroxylapatite or by
electrophoresis or immunoprecipitation.
[0046] The reverse transcriptase so produced may be used to prepare
cDNA from RNA by, for example, hybridizing an oligo(dT) primer or
other complementary primers with the mRNA. The synthesis of a
complete cDNA may be accomplished by adding the reverse
transcriptase and all four deoxynucleoside triphosphates. Using the
reverse transcriptase produced by the present invention allows for
the preparation of cDNA from mRNA without concomitant degradation
of the mRNA which results in incomplete cDNA synthesis. The
resulting RNA-DNA hybrid may be treated, for example, with alkali
or RNase H to selectively hydrolyze the RNA to leave cDNA which may
be converted to double-stranded form in a second DNA reaction
catalyzed by reverse transcriptase or other DNA polymerase. See
Old, R. W., et al., Principals of Gene Manipulation, second
edition, Studies in Microbiology, Vol. 2, University of California
Press, p. 26 (1981).
[0047] The reverse transcriptase of the invention is ideally suited
for incorporation into a kit for the preparation of cDNA from RNA.
Such a kit may comprise a carrier means being compartmentalized to
receive a close confinement therein, one or more container means,
such, as vials, tubes, and the like, each of said container means
comprising one of the separate elements of the method used to
prepare cDNA from RNA. For example, there may be provided a
container means containing reverse transcriptase having DNA
polymerase activity and substantially no RNase H activity, in
solution. Further container means may contain suitable buffers,
substrates for DNA synthesis such as the deoxynucleoside
triphosphate, oligo(dT) primer, and control RNA for use as a
standard.
[0048] The reverse transcriptase may be present in the solution at
a concentration of 200 units/ml to 400 units/ml. The
deoxynucleoside triphosphases may be present either in lyophilized
form or as part of a buffer at a concentration of 0.5 mM to 2 mM. A
suitable buffer, present at 5 times the final concentration of use,
includes 250 mM Tris-HCl (pH 7.5 to 8.3), 375 mM KCl, 15 mM
MgCl.sub.2, and 50 mM dithiothreitol. The oligo (dT) may be present
at a concentration of 5 .mu.g/ml to 20 .mu.g/ml. Control RNA, such
as 2.3 kb control RNA, may be present at a concentration of 10
.mu.g/ml to 20 .mu.g/ml.
[0049] The following examples are illustrative but not limiting of
the methods and compositions of the present invention. Any suitable
modifications and adaptations which are obvious to one of ordinary
skill in the art in recombinant DNA techniques are within the
spirit and scope of the present invention.
EXAMPLES
Materials and Methods
Plasmids and Bacterial Strains
[0050] For deletion analysis of RT, a clone of M-MLV RT was
constructed to overproduce stable RT in Escherichia coli, pRT601
(FIG. 1). Gerard, G. F., et al., DNA 5:271-279 (1986). It is a
pBR322-replicon containing the strong lambda leftward promoter, pL,
and the ribosome binding site of the lambda cII gene. (Higher copy
number derivatives of pER322, such as pUC plasmids, can also be
used.) The coding sequence for the RT gene was carefully engineered
into this plasmid to produce a protein with the amino terminus of
the viral protein and a carboxy terminus similar to the viral
enzyme. Gerard, G. F., supra.
[0051] Two bacterial strains were used to propagate clones and
express RT: K802 (Maniatis, T., et al., (1982), Molecular Cloning:
A Laboratory Handbook, pp. 504-505, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.), made lysogenic for lambda cIindlts857
Sam7, and N4830 (Gottesman, M. E., et al., J. Mol. Biol. 140:57-75
(1980)), which contains a deleted a cryptic lambda prophage
expressing the temperature sensitive cI allele indlts857. Bacteria
were grown in super broth (SB) containing 2% tryptone, 1% yeast
extract, 0.1% NaCl, pH 7.5, and 50 .mu.g/ml ampicillin.
Plasmid Construction
[0052] Standard procedures for plasmid construction were performed
as described previously (Kotewicz, M. L., et al., Gene 35:249-258
(1985); Gerard, G. F., et al., DNA 5:271-279 (1986)).
