Board of Patent Appeals and Interferences
Patent and Trademark Office (P.T.O.)
*1 EX PARTE GWYNFOR O. HUMPHREYS, CHRISTOPHER R. BAILEY AND CHRISTINE A.
MCKILLOP
Appeal No. 91-2889
February 28, 1992
HEARD: November 26, 1991
Application for Patent filed December 17, 1986, Serial No. 06/943,671. Isolation Of Genes For Biosynthesis Of Polyketide Antibiotics.
Primary Examiner--Richard A. Schwartz
Examiner--R. Peet
Before Winters, W. Smith and Garris
Examiners-in-Chief
Examiner-in-Chief
This is an appeal from the final rejection of claims 1 and 3 through 42, all the claims in the application. A copy of the claims is attached to this decision.
The references relied upon by the examiner are:
Malpartida et al. (Malpartida 1984), "Molecular Cloning Of The Whole Biosynthetic Pathway Of A Streptomyces Antibiotic And Its Expression In A Heterologous Host", Nature, Vol. 309, pages 462-464 (1984).
Rose, "Secondary Products Of Metabolism", Economic Microbiology, Vol. 3, pages 355-387 (1979). [FN1]
Among the references relied upon by appellants are:
Malpartida et al. (Malpartida), "Molecular Cloning Of The Whole Biosynthetic Pathway Of A Streptomyces Antibiotic And Its Expression In A Heterologous Host", Nature, Vol. 309, pages 462-464 (1984).
Hopwood, et al. (Hopwood), Genetic Manipulation Of Streptomyces, A Laboratory Manual, John Innes Foundation, (1985) (Hopwood I).
Pouwels et al. (Pouwels), Cloning Vectors, A Laboratory Manual, Elsevier, (1985).
Lydiate et al. (Lydiate), "The Streptomyces Plasmid SCP2*: Its Functional Analysis And Development Into Useful Cloning Vectors", Gene, Vol. 35, pages 223-235 (1985).
Hopwood, "Cloning And Analysis Of Antibiotic Biosynthetic Genes In Streptomyces", Sixth Int. Symp. On Actinomycetes Biology, pages 3-14 (1985) (Hopwood II).
Malpartida et al. (Malpartida), "Physical And Genetic Characterization Of The Gene Cluster For The Antibiotic Actinorhodin in Streptomyces Coelicolor A3(2)", Mol.Gen.Genet, Vol. 205, pages 66-73 (1986).
Stanzak et al. (Stanzak), "Cloning And Expression In Streptomyces Lividans Of Clustered Erythromycin Biosynthesis Genes From Streptomyces Erythreus", Bio/Technology, Vol. 4, pages 229-232 (March 1986).
Motamedia et al. (Motamedia), "Cloning And Heterologous Expression Of A Gene Cluster For The Biosynthesis Of Tetracenomycin C, The Anthracycline Antitumor Antibiotic Of Streptomyces Glaucescens", Proc.Natl.Acad.Sci.USA, Vol. 84, pages 4445-4449 (1987).
Malpartida et al. (Malpartida), "Homology Between Streptomyces Genes Coding For Synthesis Of Different Polyketides Used To Clone Antibiotic Biosynthetic Genes", Nature, Vol. 325, pages 818-921 (1987).
Ikeda et al. (Ikeda), "Genetic Studies Of Avermectin Biosynthesis In Streptomyces Avermitilis, Journal Of Bacteriology, Vol. 169, No. 12, pages 5615-5621 (1987).
Hallam et al. (Hallam), "Nucleotide Sequence, Transcription And Deduced Function Of A Gene Involved In Polyketide Antibiotic Synthesis In Streptomyces Coelicolor", Gene, Vol. 74, pages 305-320 (1988).
*2 Horinouchi et al. (Horinouchi), "afsB Stimulates Transcription Of The Actinorhodin Biosynthetic Pathway In Streptomyces Coelicolor A3(2) And Streptomyces Lividans", Mol.Gen.Genet, Vol. 215, pages 355-357 (1989).
Sherman et al. (Sherman), "Structure And Deduced Function Of The Granaticin-Producing Polyketide Synthase Gene Cluster Of Streptomyces Violaceoruber Tu22", The EMBO Journal, Vol. 8, No. 9, pages 2712-2725 (1989).
