Summary
In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 is mutations. These plasmids also complement the is growth defect of additional independent mutants identified as new prp6 and prp9 is alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the is complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.
Similar content being viewed by others
References
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Brody E, Abelson J (1985) The spliceosome: yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science 228:963–967
Chang T-H, Clark MW, Lustig AJ, Cusick ME, Abelson J (1988) RNAll protein is associated with the yeast spliceosome and is located in the periphery of the cell nucleus. Mol Cell Biol 8:2379–2393
Green MR (1986) Pre-mRNA splicing. Annu Rev Genet 20:671–708
Hartwell LH, McLaughlin CS, Warner JR (1970) Identification of ten genes that control ribosome formation in yeast. Mol Gen Genet 109:42–56
Hinnen A, Hicks J, Fink GR (1978) Transformation in yeast. Proc Nall Acad Sci USA 75:1929–1932
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Legrain P, Rosbash M (1989) Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57:573–583
Legrain P, Choulika A (1990) The molecular characterization of PRP6 and PRP9 yeast genes reveals a new cysteine/histidine motif common to several splicing factors. EMBO J 9:2775–2781
Legrain P, Seraphin B, Rosbash M (1988) Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol Cell Biol 8:3755–3760
Losson R, Lacroute F (1979) Interference of nonsense mutations with eukaryotic messenger RNA stability. Proc Natl Acad Sci USA 76:5134–5137
Lustig AJ, Lin R-J, Abelson J (1986) The yeast RNA gene products are essential for mRNA splicing in vitro. Cell 47:953–963
Maniatis T, Reed R (1987) The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature 325:673–678
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Mortimer RK, Schild D, Contopoulou CR, Kans JA (1989) Genetic map of Saccharomyces cerevisiae, edition 10. Yeast 5:321–403
Pikielny CW, Rymond BC, Rosbash M (1986) Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. Nature 324:341–345
Rosbash M, Harris PKW, Woolford Jr JL, Teem JL (1981) The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell 24:679–686
Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243
Rothstein RJ (1983) One step gene disruption in yeast. Methods Enzymol 101: 202–224
Seraphin B, Rosbash M (1989) Identification of functional U1 snRNA: pre-mRNA complexes committed to spliceosome assembly and splicing. Cell 59:349–358
Seraphin B, Kretzner L; Rosbash M (1988) A U1 snRNA: premRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J 7:2533–2538
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Vijayraghavan U, Abelson J (1990) PRP18, a protein required for the second reaction in pre-mRNA splicing. Mol Cell Biol 10:324–332
Vijayraghavan U, Company M, Abelson J (1989) Isolation and characterization of pre-mRNA splicing mutants. Genes Dev 3:1206–1216
Whittaker E, Lossky M, Beggs JD (1990) Affinity purification of spliceosomes reveals that the precursor RNA processing protein PRP8, a protein in the U5 small nuclear ribonucleoprotein particle, is a component of yeast spliceosomes. Proc Nall Acad Sci USA 87:2216–2219
Woolford Jr JL (1989) Yeast pre-mRNA splicing. Yeast 5:439–457
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Legrain, P., Chapon, C., Schwob, E. et al. Cloning of the two essential yeast genes, PRP6 and PRP9,and their rapid mapping, disruption and partial sequencing using a linker insertion strategy. Molec. Gen. Genet. 225, 199–202 (1991). https://doi.org/10.1007/BF00269848
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00269848