Isolation and identification of novel polymerases

Details Isolation and identification of novel polymerases Isolation and identification of novel polymerases

1999-09-07

2002-12-10

Prouty, Rebecca E. (Department: 1652)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S194000, C536S023100, C536S024320, C536S024330

Reexamination Certificate

active

06492511

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as polymerases.
BACKGROUND OF THE INVENTION
Thermophilic bacteria have received considerable attention as sources of highly active and thermostable enzymes. Recently, the most extremely thermophilic organotrophic eubacteria presently known have been isolated and characterized. These bacteria, which belong to the genus thermotoga, are fermentative microorganisms metabolizing a variety of carbohydrates (Huber, R. and Stetter, K. O., in Ballows, et al., (Ed.), The Procaryotes, 2nd Ed., Springer-Verlaz, New York, pgs. 3809-3819 (1992)).
In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation of the bacterium
Thermotoga maritima
is described.
T. maritima
is a eubacterium that is strictly anaerobic, rod-shaped, fermentative, hyperthermophilic, and grows between 55° C. and 90° C., with an optimum growth temperature of about 80° C. This eubacterium has been isolated from geothermally heated sea floors in Italy and the Azores.
T. maritima
cells have a sheath-like structure and monotrichous flagellation.
T. maritima
is classified in the eubacterium kingdom by virtue of having murein and fatty acid-containing lipids, diphtheria-toxin-resistant elongation factor 2, an RNA polymerase subunit pattern, and sensitivity to antibiotics.
Since, to date, most organisms identified from the archaeal domain are thermophiles or hyperthermophiles, archaea are also considered a fertile source of thermophilic enzymes.
SUMMARY OF THE INVENTION
The present invention provides polynucleotides and polypeptides encoded thereby which have been identified as polymerase enzymes. In accordance with one aspect of the present invention, there is provided novel enzymes, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding enzymes of the present invention including mRNAs, DNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such enzymes.
In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polypeptides expressed by the DNA in SEQ ID Nos: 1, 3, 5, 7, 9, 11.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding an enzyme of the present invention, under conditions promoting expression of said enzyme and subsequent recovery of said enzyme.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotide encoding such enzymes for polymerizing DNA.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other organisms.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.


REFERENCES:
patent: H1531 (1996-05-01), Blumentals et al.
patent: 5948666 (1999-09-01), Callen et al.

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