Gewain et al. (Gewain), "Cloning Streptomyces Avermitilis Genes For Avermectin Biosynthesis", H-113, date not given.
The claims stand rejected as follows:
I. Claims 3 through 8, 11 through 16, and 18 through 42 under 35 USC § 112, first paragraph;
II. Claims 1 and 3 through 26 under 35 USC § 112, first paragraph;
III. Claims 1, 3 through 5, 9 through 13, and 17 under 35 USC § 102(b) as anticipated by Malpartida 1984;
IV. Claims 6 through 8, 14 through 16, 18 through 23, and 29 through 42 under 35 USC § 103 as unpatentable over Malpartida 1984 in view of Rose.
The many aspects of the present invention appear to be based in large part upon the work discussed in Malpartida 1984 concerning the isolation of the entire DNA sequence corresponding to the structural genes for actinorhodin biosynthesis. Actinorhodin is an antibiotic produced by Streptomyces coelicolor (S. coelicolor). Working with a series of six actinorhodin non-producing mutants, the authors were able to isolate and identify a transformed S. coelicolor strain containing a plasmid (pIJ2303) believed to include the entire set of actinorhodin biosynthesis genes. Subsequent work including the insertion of pIJ2303 into a separate Streptomyces strain which does not produce actinorhodin confirmed that pIJ2303 contains the entire DNA sequence corresponding to the structural genes for actinorhodin biosynthesis.
Malpartida 1984 also discloses that certain Streptomyces strains produce nearly two-thirds of the known natural antibiotics and that the genes for antibiotic synthesis in such species tend to be clustered together on the chromosomes of the microorganism. The reference also acknowledges that it is generally accepted that antibiotics such as actinorhodin which are biosynthesized through the polyketide pathway are assembled on multi-enzyme complexes and that the gene clusters that encode polyketide synthetases may have had a common evolutionary origin.
On the basis of all of this information, the authors concluded that the DNA sequence corresponding to the structural genes for actinorhodin biosynthesis (pIJ2303) may possibly be used as DNA probes for the isolation of other antibiotic synthetases which are produced via the polyketide pathway.
Malpartida 1986 [FN2] identifies subclones of pIJ2303, e.g., pIJ2305 and pIJ2308, as well as regions of these subclones which contain specific genes associated with the six classes of the actinorhodin non-producing mutants used to isolate pIJ2303. For example, the structural gene(s) represented by a Class III mutant is disclosed to be contained in pIJ2305.
*3 The Malpartida references indicate that the authors were associated with the John Innes Institute. As set forth in Example I of this application, the present inventions are based upon pIJ2303, pIJ2305, and pIJ2308. Appellants acknowledge on page 8 of the Appeal Brief and page 3 of the Reply Brief that pIJ2303 was obtained from the John Innes Institute.
Employing conventional, well known techniques, appellants conducted hybridization studies of Streptomyces sp. B41-146 (a known producer of the polyketide antibiotic milbemycin) using the pIJ2305 insert as a probe to identify the genes responsible for milbemycin production. Subsequently, in order to identify the region of insert pIJ2305 which hybridized to Streptomyces sp B41-146, a 1.1 BamHI fragment of pIJ2305 was formed and found to hybridize to an 8.0 KB fragment of Streptomyces sp. B41-146 DNA. The 1.1 BamHI fragment of pIJ2305 was identified by Malpartida 1986 to contain the structural gene(s) associated with a Class III mutant.
After identifying several fragments of Streptomyces sp. B41-146 which hybridized to the probes obtained from pIJ2305, appellants constructed a composite DNA sequence which was named MC. Appellants postulate that this DNA fragment contains the genes necessary for biosynthesis of milbemycin. However, the entire MC sequence was not inserted in a non-producing milbemycin Streptomyces strain in order to determine whether this DNA fragment does in fact contain all of the structural genes needed for milbemycin production as Malpartida 1984 did with pIJ2303. Rather, appellants used fragments of the MC fragment in insertional inactivation studies of the milbemycin genes of Streptomyces sp. B41-146. As set forth in Example 6 of the present specification, these studies indicate that the specific fragments tested do contain genes essential for milbemycin production. However, it is not stated that all genes needed for milbemycin production are present on fragment MC.