Temperature Induction of E. coli Carrying pRT601 and Its
Derivatives
[0053] Cultures of, bacteria were grown in SB broth overnight at
32.degree. C. and diluted 1:20 in fresh SB in the morning. The
cells were grown at 32.degree. C. until the A.sub.590 was 0.8, and
were induced by swirling in a 65.degree. C. water bath until the
temperature reached 42.degree. C. Induction was continued for 30
minutes in a shaking water bath at 42.degree. C., and then the
cultures were incubated at 37.degree. C. and grown an additional 30
minutes.
Preparation of Cell Extracts
[0054] Unless otherwise noted, all operations were performed at
4.degree. C. Pelleted cells from one ml of culture were washed,
lysed, and centrifuged as described previously (Kotewicz, M. L.,
supra). Supernatants were removed and assayed for RNase H and DNA
polymerase activity.
Enzymes Assays
[0055] RT DNA polymerase activity in extracts was assayed
specifically by using
poly(2'-O-methylcytidylate).oligo-deoxyguanylate
[(Cm).sub.n.(dG).sub.12-18] (Gerard, G. F., et al., DNA 5:271-279
(1986), eliminating interference from cellular DNA polymerases. To
establish DNA polymerase specific activities of purified RT
preparations, activity was assayed with (A).sub.n.(dT).sub.12-18
(Houts, G. E., et al., J. Virol. 29:517-522 (1979) as described by
Gerard, G. F., et al., DNA 5:271-279 (1986). One unit of DNA
polymerase activity is the amount of enzyme that incorporates one
nmole of deoxynucleoside monophosphate into acid insoluble product
at 37.degree. C. in 10 min.
[0056] RNase H activity in crude extracts and purified enzyme was
assayed in reaction mixtures (50 .mu.l) containing 50 mM Tris-HCl
(pH 8.3), 2 mM MnCl.sub.2, 1 mM dithiothreitol, and
[.sup.3H](A).sub.n.(dT).sub.n (5 .mu.M [.sup.3H](A).sub.n, 35
cpm/p-mole; 20 .mu.M (dT).sub.n). Reactions were incubated at
37.degree. C. for 20 min and were stopped by adding 10 .mu.l of
tRNA (1 mg/ml and 20 .mu.l of cold 50% TCA. After 10 minutes on
ice, the mixture was centrifuged for 10 minutes in an Eppendorf
centrifuge. Forty .mu.l of the supernatant was counted in aqueous
scintillant. One unit of RNase H activity is the amount of enzyme
required to solubilize one mole of [.sup.3H](A).sub.n in
[.sup.3H](A).sub.n.(dT).sub.n in 10 min at 37.degree. C.
Synthesis of Poly(A)-Tailed RNA
[0057] Synthetic 2.3 kb and 6.2 kb RNAs containing a 19 nucleotide
poly(A) tail at the 3' end-were synthesized with T7 RNA polymerase
from Xac I-cut pJD2.3- and Hind III-cut pHL3X, respectively.
Reaction mixtures (0.3 ml) contained 40 mM Tris-HCl (pH 8.0), 8 mM
MgCl.sub.2, 2 mM spermidine-HCl, 5-mM dithiothreitol, 0.4 mM each
of CTP, UTP, GTP, and ATP, 20 .mu.g/ml DNA, and 2,000 units/ml T7
RNA polymerase. Uniformly labeled RNA was synthesized with all four
[.alpha.-.sup.32P]NTPs, each at 0.4 mM and 250 cpm/pmole. After 1
hr incubation at 37.degree. C., the RNA product was phenol
extracted, ethanol precipitated, and purified by
oligo(dT)-cellulose chromatography to ensure the presence of a
poly(A) tail.
Conditions for cDNA Synthesis
[0058] When assessing the effect of cDNA synthesis upon the
integrity of template RNA, reaction mixtures (50 .mu.l) contained
50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2, 10 mM
dithiothreitol, 0.5 mM each dATP, dGTP, and dTTP, 0.5 mM
[.sup.3H]dCTP (200 cpm/pmol), 50 .mu.g/ml (dT).sub.12-18, 20
.mu.g/ml 2.3 kb [.sup.32P]labeled RNA, and 4,000 units/ml RT. The
reactions were incubated at 37.degree. C. and duplicate 2.5 .mu.l
aliquots were removed at 0, 1, 5, 10, 30, and 60, min. One aliquot
was precipitated onto glass fiber filters using TCA to determine
the amount of cDNA synthesized, and the other aliquot was prepared
for glyoxal gel analysis. Carmichael, G. G., et al., Method.
