Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869

Efficient cloning of genes of Neurospora crassa

Steven J Vollmer 1,*, Charles Yanofsky 1,†
PMCID: PMC323844  PMID: 16593723

Abstract

We have constructed a genomic library of Neurospora crassa DNA in a cosmid vector that contains the dominant selectable marker for benomyl resistance. The library is arranged to permit the rapid cloning of Neurospora genes by either sib-selection or colony-hybridization protocols. Detailed procedures for the uses of the library are described. By use of these procedures, a modest number of unrelated genes have been isolated. The cloning of trp-3, the structural gene for the multifunctional enzyme tryptophan synthetase (tryptophan synthase, EC 4.2.1.20), is reported in detail; its identity was verified by restriction fragment length polymorphism mapping. The strategies described in this paper should be of use in the cloning of any gene of Neurospora, as well as genes of other lower eukaryotes.

Keywords: cosmid library, transformation, sib selection, tryptophan synthase

Full text

PDF
4869

Images in this article

4872Image
on p.4872

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akins R. A., Lambowitz A. M. General method for cloning Neurospora crassa nuclear genes by complementation of mutants. Mol Cell Biol. 1985 Sep;5(9):2272–2278. doi: 10.1128/mcb.5.9.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arends H., Sebald W. Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa. EMBO J. 1984 Feb;3(2):377–382. doi: 10.1002/j.1460-2075.1984.tb01815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berlin V., Yanofsky C. Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa. Mol Cell Biol. 1985 Apr;5(4):849–855. doi: 10.1128/mcb.5.4.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buxton F. P., Radford A. Cloning of the structural gene for orotidine 5'-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli. Mol Gen Genet. 1983;190(3):403–405. doi: 10.1007/BF00331067. [DOI] [PubMed] [Google Scholar]
  5. Case M. E., Schweizer M., Kushner S. R., Giles N. H. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5259–5263. doi: 10.1073/pnas.76.10.5259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chia W., Scott M. R., Rigby P. W. The construction of cosmid libraries of eukaryotic DNA using the Homer series of vectors. Nucleic Acids Res. 1982 Apr 24;10(8):2503–2520. doi: 10.1093/nar/10.8.2503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  8. DEMOSS J. A. Studies on the mechanism of the tryptophan synthetase reaction. Biochim Biophys Acta. 1962 Aug 13;62:279–293. doi: 10.1016/0006-3002(62)90041-0. [DOI] [PubMed] [Google Scholar]
  9. Dhawale S. S., Marzluf G. A. Transformation of Neurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA. Curr Genet. 1985;10(3):205–212. doi: 10.1007/BF00798750. [DOI] [PubMed] [Google Scholar]
  10. Grant D. M., Lambowitz A. M., Rambosek J. A., Kinsey J. A. Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid. Mol Cell Biol. 1984 Oct;4(10):2041–2051. doi: 10.1128/mcb.4.10.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  13. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. KAPLAN S., SUYAMA Y., BONNER D. M. FINE STRUCTURE ANALYSIS AT THE TD LOCUS OF NEUROSPORA CRASSA. Genetics. 1964 Jan;49:145–158. doi: 10.1093/genetics/49.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krumlauf R., Marzluf G. A. Characterization of the sequence complexity and organization of the Neurospora crassa genome. Biochemistry. 1979 Aug 21;18(17):3705–3713. doi: 10.1021/bi00584a011. [DOI] [PubMed] [Google Scholar]
  16. Krumlauf R., Marzluf G. A. Genome organization and characterization of the repetitive and inverted repeat DNA sequences in Neurospora crassa. J Biol Chem. 1980 Feb 10;255(3):1138–1145. [PubMed] [Google Scholar]
  17. Legerton T. L., Yanofsky C. Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa. Gene. 1985;39(2-3):129–140. doi: 10.1016/0378-1119(85)90306-3. [DOI] [PubMed] [Google Scholar]
  18. Lester G. Regulation of tryptophan biosynthetic enzymes in Neurospora crassa. J Bacteriol. 1971 Jul;107(1):193–202. doi: 10.1128/jb.107.1.193-202.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matchett W. H., DeMoss J. A. The subunit structure of tryptophan synthase from Neurospora crassa. J Biol Chem. 1975 Apr 25;250(8):2941–2946. [PubMed] [Google Scholar]
  20. Metzenberg R. L., Stevens J. N., Selker E. U., Morzycka-Wroblewska E. Identification and chromosomal distribution of 5S rRNA genes in Neurospora crassa. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2067–2071. doi: 10.1073/pnas.82.7.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miles E. W. Tryptophan synthase: structure, function, and subunit interaction. Adv Enzymol Relat Areas Mol Biol. 1979;49:127–186. doi: 10.1002/9780470122945.ch4. [DOI] [PubMed] [Google Scholar]
  22. Münger K., Germann U. A., Lerch K. Isolation and structural organization of the Neurospora crassa copper metallothionein gene. EMBO J. 1985 Oct;4(10):2665–2668. doi: 10.1002/j.1460-2075.1985.tb03985.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paietta J. V., Marzluf G. A. Gene disruption by transformation in Neurospora crassa. Mol Cell Biol. 1985 Jul;5(7):1554–1559. doi: 10.1128/mcb.5.7.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paietta J., Marzluf G. A. Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa. Curr Genet. 1985;9(5):383–388. doi: 10.1007/BF00421609. [DOI] [PubMed] [Google Scholar]
  25. Perkins D. D., Radford A., Newmeyer D., Björkman M. Chromosomal loci of Neurospora crassa. Microbiol Rev. 1982 Dec;46(4):426–570. doi: 10.1128/mr.46.4.426-570.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  27. Russell P. J., Wagner S., Rodland K. D., Feinbaum R. L., Russell J. P., Bret-Harte M. S., Free S. J., Metzenberg R. L. Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora. Mol Gen Genet. 1984;196(2):275–282. doi: 10.1007/BF00328060. [DOI] [PubMed] [Google Scholar]
  28. SUYAMA Y., LACY A. M., BONNER D. M. A GENETIC MAP OF THE TD LOCUS OF NEUROSPORA CRASSA. Genetics. 1964 Jan;49:135–144. doi: 10.1093/genetics/49.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schechtman M. G., Yanofsky C. Structure of the trifunctional trp-1 gene from Neurospora crassa and its aberrant expression in Escherichia coli. J Mol Appl Genet. 1983;2(1):83–99. [PubMed] [Google Scholar]
  30. Stohl L. L., Akins R. A., Lambowitz A. M. Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid. Nucleic Acids Res. 1984 Aug 10;12(15):6169–6178. doi: 10.1093/nar/12.15.6169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stohl L. L., Lambowitz A. M. Construction of a shuttle vector for the filamentous fungus Neurospora crassa. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1058–1062. doi: 10.1073/pnas.80.4.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vapnek D., Hautala J. A., Jacobson J. W., Giles N. H., Kushner S. R. Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3508–3512. doi: 10.1073/pnas.74.8.3508. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES