Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-14
2001-03-27
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S471000, C435S320100, C435S252300, C536S023100, C536S024100
Reexamination Certificate
active
06207377
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to recombinant DNA molecules encoding plasmid DNA replication origins in Thermus, as well as to shuttle vectors which contain the same.
Many species of bacteria contain small circular extrachromosomal genetic elements, known as plasmids. Plasmids have been found in a number of bacteria which live in extreme environments, including the thermophiles, which live at high temperatures of more than 55° C. (Munster et al.,
Appl. Environ. Microbiol.
50:1325-1327 (1985); Kristjansson and Stetter, in ‘Thermophilic Bacteria’, Kristjansson, ed., p. 1-18 (1992)). However, most thermophile plasmids remain ‘cryptic’ in that functional genes have not been isolated from them, hence leaving their functional significance speculative (Hishinuma et al.,
J. Gen. Microbiol.
104:193-199 (1978); Eberhard et al.,
Plasmid
6:1-6 (1981); Vasquez et al.,
FEBS Lett.
158:339-342 (1983)). Common genes found in plasmids include those encoding plasmid replication and cellular maintenance, antibiotic resistance, bacteriocin production, sex determination, and other cellular functions (Kornberg and Baker, ‘DNA Replication’, 2
nd
ed. (1991)).
It is often particularly difficult to cultivate thermophilic bacteria within the laboratory. They require high temperatures and often-unknown environmental conditions for acceptable growth (Kristjansson and Stetter, in ‘Thermophilic Bacteria’, Kristjansson, ed., p. 1-18 (1992)). However, with the advent of genetic engineering, it is now possible to clone genes from thermophiles into more easily cultivatable laboratory organisms, such as
E. coli
(Kristjansson,
Trends Biotech.
7:349-353 (1989); Coolbear et al.,
Adv. Biochem. Eng. Biotech.
45:57-98 (1992)). The expression of such genes can be finely controlled within
E. coli.
A
Thermus
-
E. coli
shuttle vector would be desirable if one needs to have the convenience of cloning in
E. coli,
isolation of DNA from
E. coli
for further manipulations and subsequently gene selection and expression in Thermus. Such
Thermus
-
E. coli
shuttle vectors could be used to screen, select and express thermostable proteins in Thermus. Using these vectors, a gene could, for example, be mutated within a mesophile, transferred to a thermophile, and then its encoded protein selected for increased thermostability. In this way, mesophile-thermophile shuttle-vectors can be used to conduct directed evolution, or protein engineering, on desirable gene products.
There is commercial incentive to produce thermostable proteins which are usually more thermostable in denaturing conditions then mesophilic counterparts (Wiegel and Ljungdahl,
CRC Crit. Rev. Biotech.
3:39-108 (1984); Kristjansson,
Trends Biotech.
7:349-353 (1989); Coolbear et al.,
Adv. Biochem. Eng. Biotech.
45:57-98 (1992)). These thermostable enzymes can also be used in a variety of assays, such as PCR, restriction enzyme-mediated PCR, thermo-cycle DNA sequencing and strand-displacement amplification, in which high temperatures are desirable. The shuttle vectors of the present invention should facilitate production of such thermostable proteins.
SUMMARY OF THE INVENTION
The present invention relates to recombinant DNA molecules encoding plasmid DNA replication origins in Thermus, as well as to shuttle vectors which contain the same.
Mesophile-thermophile shuttle vectors require origins of replication (oris) to be genetically maintained and transferred within each bacterial species. To construct appropriate mesophile-thermophile shuttle-vectors, restriction digested thermophile plasmid DNA fragments were ligated into the mesophilic vector pUC19-Km
R
(the thermostable Km
R
marker can be selected at 50°-65° C.). Plasmid pUC19 uses the ColEI ori to replicate within
E. coli
and does not replicate within the plasmid-accepting thermophile
Thermus thermophilus
HB27 or HB27 Pro
−
(Koyama et al.,
J. Bacteriol.
166:338-340 (1986)). We reasoned that the introduction of plasmid DNA from related Thermus species, which contained a complete thermophilic ori, would confer plasmid replication within HB27.