Enzymol. 65:380-391 (1980). The glyoxalated RNA was fractionated on
a 1% agarose gel, dried, and autoradiographed. In some cases, 10
units of E. coli RNase H were added to the reaction mixture after
60 min and the incubation continued for 10 more min before aliquots
were taken.
[0059] When measuring the ability of RT to ssynthesize a cDNA copy
of long RNA, reaction mixtures (10 .mu.l) contained the same buffer
and salts, 0.5 mM each of dATP, dGTP, dTTP, and
[.alpha.-.sup.32P]dCTP (600 cpm/pmole), 50 .mu.g/ml actinomycin D,
50 .mu.g/ml (dT).sub.12-18 100 .mu.g/ml 6.2 kb poly(A)-tailed RNA,
and 20,000 units/ml RT. After 1 hr at 37.degree. C., the product in
an aliquot (1 .mu.l) was precipitated with TCA, counted, and the
remaining DNA size fractionated on an alkaline 1.4% agarose gel
according to McDonnel, M. W, et al., J. Mol. Biol. 110:119-146
(1977>.
Purification of RT
[0060] Cells were grown to an A.sub.590 of 3 in TYN and ampicillin
medium (Gerard, G. F., et al., DNA 5:271-279 (1986)) at 30.degree.
C., induced at 43.degree. C. for 45 min, and then grown at
36.degree. C. for 3.5 hr before harvesting. RT was extracted from
100 g of cells as described (Gerard, G. F., supra) with the
following exceptions. RT was precipitated by addition of solid
(NH.sub.4).sub.2SO.sub.4 to 40% saturation. The
(NH.sub.4).sub.2SO.sub.4 pellet was dissolved in 50 ml of 20 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl, 5% glycerol, 1 mM
dithiothreitol, and 0.01% n-octyl-.beta.-D-glucopyranoside, the
suspension was, clarified by centrifugation at 10,000.times.g for
10 min, and the supernatant was desalted on a 320 ml (5.times.16
cm) Sephadex G-25 column run in buffer A (20 mM Tris-HCl, pH 7.5, 1
mM dithiothreitol, 1 mM EDTA, 5% glycerol, 0.01% NP-40) plus 0.1 M
NaCl. After phosphocellulose chromatography, the RT peak was
pooled, diluted with an equal volume of buffer A, and
chromatographed on a 21-ml heparin-agarose column (1.5.times.12 cm)
equilibrated in buffer A plus 0.1 M NaCl. The RT peak from the
heparin-agarose column was chromatographed on a Mono-S HR 5/5
column equilibrated in buffer A (Gerard, G. F., supra).
Results
Construction of Reverse Transcriptase Gene Deletions
[0061] Deletions of the M-MLV RT gene were constructed by cutting
pRT601. (FIG. 1) at selected restriction sites within the RT gene,
discarding the excised fragment, and joining the DNA ends in such a
way that the translation reading frame of the gene was maintained.
pRTdBam-Bam was constructed by deleting the Bam HI fragment between
nucleotide positions 1,654 and 1,960 (FIGS. 1 and 2). Ligation of
the Bam HI half sites at positions 1,654 and 1,960 maintained the
translation reading frame across the site.
[0062] A deletion at the carboxy terminus of M-MLV RT (pRTdEcoRV-C)
was constructed by deleting all of the 3' end of the gene
downstream of the Eco. RV site at position 2,513 (FIGS. 1 and 2).
To construct pRTd-EcoRV-C, a Sca I (position 6,238) to Eco RV
(position 2,513) fragment of pRT601 containing the 5' portion of
the RT gene was ligated to a Sca I-Eco RI fragment derived from
plasmid PBRT (Gerard, G. F., Myra). The 3,211 base pair pBRT Sca
I-Eco RI fragment contained the pBR322 origin of replication and a
universal translation terminator sequence just inside the Eco RI
site. The Eco RI site was repaired with DNA polymerase I Klenow
fragment before ligation.
[0063] The plasmid of pRTdEcoRV-C was deposited in E. coli under
the terms of the Budapest Treaty at the American Type Culture
Collection (ATCC), Rockville, Md., and given accession number
67555.
