Abstract
The filamentous fungus Neurospora crassa undergoes a complex program of sexual development to form a fruiting body composed of several kinds of specialized tissue. Subtractive hybridization was used to isolate genes that are expressed preferentially during this sexual phase. Many such sexual development (sdv) genes were identified in a cosmid library of Neurospora genomic DNA. Fourteen of the sdv genes were subcloned, and their expression in mutant strains and under crossing and vegetative growth conditions was examined. All of the regulated transcripts were less abundant (and in many cases not detectable) in strains grown under vegetative (high nitrogen) conditions, suggesting that nitrogen starvation is required for their synthesis. The expression of most of the sdv genes also required a functional A mating type product, even under crossing growth conditions, suggesting that this product functions as a master control in sexual development. To determine if the products of the sdv genes play essential roles in the sexual cycle, a reverse-genetic approach (based on RIP (repeat-induced point mutation)-mediated gene disruptions) was used to create mutations in the genes. A mutant strain (asd-1) with a recessive crossing defect (apparently caused by the RIP process) was isolated; in this strain, early development is normal and many asci are formed, but ascospores are never delineated. A second recessive mutant strain (asd-2) was apparently created by ectopic integration of the transforming DNA into a gene required for the sexual process; in this strain the sexual process was blocked at an early stage, and the ascogenous tissue underwent little development.
Full Text
The Full Text of this article is available as a PDF (5.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Asch D. K., Kinsey J. A. Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene. Mol Gen Genet. 1990 Mar;221(1):37–43. doi: 10.1007/BF00280365. [DOI] [PubMed] [Google Scholar]
- Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Cambareri E. B., Jensen B. C., Schabtach E., Selker E. U. Repeat-induced G-C to A-T mutations in Neurospora. Science. 1989 Jun 30;244(4912):1571–1575. doi: 10.1126/science.2544994. [DOI] [PubMed] [Google Scholar]
- Case M. E. Genetical and molecular analyses of qa-2 transformants in Neurospora crassa. Genetics. 1986 Jul;113(3):569–587. doi: 10.1093/genetics/113.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Fincham J. R., Connerton I. F., Notarianni E., Harrington K. Premeiotic disruption of duplicated and triplicated copies of the Neurospora crassa am (glutamate dehydrogenase) gene. Curr Genet. 1989 May;15(5):327–334. doi: 10.1007/BF00419912. [DOI] [PubMed] [Google Scholar]
- Glass N. L., Grotelueschen J., Metzenberg R. L. Neurospora crassa A mating-type region. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4912–4916. doi: 10.1073/pnas.87.13.4912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
- Harding R. W., Melles S. Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants. Plant Physiol. 1983 Aug;72(4):996–1000. doi: 10.1104/pp.72.4.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson P. D., Felsenfeld G. In vivo footprinting of specific protein-DNA interactions. Methods Enzymol. 1987;152:735–755. doi: 10.1016/0076-6879(87)52077-8. [DOI] [PubMed] [Google Scholar]
- Kinnaird J. H., Keighren M. A., Kinsey J. A., Eaton M., Fincham J. R. Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe. Gene. 1982 Dec;20(3):387–396. doi: 10.1016/0378-1119(82)90207-4. [DOI] [PubMed] [Google Scholar]
- Kuiper M. T., Akins R. A., Holtrop M., de Vries H., Lambowitz A. M. Isolation and analysis of the Neurospora crassa Cyt-21 gene. A nuclear gene encoding a mitochondrial ribosomal protein. J Biol Chem. 1988 Feb 25;263(6):2840–2847. [PubMed] [Google Scholar]
- Lucas M. C., Jacobson J. W., Giles N. H. Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa. J Bacteriol. 1977 Jun;130(3):1192–1198. doi: 10.1128/jb.130.3.1192-1198.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mann B. J., Akins R. A., Lambowitz A. M., Metzenberg R. L. The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1. Mol Cell Biol. 1988 Mar;8(3):1376–1379. doi: 10.1128/mcb.8.3.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Miller H. Practical aspects of preparing phage and plasmid DNA: growth, maintenance, and storage of bacteria and bacteriophage. Methods Enzymol. 1987;152:145–170. doi: 10.1016/0076-6879(87)52016-x. [DOI] [PubMed] [Google Scholar]
- Newmeyer D. A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can J Genet Cytol. 1970 Dec;12(4):914–926. doi: 10.1139/g70-115. [DOI] [PubMed] [Google Scholar]
- Rothschild H., Suskind S. R. Protoperithecia in Neurospora crassa: technique for studying their development. Science. 1966 Dec 9;154(3754):1356–1357. doi: 10.1126/science.154.3754.1356. [DOI] [PubMed] [Google Scholar]
- Sargent T. D. Isolation of differentially expressed genes. Methods Enzymol. 1987;152:423–432. doi: 10.1016/0076-6879(87)52049-3. [DOI] [PubMed] [Google Scholar]
- Schweizer M., Case M. E., Dykstra C. C., Giles N. H., Kushner S. R. Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5086–5090. doi: 10.1073/pnas.78.8.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Selker E. U., Garrett P. W. DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6870–6874. doi: 10.1073/pnas.85.18.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Staben C., Yanofsky C. Neurospora crassa a mating-type region. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4917–4921. doi: 10.1073/pnas.87.13.4917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- THRELKELD S. F. PANTOTHENIC ACID REQUIREMENT FOR SPORE COLOR IN NEUROSPORA CRASSA. Can J Genet Cytol. 1965 Mar;7:171–173. doi: 10.1139/g65-022. [DOI] [PubMed] [Google Scholar]
- Vollmer S. J., Yanofsky C. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]