Claims 1 and 3 through 8 on appeal are directed to a method of isolating a gene involved in the biosynthesis of a first polyketide antibiotic which involves the use of nucleic acid probes which comprise at least a part of a gene involved in the biosynthesis of a second polyketide antibiotic. For example, part of pIJ2303, e.g., pIJ2305, is used as a probe in order to identify a part of a gene involved in the biosynthesis of milbemycin.
Claims 9 through 23 are directed to methods for producing a first polyketide antibiotic in a naturally non-producing strain of a bacterium of the genus Streptomyces which involves obtaining the complete gene cluster of the first polyketide antibiotic. The specific steps involved in obtaining the complete gene cluster are acknowledged to be conventional and well known. The key to this method appears to be the availability of a nucleic acid sequence containing at least a part of a gene involved in the biosynthesis of a second polyketide antibiotic for use as a probe. It should be noted that these claims require the positive step of introducing the clone fragment thought to contain the complete cluster of biosynthetic genes responsible for production of the first polyketide antibiotic into a non-producing strain of Streptomyces and producing the first polyketide antibiotic in the normally non-producing Streptomyces strain.
*4 Claims 29 through 42 are directed to DNA fragments, hybridization probes, vectors, and transformed bacterial strains used in the hybridization and antibiotic production methods.
Claims 24 through 28 are directed to a separate aspect of the present invention. As set forth in Malpartida 1984 and Malpartida 1986, the Class II gene involved in the biosynthesis of actinorhodin production in S. coelicolor is apparently involved in regulation of the polyketide pathway. As set forth in Example 4 of the present application, appellants have found that transforming a Streptomyces strain which contains the genes for production of actinorhodin but is not capable of normally producing the antibiotic with certain DNA fragments from Streptomyces sp. B41-146 results in the normally non-producing strain being capable of producing actinorhodin. Using the nomenclature of Malpartida, appellants identify the gene included in these DNA fragments which is responsible for "turning on" actinorhodin production in the normally non-producing streptomyces strain as milbemycin gene II.
ISSUES
The issues presented in this appeal are the correctness of the examiner's rejections.
OPINION
REJECTION I
The examiner has concluded that one skilled in the art is enabled to practice claims 3 through 8, 11 through 16, and 18 through 42 only if certain of the biological material used by appellants is deposited per the procedures set forth in MPEP § 608.01(p)C. In reaching this conclusion, the examiner has expressed concern in regard to whether the written description of the present invention is sufficiently repeatable and whether certain of the starting materials are readily available to the public. In expressing these concerns on pages 3-6 of the Examiner's Answer, the examiner has not specifically stated which of the myriad biological materials involved in the present invention should be deposited. In arguing this rejection in the Appeal Brief, appellants have indicated that plasmid pIJ2303 and the purported milbemycin gene cluster (fragment MC) are the biological materials which the examiner has required to be deposited.
As set forth above, appellants acknowledge that the present invention does employ DNA probes obtained from pIJ2303 but assert that this plasmid is available to the public from the John Innes Institute. Appellants rely upon publications bearing publication dates both before and after the present filing date [FN3] which refer to either pIJ2303 or other plasmids which contain various of the actinorhodin genes identified by Malpartida 1984 and 1986, arguing that these references indicate that pIJ2303, its actinorhodin gene insert, or functional equivalents thereof have been made available to a variety of universities and industrial laboratories.
Appellants have also traced the manner in which pIJ2303 was developed as documented in the prior art. See the paragraph bridging pages 3-4 of the Reply Brief, relying upon, inter alia, Hopwood I. As explained by appellants, the genesis of this plasmid can be traced to ultraviolet mutagenesis of certain bacterium. Appellants assert that mutagenesis is a well-known and reproducible technique so that pIJ2303 can be reconstructed by those skilled in this art if the plasmid is not publicly available. Appellants also refer to disclosures in other references, e.g., Malpartida 1984, that the mutant strains from which pIJ2303 was developed are maintained at the John Innes Institute so that it may be implied that these mutant strains are available to researchers interested in reconstructing this plasmid.