[0064] pRT603 was constructed as described (Gerard, G. R., supra)
which encodes an RT that contains 73 fewer amino acids than pRT601,
all deleted from the carboxy terminus (FIG. 2).
DNA Polymerase and RNase H Levels in Cells Bearing Deletion
Plasmids
[0065] Alteration of as little as 3 amino acids at the carboxy end
of M-MLV RT can influence markedly the stability of the protein in
E. coli (Gerard, G. F. supra). This must be taken into
consideration in making correlations between cloned RT deletions
and enzymatic activities in E. coli extracts. Both DNA polymerase
and RNase. H activity must be assayed and relative enzyme levels
compared. For example, pRT603 codes for an RT with 73 fewer amino
acids at the carboxy terminus than pRT601 RT (Gerard, G. F., supra;
FIG. 2). The level of DNA polymerase activity in E. coli extracts
of pRT603 RT is reduced 5 fold relative to pRT601. (Gerard, G. F.
supra). However, the DNA polymerase and RNase H specific activities
of purified pRT601 and pRT603 RT are comparable (Table 2). The
reduced DNA polymerase activity in E. coli extracts of pRT603 RT is
not due to a selective effect of the deletion on DNA polymerase
activity, but rather to a reduction in the stability of pRT603 RT
relative to pRT601 RT in cells (t.sub.1/2 of 7 min versus 33 min)
(Gerard, G. F. supra). Therefore, deletions within 70 amino acids
of the RT carboxy terminus do not affect either RNase H or DNA
polymerase activity.
[0066] In contrast, the DNA polymerase activity of pRTdBam-Bam RT
was eliminated totally without affecting RNase H activity (Table 1)
by the deletion of 102 amino acid residues between amino acids 212
and 314 (FIG. 2). Introduction of a more extensive deletion of 180
amino acids at the carboxy end of RT in pRTdEcoRV-C RT (FIG. 2)
yielded extracts with RT DNA polymerase levels unchanged compared
to pRT601 extracts, but with RNase H levels reduced 7.5 fold (Table
1). The residual RHase H activity in pRTdEcoRV-C extracts could be
due to E. coli RNase H, the 5'.fwdarw.3' exonuclease of DNA
polymerase I, or a small amount of residual RT-coded RNase H
activity. To resolve this issue, pRTdEcoRV-C RT was purified and
compared to RT encoded by pRT601.
Purification and Properties of pRTdEcoRV-C RT
[0067] M-MLV reverse transcriptase encoded by pRTdEcoRV-C, pRT601,
and pRT603 were purified as described in Materials and Methods. A
summary of the purification of pRTdEcoRV-C RT is presented in Table
3. Three column steps produced a nearly homogeneous mutant enzyme
with the same DNA polymerase specific activity as pRT601 RT with
the template-primer (Cm).sub.n.(dG).sub.12-18 (Table 2). With
(A).sub.n.(dT).sub.12-18, the mutant enzyme had one-fourth the DNA
polymerase activity of pRT601 RT (Table 2). RNase H activity of
purified pRTdEcoRV-C RT was undetectable using
[.sup.3H](A).sub.n.(dT).sub.n as the substrate. Most RNase H
activity in extracts was eliminated from mutant RT by precipitation
of the enzyme with 40% (NH.sub.4).sub.2SO.sub.4 (Table 3). Under
these conditions, DNA polymerase I remains soluble (Richardson, C.,
et al., J. Biol. Chem. 239:222-230 (1964)), as does most of the
RNase H activity in the extract. As judged by SDS-polyacrylamide
gel electrophoresis, pRTdEcoRV-C RT purified through the Mono-S
column was greater than 90% pure and had a molecular weight of
56,000 (FIG. 3), consistent with the molecular weight (57,000)
predicted by the DNA sequence.
[0068] A number of enzymatic properties of purified pRTdEcoRV-C RT
and pRT601 RT were compared and were found to be similar. These
included half life at 37.degree. C., monovalent and divalent metal
ion optima, fidelity of dNTP incorporation with homopolymer
templates, and insensitivity to stimulation by polyanions. The
abilities of the two enzymes to synthesize heteropolymeric DNA were
also compared. FIG. 4 shows that pRTdEcoRV-C RT catalyzed the
synthesis of full-length cDNA from 6.2 kb RNA more efficiently than
pRT601 RT. The amount of cDNA synthesized from 1 .mu.g of RNA was
0.28 .mu.g (34% full-length) and 0.24 .mu.g (24% full-length) with
pRTdEcoRV-C RT and pRT601 RT, respectively.