The thermophilic eubacterium Thermus species YS45 (Raven et al.,
Nucl. Acids Res.
21:4397 (1993)) contains two cryptic plasmids, and grows between 55° C. and 70° C. These two Thermus plasmids were named pTsp45S and pTsp45L. These plasmids were digested with a variety of restriction endonucleases to produce fragments that can be cloned into pUC19-derived vectors. A pUC19-derived plasmid with a 4.2-kb XbaI fragment of the small plasmid (pTsp45S, 5.8 kb) of YS45 replicated within HB27. Therefore this XbaI fragment must contain a thermophilic ori. Subsequent deletion analysis revealed that only 2.3 kb (an NheI fragment) within the 4.2 kb was necessary for thermophilic plasmid replication, and that it encodes a replication protein (RepT). The repT gene encodes the 341 amino acid protein, RepT, with predicted molecular mass of 38.2 kDa.
A second Thermus plasmid replication origin from pTsp45L was defined within a 9 kb SphI fragment. This fragment encodes a gene (parA) for plasmid replication and partition. It also contains direct repeats of 5′ RRCTTTTYYY 3′ (SEQ ID NO:1), 5′ RRYTTTG 3′ (SEQ ID NO:2), and an inverted repeat of
5′ TTAACCTTTTTTCAAGAAAAAGAGATAA 3′ (SEQ ID NO:3)
3′ AATTGGAAAAAAGTT CTTTTTCTCTATT 5′
(COMPLEMENT OF SEQ ID NO:3)
The direct repeats and inverted repeats are important for pTsp45L plasmid replication. Deletion of these repeats abolished replication activity in Thermus.
REFERENCES:
patent: 5786174 (1998-07-01), Weber et al.
Munster et al, Appl. Environ. Microbiol., 50:1325-1327 (1985).
Kristjansson et al., ‘Thermophilic Bacteria’, Kristjansson, ed., p. 1-18 (1992).
Hishinuma et al., J. Gen. Microbiol. 103:193-199 (1978).
Eberhard et al., Plasmid 6:1-6 (1981).
Vasquez et al., FEBS Lett. 158:339-342 (1983).
Kristjansson, Trends Biotech. 7:349-353 (1989).
Coolbear et al., Adv. Biochem. Eng. Biotech. 45:57-98 (1992).
Wiegel et al., CRC Crit. Rev. Biotech. 3:39-108 (1984).
Koyama et al., J. Bacteriol. 166:338-340 (1986).
Raven et al., Nucl. acids Res. 21:4397 (1993).
Oshima et al., J. Sys. Bacteriol. 24:102-112 (1974).
Wayne et al., Gene 195:321-328 (1997).
Sambrook et al, ‘Molecular Cloning A Laboratory Manual’, 2nd ed. (1989), pp. 17.29-17.33.
Hartmann et al., J. Bacteriol., 171:2933-2941 (1989).
Maseda et al., FEMS Microbiol. Lett. 128:127-134 (1985).
McMacken et al., DNA Replication (Chapter 39) p. 586-587 inEscherichia coliandSalmonella typhimmarium, Amer. Soc. for Microbiol., Washington DC (1987).
The Tsp45I restriction-modification system is plasmid-borne within its thermophilic host. Wayne et al. Gene. vol. 202:83-88, Dec. 1997.*
Towards a unified grammatical model of sigma 70 and sigma 54 bacterial promoters. Collado-Vides, J. Biochimie vol. 78:251-363, Jun. 1996.*
DNA micorloops and microdomains: A general mechanism for transcription activation by torsional transmission. Travers et al. J. Mol. Biol. vol. 279:1027-1043, Aug. 1998.
Wayne Jay
Xu Shuang-yong
New England Biolabs Inc.
Sandals William
Schwartzman Robert A.
Williams Gregory D.
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