*5 Alternatively, appellants argue that the nucleotide sequence of the 1.1 BamHI fragment containing the gene associated with Class III mutants was published subsequent to the filing date of this application (Hallam).
We have carefully considered appellants' position but agree with the examiner that the written description of this application does not enable one skilled in the art to practice the inventions encompassed by the rejected claims absent access to pIJ2303 and that this plasmid must be deposited under appropriate conditions.
In regard to reconstructing pIJ2303, we point out that appellants have not provided any evidence on this record that ultraviolet mutagenesis of bacteria is a reproducible phenomenon so that the mutant bacteria needed in order to reconstruct pIJ2303 may be obtained without undue experimentation. Attorney's argument in a brief does not take the place of factual evidence. In re Greenfield, 571 F.2d 1185, 197 USPQ 227 (CCPA 1978).
As to appellants' arguments concerning the apparent availability to the public of pIJ2303 and/or its parent mutant strains from the John Innes Institute, we point out that appellants receipt of pIJ2303 from the John Innes Institute as well as the ability of others to obtain this or similar material from this establishment prior to and after the present filing date does not establish that upon issuance of a patent on this application that such material would continue to be accessible to the public from the John Innes Institute. Appellants have not made of record any of the facts and circumstances surrounding their obtention of material from the John Innes Institute nor is there any evidence as to what its policy is in this regard, either now or in the future if a patent were to issue on this application. On this record, there is no assurance that the John Innes Institute would allow unlimited access to pIJ2303 if this application were to mature into a patent.
In response to appellants' reliance upon the later published Hallam reference for its disclosure of the nucleotide sequence of one of the genes involved in the present invention, the examiner argues that such a later published reference is not available for appellants' use to establish enablement, citing In re Glass, 492 F.2d 1228, 181 USPQ 31 (CCPA 1974). Appellants respond that under the circumstances of this application, the later published reference may be relied upon in this manner, citing In re Lundak, 723 F.2d 1216, 227 USPQ 90 (Fed.Cir.1985). We do not find it necessary to resolve this specific issue since, assuming arguendo, Hallam is properly relied upon by appellants, we do not find that it relieves appellants of their burden of depositing pIJ2303.
At best, Hallam discloses the nucleotide sequence of a single gene involved in the present invention. Appellants have not explained on this record how this disclosure in and of itself enables practice of the present invention throughout the breadth of the rejected claims. Appellants direct attention to dependent claims 4 and 12 which are stated to be limited to the use of the subsequently published nucleotide sequence. We disagree. These claims only require the use of "the nucleic acid sequence of at least a part of the actinorhodin gene III" and are not limited to the use of the subsequently published nucleotide sequence as argued.
*6 From this record, it appears that the only manner in which appellants can satisfy their burden of assuring public access to this needed biological material upon issuance of a patent is by themselves undertaking the responsibility of making an appropriate deposit of this material. Therefore, we conclude that appellants must deposit pIJ2303 in order to comply with the enablement requirement of 35 USC § 112, first paragraph.
As to the examiner's concern that fragment MC must also be deposited, we note that this fragment was obtained by using what are acknowledged to be well known techniques using pIJ2303. The examiner has not explained why one having access to pIJ2303 upon issuance of a patent on this application would not be enabled to obtain fragment MC using these well known techniques. Absent a cogent explanation from the examiner, we do not find that appellants need deposit fragment MC.
REJECTION II
We reverse this rejection as it applies to claims 1 and 3 through 8 and affirm this rejection as it applies to claims 9 through 26.
From the statement of the rejection it appears that the examiner's concern in separately rejecting these claims under this section of the statute is in regard to their breadth. In other words, assuming public access to pIJ2303 upon issuance of a patent based upon this application, those skilled in the art would not be enabled to practice these claims throughout their scope without undue experimentation. See In re Wands, 858 F.2d 731, 8 USPQ2d 1400 (Fed.Cir.1988).
