Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-26
2001-08-07
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S091200, C435S091500, C435S006120
Reexamination Certificate
active
06271004
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of recombinant genetics.
BACKGROUND OF THE INVENTION
Reverse transcriptase has been widely used in reverse transcription reactions of RNA to DNA. However, a reverse transcriptase has RNase H activity besides its RNA-dependent DNA polymerase activities and it is not stable at elevated temperature.
Chaperonins as thermal stabilizing factors. Chaperonins are a group of molecular chaperones and are a subset of the heat shock proteins (HSPs), whose members are widely distributed from prokaryotes to eukaryotes and first came to light because of their specific induction during the cellular response of all organisms to heat shock. It is now clear that the majorities of those proteins are expressed constitutively and abundantly in the absence of any stress, and genetic studies show that many of them are essential for cell viability under normal growth conditions (1,2,3). According to structure, molecular mass, and function, HSPs have previously been divided into several families (3); the stress-70 protein family, the stress-90 protein family, and the chaperonin family. In hyperthermophilic archaea which can grow above 80° C., chaperonin play an essential role in hindering protein denaturation (4,5,6). Many of the HSPs are or may be involved in de novo protein folding and assembling of proteins (2). One particular gene of interest for this invention is the gene encoding the Beta-subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus (7), FIGS.
1
,
2
.
Reverse Transcriptase—a multi functional polypeptide
The reverse transcriptase (RT) of the retrovirus Moloney murine leukemia virus (MMLV) is an essential enzyme involved in its life cycle and is commonly used as a reagent in modern molecular biology. Like other retroviral reverse transcriptases, the 76 kDa polypeptide from MMLV contains two separable activities: a DNA polymerase function encoded within its N-terminal portion and a ribonuclease H (RNAseH) function encoded in its C -terminus (FIGS.
3
)(
8
-
9
). The combination of these two activities allows RT to convert single-stranded RNA into the double-stranded DNA needed for integration into the host chromosome. The RNaseH domain is responsible for the hydrolysis of the RNA portion of RNA-DNA hydrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site (10). Today high resolution structures of the three members of this ubiquitous family have been determined by X-ray crystallography (11-14). The RT DNA polymerase activity is responsible for transcribing viral RNA into double-stranded DNA (15). RT is used extensively in recombinant DNA technology to synthesize cDNA from mRNA. One major problem with cDNA synthesis is to gain full-length cDNA from the RNA template, due to the tree dimension structure of mRNA molecules (16). One solution to this problem would be to increase the temperature during the cDNA synthesis to unfolded the different tree dimension structures of mRNA molecules. However since the RT DNA polymerase activity are decreasing relative fast at temperature above 65° C., due to thermal denaturation of the reverse transcriptase, this solution has not been possible so far. Another potential problem is the RNAseH activity of RT polypeptide. The mRNA poly(A)-oligo(dT) hybrid used as a primer for first-strand cDNA synthesis is degraded by RT RNAseH. Thus, at the outset of cDNA synthesis, a competition is established between RNaseH mediated deadenylation of mRNA and initiation of DNA synthesis, which reduces the yield of cDNA product (17). To removed the unwanted RNAseH activity the RT polypeptide from MMLV has been genetically changed either by entire removal of RNASEH C-terminal domain of the polypeptide or by point mutating essential amino acid, and thereby reducing the RNAseH activity (18).
BRIEF SUMMARY OF THE INVENTION
The present invention encompasses a surprising solution to the problems described above, by combining the stabilising effect of a thermostable Chaperonin with a reverse transcriptase.
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Display Systems Biotech A/S
Frommer & Lawrence & Haug LLP
Horlick Kenneth R.
Kowalski Thomas J.
Spiegler Alexander H.
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