[0069] To confirm that pRTdEcoRV-C RT completely lacked, RNase H
activity, the integrity of a uniformly .sup.32P-labeled RNA
template after conversion to hybrid form during RT-catalyzed DNA
synthesis was examined. FIG. 5 shows that with pRT601 RT, the
full-length 2.3 kb RNA template was degraded totally after 5 min of
synthesis In contrast, with pRTdEcoRV-C RT the RNA was intact even
after 60 min. The amount of cDNA synthesized after 60 min from 1
.mu.g of RNA was 0.67 and 0.76 .mu.g with pRT601 and pRTdEcoRV-C
RT, respectively. When 10 units of E. coli RNase H were added to
the pRTdEcoRV-C RT reaction after 60 min of incubation, all of the
RNA was degraded, confirming the hybrid state of the RNA. In
addition, 15 .mu.g (1,200 units) of pRTdEcoRV-C RT solubilized no
radioactivity from a [.sup.3H](A).sub.n.(dT).sub.n substrate in
which the [.sup.3H](A).sub.n had a specific activity of 2,200
cpm/pmole (Materials and Methods).
[0070] Experiments with a frameshift mutant of MLV producing a 47K
RT molecule truncated at the carboxy terminus (Levin, J. G. et al.,
J. Virol. 51:470-478 (1984)) and with antibodies to synthetic
peptides modeled to Rous sarcoma virus pol gene sequences
(Grandgenett, D. et al., J. Biol. Chem. 260:8243-8249 (1985))
suggest the RNase H activity of RT resides within the
amino-terminal portion of the molecule. Conversely, the extensive
homology found between the amino acids of E. coli RNase H and the
153-residue segment at the carboxy-terminal end of M-MLV RT
(Johnson, M. S. et al. Proc Natl. Acad. Sci. (USA) 83:7648-7652
(1986)) suggests the RNase H activity resides within the
carboxy-terminal portion of RT.
[0071] By deleting large segments (100 to 200 codons) of the M-MLV
RT gene, the regions within the RT molecule responsible for DNA
polymerase and RNase H activity have been identified. DNA
polymerase was mapped to the amino half of the molecule, and RNase
H to within 200 amino acids of the carboxy end, confirming the
predictions based upon amino acid homology (Johnson, M. S. et al.,
supra). In this context, the results with one RT clone, pRT603.
(FIG. 2), are of interest. The RT protein encoded by pRT603 is
missing the carboxy half of the 153 amino acid segment of RT
homologous to E. coli RNase H, which includes 20 of 48 homologous
amino acids. Yet, pRT603 RT has normal levels of RNase H activity.
These missing, homologous residues apparently are not required for
catalysis, and might serve a nucleic acid binding or structural
role. Consistent with the latter, a single amino acid change at a
position 12 residues from the carboxy end of E. coli RNase H
produces a 10-fold reduction in RNase H specific activity (Kanaya,
S. et al., J. Bacteriol. 154:1021-1026 (1983)). This reduction
appears to be the result of altered protein conformation (Kanaya,
S. et al., supra).
[0072] If the RT polymerase and nuclease active sites reside on
separate structural domains, it should be possible theoretically to
isolate two separate protein fragments, each with a single
activity. A 24K to 30K proteolytic fragment of RT possessing only
RNase H activity has been isolated (Lai, M. H. T. et al., J. Virol.
25:652-663 (1978); Gerard, G. F., J. Virol. 26:16-28 (1978);
Gerard, G. F., J. Virol. 37:748-754 (1981)), but unfortunately, the
location of the RNase H fragment in the parent RT polypeptide has
not been established, and no analogous DNA polymerase containing
fragment has ever been found. The results presented here show that
of the 684 amino acids in pRT601 RT, residues between amino acid
212 and 314 are required for DNA polymerase activity, and residues
between amino acid 503 and 611 are required for RNase H activity.