Claims 1 and 3 through 8 are directed to a conventional method of hybridization in order to isolate a gene involved in the biosynthesis of a first polyketide antibiotic. Appellants and the examiner agree that the manipulative steps involved in these claims are well known in this art. It is not clear from the examiner's statement of the rejection specifically what concerns he has in regard to claims 1 and 3 through 8. Having isolated the complete gene cluster for actinorhodin biosynthesis in pIJ2303, Malpartida 1984 states that this plasmid may be possibly useful as DNA probes for the isolation of other antibiotic synthetases. Hopwood II also suggests the use of cloned DNA such as that of pIJ2303 as a probe to isolate the genes encoding for other polyketide synthetases. See Strategy 7 in Table 1 of Hopwood II.
In view of this disclosure of Malpartida 1984 and Hopwood II to use the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of a second polyketide antibiotic as a probe to screen a clone library of genomic DNA from a microorganism which produces a first polyketide antibiotic, it is difficult to determine precisely which step(s) of claims 1 and 3 through 8 is not enabled or would require undue experimentation.
Claims 9 through 23 are directed to methods for producing a polyketide antibiotic in a naturally non-producing strain of a bacterium of the genus Streptomyces. As seen from claim 9(a)(vii), an essential step of the claimed method is to identify and isolate a DNA fragment which comprises the complete gene cluster for producing the desired polyketide antibiotic by actual production of this polyketide antibiotic in a non-producing strain of Streptomyces. Claim 22, while limited to the use of DNA fragment MC in a naturally non-milbemycin producing strain of a bacterium of the genus Streptomyces, contains a similar requirement.
*7 As set forth in In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970):
[Section 112] requires that the scope of the claims must bear a reasonable correlation to scope of enablement provided by the specification to persons of ordinary skill in the art.
* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
In cases involving unpredictable factors, such as most chemical reactions and physiological activity, the scope of enablement varies inversely with degree of unpredictability of the factors involved.
Here, the rejected claims involve the isolation and identification of the complete gene cluster responsible for biosynthesis of a polyketide antibiotic and the expression of this gene cluster in a Streptomyces strain which does not normally produce this antibiotic. Due to the unpredictability of such physiological activity, we conclude that the examiner has correctly shifted the burden of proof to appellants to establish through objective evidence that the methods encompassed by claims 9 through 23 are enabled throughout their scope.
Appellants rely upon the working examples of the present specification as rebuttal evidence. We have carefully considered this evidence but do not find it is probative of enablement. First, the work set forth in the working examples is directed only to the obtention of the gene cluster for the biosynthesis of milbemycin using the actinorhodin gene cluster as the probe. However, we agree with the examiner that even this single limited showing does not establish that fragment MC represents a gene cluster responsible for milbemycin production as asserted. Example 6 of the specification only used fragments of fragment MC in homologous recombination experiments. The conclusions reached in Example 6 amount to only statements that the tested fragments of fragment MC "contain genes essential for milbemycin production." It is only inferred from this work that fragment MC contains all of the genes required for milbemycin production in a non-producing Streptomyces strain as required by these claims on appeal. From this record, it appears that those skilled in this art would conclude that a clone fragment contains the complete cluster of genes needed for biosynthesis of a polyketide antibiotic in a normally non-producing Streptomyces strain only when its introduction into a non-producing strain results in the production of the desired polyketide antibiotic as in Malpartida 1984.
Appellants rely upon Motamedi and Stanzak as successful examples of introducing gene clusters for the biosynthesis of a first polyketide antibiotic into a non-producing Streptomyces strain for the polyketide antibiotic and obtaining expression of the desired antibiotic. However, appellants have not established on this record that the gene clusters of these references were obtained using the probing procedures required by the rejected claims. [FN4] It is of interest to note however that the conclusions reached by the authors of these two references concerning production of a polyketide antibiotic in a normally non-producing Streptomyces strain are based upon incorporation of the purported gene cluster in such a strain and the subsequent observation of antibiotic production in the transformed strain. This is further evidence that those skilled in this art would not readily accept the procedure set forth in Example 6 of the present specification as evidence that the fragment MC does in fact contain the complete set of genes needed for a biosynthesis of milbemycin in a non-producing Streptomyces strain.
*8 We also affirm this rejection as it pertains to claims 24 through 26. There is no evidence of record which establishes that milbemycin Gene II is a universal activator in regard to either the desired polyketide antibiotic or the normally inactive Streptomyces strain as encompassed by these claims.
