Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-05-22
2001-10-02
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091200, C435S173300, C536S022100, C536S024300
Reexamination Certificate
active
06297007
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to a method for the isolation of biosynthesis genes for antibiotics and other bioactive molecules from complex natural sources such as humus, soil and lichens.
Antibiotics play an important role in man's efforts to combat disease and other economically detrimental effects of microorganisms. Traditionally, antibiotics have been identified by screening microorganisms, especially those found naturally in soil, for their ability to produce an antimicrobial substance. In some cases, the gene or genes responsible for antibiotic synthesis have then been identified and cloned into producer organisms which produce the antibiotic in an unregulated manner for commercial applications. However, it has been estimated that less than 1% of the microorganisms present in soil are culturable. Torsvik et al.,
Appl. Environ. Microbiol.
56: 782-787 (1990). Thus, much of the genetic diversity potentially available in soil microorganisms is unavailable through traditional techniques.
As pathogenic microorganisms become increasingly resistant to known antibiotics, it would, however, be highly desirable to be able to access the reservoir of genetic diversity found in soil, and to facilitate the exploration of new species of antibiotics which may be made by the vast numbers of unculturable organisms found there. It would further be desirable to have access to novel biosynthetic enzymes and the genes encoding such enzymes, which could be used in recombinant organisms for antibiotic production or for in vitro enzymatic synthesis of desirable compounds. Thus, it is an object of the present invention to provide a method and compositions for isolating DNA and DNA fragments encoding enzymes relevant to the production of pharmaceutically active molecules such as antibiotic biosynthesis enzymes.
SUMMARY OF THE INVENTION
We have now identified degenerate primers which hybridize with various classes of antibiotic biosynthesis genes, and have used such primers to amplify fragments of DNA from soil and lichen extracts. Cloning and sequencing of the amplified products showed that these products included a variety of novel and previously uncharacterized antibiotic biosynthesis gene sequences, the products of which have the potential to be active as antibiotics, immunosuppressors, antitumor agents, etc. Thus, antibiotic biosynthesis genes can be recovered from soil by a method in accordance with the present invention comprising the steps of:
(a) combining a soil-derived sample with a pair of amplification primers under conditions suitable for polymerase chain reaction amplification, wherein the primer set is a degenerate primer set selected to hybridize with conserved regions of known antibiotic biosynthetic pathway genes, for example Type I and Type II polyketide synthase genes, isopenicillin N synthase genes, and peptide synthetase genes;
(b) cycling the combined sample through a plurality of amplification cycles to amplify DNA complementary to the primer set; and
(c) isolating the amplified DNA.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, antibiotic biosynthesis genes can be recovered from soil and lichens by a method comprising the steps of:
(a) combining a humic or lichen-derived sample with a pair of amplification primers under conditions suitable for polymerase chain reaction amplification, wherein the primer set is a degenerate primer set selected to hybridize with conserved regions of an antibiotic biosynthesis gene;
(b) cycling the combined sample through a plurality of amplification cycles to amplify DNA complementary to the primer set; and
(c) isolating the amplified DNA.
As used in the specification and claims of this application, the term “humic or lichen-derived sample” encompasses any sample containing the DNA found in lichens or in samples of humic materials including soil, mud, peat moss, marine sediments, and effluvia from hot springs and thermal vents in accessible form for amplification, substantially without alteration of the natural ratios of such DNA in the sample. One exemplary form of a humic sample is a sample obtained by performing direct lysis as described by Barns et al.,
Proc. Nat'l Acad. Sci. USA
91:1609-1613 (1994) on a soil sample and then purifying the total DNA extract by column chromatography. Related extraction methods can be applied to the isolation of community DNA from other environmental sources. See, Trevors et al., eds.
Nucleic Acids in the Environment
, Springer Lab Manual (1995). Lichen-derived samples may be prepared from foliose lichens by the method of fungal DNA extraction described by Miao et al.,
Mol. Gen. Genet.
226: 214-223 (1991). Specific non-limiting procedures for isolation of DNA from humic and lichen samples are set forth in the examples herein.
The humic or lichen-derived sample is combined with at least one, and optionally with several pairs of amplification primers under conditions suitable for polymerase chain reaction amplification. Polymerase chain-reaction (PCR) amplification is a well known process. The basic procedure, which is described in U.S. Pat. No. 4,683,202 and 4,683,195, which are incorporated herein by reference, makes uses of two amplification primers each of which hybridizes to a different one of the two strands of a DNA duplex. Multiple cycles of primer extension using a polymerase enzyme and denaturation are used to-produce additional copies of the DNA in the region between the two primers. In the present invention, PCR amplification can be performed using any suitable polymerase enzyme, including Taq polymerase and Thermo Sequenase™.
The amplification primers employed in the method of the invention are degenerate primer sets selected to hybridize with conserved regions of known antibiotic biosynthetic genes, for example Type I and Type II polyketide synthase genes, isopenicillin N synthase genes, and peptide synthetase genes. Each degenerate primer set of the invention includes multiple primer species which hybridize with one DNA strand, and multiple primer species which hybridize with the other DNA strand. All of the primer species within a degenerate primer set which bind to the first strand are the same length, and hybridize with the same target region of the DNA. These primers all have very similar sequences, but have a few bases different in each species to account for the observed variations in the target region. For this reason, they are called degenerate primers. Similarly, all of the primers within a degenerate primer set which bind to the second strand are the same length, hybridize with the same target region of the DNA, and have very similar sequences with a few bases different in each species to account for the observed variations in the target region.
The degenerate primer sets of the invention are selected to hybridize to highly conserved regions of known antibiotic biosynthesis genes in such a way that they flank a region of several hundred (e.g. 300) or more base pairs such that amplification leads to the selective reproduction of DNA spanning a substantial portion of the antibiotic biosynthesis gene. Selection of primer sets can be made based upon published sequences for classes of antibiotic biosynthesis genes.
For example, for amplification of Type I polyketide synthase genes, we have designed primers based upon the conserved sequences of six beta-ketoacyl carrier protein synthase domains of the erythromycin gene cluster. Donadio et al .,
Science
252: 675-679 (1991); Donadio and Staver,
Gene
126: 147-151 (1993). These primers have the sequences
5′-GC(C/G) (A/G)T(G/C) GAC CCG CAG CG CGC-3′ [SEQ ID No. 1]
and
5′-GAT (C/G)(G/A)C GTC CGC (G/A)TT (C/G)GT (C/G)CC-3′ [SEQ ID No. 2].
The expected size of the PCR product is 1.2 kilobase pairs. Other degenerate primer sets for Type I and Type II polyketide synthetase genes could be determined from sequence information available in Hutchinson and Fujii,
Ann. Rev. Microbiol.
49: 201-238 (1995).
Type II polyketide synthase ge
Ho Yap Wai
Miao Vivian
Tong Seow Kah
Waters Barbara
Chakrabarti Arun
Fredman Jeffrey
Pennie & Edmonds LLP
TerraGen Diversity Inc.
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