They also demonstrate for the first time that the RT DNA polymerase
activity can exist independently of RNase H activity on an RT
protein fragment. Purified pRTdEcoRV-C RT appeared to be totally
devoid of RNase H activity, based upon two sensitive assays, and to
have full DNA polymerase activity. However, these results do not
rule out the possibility that the two active centers share a
portion(s) of the RT molecule.
[0073] Demonstration of a separate structural domain for the RNase
H active center was attempted by constructing two amino-terminal
deletion derivatives of pRT601. The first derivative contained
sequences for the Eco RV site at position 2513 to the .sub.3' end
of the RT gene (see FIG. 2), and the second contained sequences
from an Mco I site at position 2302 to the 3' end of the RT gene.
Unfortunately, neither clone produced detectable RNase H activity
in E. coli crude extracts. Such negative results are difficult to
interpret because the proteins might be unable to fold in an active
form, or might be extremely labile.
[0074] Deletion of the carboxyterminal one-fourth of the M-MLV RT
molecule did not disrupt the ability of the protein to fold in an
active conformation. pRTdEcoRV-C. RT copied heteropolymeric RNA,
more efficiently than intact RT. Yields of cDNA from 1 .mu.g of 2.3
kb and 6.2 kb RNA were 0.76 .mu.g (50% full-length) and 0.28 .mu.g
(34% full-lengthy, respectively. Also, the truncated and intact
enzymes had the same DNA polymerase specific activity with
(Cm).sub.n.(dG).sub.12-18. However, the truncated enzyme copied
(A).sub.n.(dT).sub.12-18, only one fourth as efficiently as the
parent RT. The origin of this difference has not yet been
established. TABLE-US-00003 TABLE 1 DNA polymerase and RNase H
activity in extracts of heat induced E. coli F802 (lambda) bearing
pRT601 or one of its derivatives. DNA polymerase RNase H
Activity.sup.a Activity.sup.b (cpm incorporated/ (cpm solubilized/
Plasmid 2.5 .mu.l extract) 2.5 .mu.l extract) pRT601 10,977 2,020
pRTdBam-Bam 179 1,564 pRTdEcoRV-C 10,038 268 .sup.aReverse
transcriptase DNA polymerase activity was assayed with (Cm).sub.n
(dG).sub.12-18 (Materials and Methods). .sup.bRNase H activity was
assayed with [.sup.3H](A).sub.n (dT).sub.n (Materials and
Methods).
[0075] TABLE-US-00004 TABLE 2 Comparison of activities of purified
RT coded by pRT601, pRT603, and pRTdEcoRV-C DNA polymerase Activity
with (Cm).sub.n (dG).sub.12-18 (A).sub.n (dT).sub.12-18 RNase H
Activity Enzyme (Units/mg) (Units/mg) (Units/mg) pRT601 21,700
350,000 2,670 pRT603 ND.sup.a 230,000 1,100 pRTdEcoRV-C 17,500
81,000 --.sup.b .sup.aND, not determined .sup.bNo activity was
detected
[0076] TABLE-US-00005 TABLE 3 Summary of the purification of
pRTdEcoRV-C RT DNA Polymerase Activity.sup.a RNase H Activity Total
Specific Specific Protein.sup.c Total Activity Yield Total Activity
Yield Fraction (mg) (Units) .times. 10.sup.3 (Units/mg) .times.
10.sup.3 (%) (Units) .times. 10.sup.3 (Units/mg) .times. 10.sup.3
(%) Crude lysate 7,913 255 0.03 100 80 0.01 100 Polymin P
Supernatant 2,735 323 0.12 127 157 0.06 196
(NH.sub.4).sub.2SO.sub.4 pellet 63 168 1.38 66 6.0 0.10 7
Phosphocellulose pool 8.8 167 19.0 66 2.0 0.23 3 Heparin-agarose
pool 6.5 111 17.1 44 --.sup.b -- -- Mono S pool 3.1 55 17.5 22
--.sup.b -- -- .sup.aDNA polymerase activity was assayed with
(Cm).sub.n (dG).sub.12-18 .sup.bNo activity could be detected
.sup.cProtein concentrations were determined using bovine serum
albumin as standard according to Lowry, O. H., et al., J. Biol.
Chem. 239: 222-230 (1964).
* * * * *