The examiner has not included any of claims 29 through 42 in this rejection. While it may be that access to pIJ2303 will enable one skilled in the art to obtain the numbered fragments set forth in claims such as claim 42 per the teachings of the present disclosure, it is not clear why one would be enabled to practice generic claims such as claims 29, 30, 37, and 41 throughout their scope. It is also not clear why claim 28 is not included in this rejection since the production of an avermectin in the manner required by this claim has not been shown to be an enabled species of claim 24. We decline to exercise our discretion under 37 CFR § 1.196(b) and enter new grounds of rejection. However, these issues should be considered by appellants and the examiner if prosecution of this subject matter is continued in a continuing application.
REJECTION III
We affirm this rejection as it applies to claims 1 and 3 through 5 and reverse the rejection as it applies to claims 9 through 13 and 17.
In discussing the enablement issue as raised in regard to hybridization claims 1 and 3 through 5, we found that the suggestion of Malpartida 1984 to use the gene cluster found in pIJ2303 as probes to isolate other polyketide antibiotic synthetases was capable of being implemented by those skilled in the art since the needed procedures were well known and conventional. On this basis, we agree with the examiner's conclusion that Malpartida 1984 is an anticipation of the procedure set forth in claims 1 and 3 through 5. By "suggesting" that pIJ2303 be used as a DNA probe for isolation of other polyketide antibiotic synthetases, Malpartida 1984 is describing the conventional steps known at that time for implementing such a procedure. Claims 1 and 3 through 5 call for nothing more.
Appellants argue that there is no enabling teaching in Malpartida 1984 for their invention. However, appellants have not explained on this record what aspect of claims 1 and 3 through 5 is not disclosed in Malpartida 1984. As set forth in In re Donohue, 632 F.2d 123, 207 USPQ 196 (CCPA 1980):
For a publication to constitute an anticipation of an invention and, thus, to bar the grant of a patent under 35 USC 102, it must be capable, when taken in conjunction with the knowledge of those skilled in the art to which it pertains, of placing that invention in the possession of the public. In re LeGrice, 49 CCPA 1123, 1145, 301 F.2d 929, 944, 133 USPQ 365, 378 (1962), In re Brown, 51 CCPA 1254, 1259, 329 F.2d 1006, 1011, 141 USPQ 245, 249 (1964).
*9 Appellants argue that a prior art reference must not require independent experimentation in order to practice the invention to be considered an anticipation. Assuming this to be the case, appellants has not set forth what experimentation is needed. The fact that Malpartida 1984 may not have actually reduced to practice the suggested probing procedure has no bearing on whether that procedure is described in the reference. See In re Sivaramakrishnan, 673 F.2d 1383, 213 USPQ 441 (CCPA 1982) and cases cited therein.
We reverse this rejection as it applies to claims 9 through 13 and 17 since Malpartida 1984 only discloses the use of pIJ2303 as a probe for isolating other polyketide antibiotic synthetases and does not describe the more comprehensive method of producing polyketide antibiotics required by these claims.
REJECTION IV
We reverse this rejection. As set forth above in regard to the anticipation rejection, Malpartida 1984 only discloses that pIJ2303 can be used to locate other polyketide antibiotic synthetases using well known hybridization techniques. Whether such hybridization studies would necessarily lead to the obtention of all of the genes required for the biosynthesis of another polyketide antibiotic in a Streptomyces strain which would normally not produce it is a matter of much conjecture. As set forth above in regard to the enablement rejection for these claims, we have concluded that those skilled in the art would not be enabled as of the filing date of this application to practice the methods called for by these claims throughout their scope. The examiner's rejection is not specific as to how one of ordinary skill in the art would have found it obvious to practice any specific method within the scope of these claims as of the filing date of this application. In this regard, we note that the examiner has not explained with any specificity on this record how Rose, either as discussed on page 2 of the present specification or from a consideration of the entire reference as supplied by appellants, would have suggested in combination with Malpartida 1984 the methods set forth by these claims.
The decision of the examiner is affirmed. [FN5]
No time period for taking any subsequent action in connection with this appeal may be extended under 37 CFR 1.136(a). See the final rule notice, 54 F.R. 29548 (July 13, 1989), 1105 O.G. 5 (August 1, 1989).
