Cloned genes encoding reverse transcriptase lacking RNase H activity

• US 5,405,776 A
• Filed: 01/24/1992
• Issued: 04/11/1995
• Est. Priority Date: 01/13/1988
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Kotewicz, Michael L., Gerard, Gary F.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/825,260
• Time in Patent Office: 1,173 Days
• Field of Search: 435/69.1, 435/69.7, 435/172.3, 435/193, 435/194, 435/252.3, 435/252.33, 435/320.1, 536/23.2, 536/23.4
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit

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.

Claims

1. An isolated DNA compound comprising a DNA sequence encoding a polypeptide having DNA polymerase activity and substantially no RNase H activity wherein said polypeptide may be used for the preparation of full length cDNA without significant degradation of the mRNA template during first strand synthesis wherein said DNA compound is derived from an organism selected from the group consisting of a retrovirus, yeast, Neurospora, Drosophila, primates and rodents.

2. The DNA compound of claim 1, wherein said organism is a retrovirus selected from the group consisting of Moloney murine leukemia virus (M-MLV), human T-cell leukemia virus type I (HTLV-I), bovine leukemia virus (BLV), Rous sarcoma virus (RSV)and human immunodeficiency virus (HIV).

3. The DNA compound of claim 1, wherein said organism is M-MLV, comprising the following DNA sequence:

##STR3## or the degenerate variants thereof.

4. The DNA compound of claim 1, wherein said organism is M-MLV, comprising the following DNA sequence:

##STR4##

6. A vector containing the DNA compound of claim 1 or 5.

7. The vector of claim 6 designated pRTdEcoRV-C which has been deposited at the American Type Culture Collection, Rockville Md. under terms of the Budapest Treaty and given accession number 67555.

8. A host cell transformed with the vector of claim 6.

9. A method of producing reverse transcriptase having DNA polymerase activity and substantially no RNase H activity comprising culturing the transformed host of claim 8 under conditions which produce reverse transcriptase, and isolating the reverse transcriptase so produced.

5. An isolated DNA compound comprising a DNA sequence which encodes a fusion protein which comprises reverse transcriptase having DNA polymerase activity and substantially no RNase H activity and a second protein comprising a hydrophobic leader peptide or a stabilizing peptide.