AFFIRMED
BOARD OF PATENT APPEALS AND INTERFERENCES
Sherman D. Winters
Examiner-in-Chief
William F. Smith
Examiner-in-Chief
Bradley R. Garris
Examiner-in-Chief
FN1. The examiner's consideration of this reference appears to be based upon appellants' summary thereof on page 2, lines 20-29 of the specification. We have considered the original reference as it was provided by appellants on October 31, 1991 (Paper No. 25).
FN2. As the record now stands, Malpartida 1986 is prior art to the claims on appeal. Appellants have not submitted copies of the two United Kingdom documents priority of which is claimed in the original declaration of this application under 35 USC § 119. Therefore, the examiner has not determined whether any of the claims on appeal enjoy the benefit of the earlier filing date of either of these documents.
FN3. See, e.g., Malpartida 1987, Horinouchi, and Lydiate.
FN4. Appellants have not established on this record that Motamedi is available evidence for establishing enablement of these claims since it was published after the present filing date. In re Glass, supra.
FN5. We note that claim 31 is improperly dependent on claim 30. This matter should also be addressed if prosecution is continued on this subject matter in a continuing application.
*10 1. A method for isolating a gene involved in the biosynthesis of a first polyketide antibiotic which comprises the steps of:
a. preparing a clone library wherein each clone contains a fragment of DNA from a microorganism which is a bacterium of the genus Streptomyces which produces said first polyketide antibiotic;
b. screening said clone library for hybridization to a nucleic acid probe molecule which comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of a second polyketide antibiotic in a bacterium of the genus Streptomyces; and
c. selecting those clones which hybridize to said nucleic acid probe molecule thereby isolating a clone which contains a fragment of DNA which comprises said gene involved in the biosynthesis of said first polyketide antibiotic.
3. A method according to claim 1 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of actinorhodin.
4. A method according to claim 3 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of the actinorhodin Gene III.
5. A method according to claim 3 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of the actinorhodin Gene I.
6. A method according to claim 1 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of milbemycin.
7. A method according to claim 6 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of the milbemycin Gene III.
8. A method according to claim 6 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of the milbemycin Gene I.
9. A method for producing a first polyketide antibiotic in a naturally non-producing strain of a bacterium of the genus Streptomyces which comprises the steps of:
a. isolating a DNA fragment which comprises the clustered biosynthetic genes for said first polyketide antibiotic from a strain of a bacterium of the genus Streptomyces which produces said first polyketide antibiotic wherein said isolation comprises the steps of:
i. preparing a clone library wherein each clone contains a fragment of DNA from a microorganism which produces said first polyketide antibiotic;
ii. screening said clone library for hybridization to a nucleic acid probe molecule which comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of a second polyketide antibiotic;
iii. selecting those clones which hybridize to said nucleic acid probe molecule thereby isolating a clone which comprises a first DNA fragment which comprises at least part of the biosynthetic gene cluster of said first polyketide antibiotic;
iv. testing said first selected DNA fragment which comprises at least part of the biosynthetic gene cluster of said first polyketide antibiotic for the ability to direct synthesis of said first polyketide antibiotic in said non-producing strain of Streptomyces;
*11 v. rescreening said clone library or a second clone library with a nucleic acid probe which comprises the right or left terminal region of said first selected DNA fragment;
vi. selecting those clones which hybridized to the right or left terminal region of said first selected DNA fragment thereby isolating a clone which comprises a second DNA fragment which comprises chromosomal DNA sequences to the right or left of said first selected clone;
vii. repeating said testing, rescreening and selection steps employing said second selected DNA fragment and subsequent selected DNA fragments until said DNA fragment which comprises the clustered biosynthetic genes for said first polyketide antibiotic is isolated as ascertained by production of said first polyketide antibiotic in said nonproducing strain of Streptomyces in said testing step; and
b. introducing said cloned fragment containing said cluster of biosynthetic genes into said non-producing strain of a bacterium of the Streptomyces, thereby producing said polyketide antibiotic in said non-producing strain of Streptomyces.
10. A method according to claim 9 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of a gene involved in an early step of the biosynthesis of said second polyketide antibiotic.
11. A method according to claim 9 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of actinorhodin.
12. A method according to claim 11 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of actinorhodin Gene III.
13. A method according to claim 11 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of actinorhodin Gene I.
14. A method according to claim 9 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of a gene involved in the biosynthesis of milbemycin.
15. A method according to claim 14 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of milbemycin Gene III.
16. A method according to claim 14 wherein said nucleic acid probe comprises the nucleic acid sequence of at least a part of milbemycin Gene I.
17. A method according to claim 9 wherein said naturally non-producing strain of a bacterium of the genus Streptomyces is selected from the group of strains consisting of strains of Streptomyces lividans, strains of Streptomyces ambofaciens, strains of Streptomyces coelicolor and strains of Streptomyces avermitilis.
18. A method according to claim 9 wherein said first polyketide antibiotic is a milbemycin.
19. A method according to claim 18 wherein said naturally non-producing strain of a bacterium of the genus Streptomyces is a strain of Streptomyces lividans.
20. A method according to claim 9 wherein said first polyketide antibiotic is an avermectin.
21. A method according to claim 20 wherein said naturally non-producing strain of a bacterium of the genus Streptomyces is a strain of Streptomyces lividans.
*12 22. A method for producing a milbemycin in a naturally non-milbemycin producing strain of a bacterium of the genus Streptomyces which comprises introducing into said non-producing strain a DNA fragment which comprises the DNA fragment MC.
23. A method according to claim 22 wherein said naturally non-milbemycin producing strain is a strain of Streptomyces lividans.
24. A method for activating expression of a polyketide antibiotic biosynthetic gene cluster in a bacterial strain of the genus Streptomyces which strain contains the biosynthetic genes sufficient for production of said polyketide antibiotic but lacks a functional polyketide antibiotic biosynthesis activator gene which comprises introducing into said bacterial strain a DNA fragment which comprises milbemycin Gene II.
25. A method according to claim 24 wherein said bacterial strain is a strain of Streptomyces lividans.
26. A method according to claim 25 wherein said polyketide antibiotic is actinorhodin.
27. A method according to claim 24 wherein said bacterial strain is a strain of Streptomyces coelicolor and said polyketide antibiotic is actinorhodin.
28. A method according to claim 24 wherein said bacterial strain is a strain of Streptomyces avermitilis and said polyketide antibiotic is an avermectin.
29. A vector which comprises a DNA fragment consisting essentially of the milbemycin biosynthetic gene cluster.
30. A bacterial strain containing the vector of claim 29.
31. A vector according to claim 30 wherein said DNA fragment is the fragment MC.
32. A bacterial strain containing the vector of claim 31.
33. A vector which comprises a DNA molecule consisting essentially of the DNA sequence encoding a milbemycin gene selected from the group consisting of milbemycin Gene I, milbemycin Gene II, and milbemycin Gene III.
34. A bacterial strain containing the vector of claim 33.
35. A vector according to claim 29 wherein said DNA fragment is a DNA fragment selected from the group of DNA fragments consisting of fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8a, fragment 8b, fragment 10, fragment 12, fragment 14, fragment 16, fragment 62, and fragment 64.
36. A bacterial strain containing the vector of claim 35.
37. A hybridization probe molecule consisting essentially of the milbemycin biosynthetic gene cluster.
38. A hybridization probe molecule that is fragment MC.
39. A hybridization probe consisting essentially of the DNA sequence encoding a milbemycin gene selected from the group of milbemycin genes consisting of milbemycin Gene I, milbemycin Gene II and milbemycin gene III.
40. A hybridization probe that is a DNA fragment selected from the group of DNA fragments consisting of fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8a, fragment 8b, fragment 10, fragment 12, fragment 14, fragment 16, fragment 62, and fragment 64.
*13 41. A hybridization probe, the sequence of which is derived from the sequence of a fragment selected from the group of fragments consisting of fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8a, fragment 8b, fragment 10, fragment 12, fragment 14, fragment 16, fragment 62, and fragment 64.
42. A DNA fragment selected from the group of DNA fragments consisting of fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8a, fragment 8b, fragment 10, fragment 12, fragment 14, fragment 16, fragment 62, fragment 64 and fragment MC.
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