Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1986 Dec;6(12):4516–4525. doi: 10.1128/mcb.6.12.4516

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

K G Coleman, H Y Steensma, D B Kaback, J R Pringle
PMCID: PMC367236  PMID: 3540615

Abstract

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Full text

PDF
4516

Images in this article

4518Image
on p.4518
4519Image
on p.4519
4520Image
on p.4520

Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  3. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  4. Birkenmeyer L. G., Hill J. C., Dumas L. B. Saccharomyces cerevisiae CDC8 gene and its product. Mol Cell Biol. 1984 Apr;4(4):583–590. doi: 10.1128/mcb.4.4.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  6. Breter H. J., Ferguson J., Peterson T. A., Reed S. I. Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39. Mol Cell Biol. 1983 May;3(5):881–891. doi: 10.1128/mcb.3.5.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  8. Burke R. L., Tekamp-Olson P., Najarian R. The isolation, characterization, and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae. J Biol Chem. 1983 Feb 25;258(4):2193–2201. [PubMed] [Google Scholar]
  9. Chisholm G., Cooper T. cis-Dominant mutations which dramatically enhance DUR1,2 gene expression without affecting its normal regulation. Mol Cell Biol. 1984 May;4(5):947–955. doi: 10.1128/mcb.4.5.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
  11. Crowley J. C., Kaback D. B. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene. J Bacteriol. 1984 Jul;159(1):413–417. doi: 10.1128/jb.159.1.413-417.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Falco S. C., Rose M., Botstein D. Homologous Recombination between Episomal Plasmids and Chromosomes in Yeast. Genetics. 1983 Dec;105(4):843–856. doi: 10.1093/genetics/105.4.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gafner J., Robertis E. M., Philippsen P. Delta sequences in the 5' non-coding region of yeast tRNA genes. EMBO J. 1983;2(4):583–591. doi: 10.1002/j.1460-2075.1983.tb01467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gallwitz D., Donath C., Sander C. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product. Nature. 1983 Dec 15;306(5944):704–707. doi: 10.1038/306704a0. [DOI] [PubMed] [Google Scholar]
  15. Genbauffe F. S., Chisholm G. E., Cooper T. G. Tau, sigma, and delta. A family of repeated elements in yeast. J Biol Chem. 1984 Aug 25;259(16):10518–10525. [PubMed] [Google Scholar]
  16. Gottlin-Ninfa E., Kaback D. B. Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jun;6(6):2185–2197. doi: 10.1128/mcb.6.6.2185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hereford L. M., Rosbash M. Number and distribution of polyadenylated RNA sequences in yeast. Cell. 1977 Mar;10(3):453–462. doi: 10.1016/0092-8674(77)90032-0. [DOI] [PubMed] [Google Scholar]
  18. Hereford L., Fahrner K., Woolford J., Jr, Rosbash M., Kaback D. B. Isolation of yeast histone genes H2A and H2B. Cell. 1979 Dec;18(4):1261–1271. doi: 10.1016/0092-8674(79)90237-x. [DOI] [PubMed] [Google Scholar]
  19. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holm C., Meeks-Wagner D. W., Fangman W. L., Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. doi: 10.1016/0378-1119(86)90293-3. [DOI] [PubMed] [Google Scholar]
  21. Hsiao C. L., Carbon J. Direct selection procedure for the isolation of functional centromeric DNA. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3760–3764. doi: 10.1073/pnas.78.6.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ivy J. M., Klar A. J., Hicks J. B. Cloning and characterization of four SIR genes of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Feb;6(2):688–702. doi: 10.1128/mcb.6.2.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaback D. B., Angerer L. M., Davidson N. Improved methods for the formation and stabilization of R-loops. Nucleic Acids Res. 1979 Jun 11;6(7):2499–2317. doi: 10.1093/nar/6.7.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaback D. B., Bhargava M. M., Halvorson H. O. Letter: Location and arrangement of genes coding for ribosomal RNA in Saccharomyces cerevisiae. J Mol Biol. 1973 Oct 5;79(4):735–739. doi: 10.1016/0022-2836(73)90076-4. [DOI] [PubMed] [Google Scholar]
  25. Kaback D. B., Feldberg L. R. Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation. Mol Cell Biol. 1985 Apr;5(4):751–761. doi: 10.1128/mcb.5.4.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kaback D. B., Oeller P. W., Yde Steensma H., Hirschman J., Ruezinsky D., Coleman K. G., Pringle J. R. Temperature-sensitive lethal mutations on yeast chromosome I appear to define only a small number of genes. Genetics. 1984 Sep;108(1):67–90. doi: 10.1093/genetics/108.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kawasaki G., Fraenkel D. G. Cloning of yeast glycolysis genes by complementation. Biochem Biophys Res Commun. 1982 Oct 15;108(3):1107–1122. doi: 10.1016/0006-291x(82)92114-3. [DOI] [PubMed] [Google Scholar]
  29. Kuo C. L., Campbell J. L. Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol Cell Biol. 1983 Oct;3(10):1730–1737. doi: 10.1128/mcb.3.10.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Larkin J. C., Woolford J. L., Jr Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene. Nucleic Acids Res. 1983 Jan 25;11(2):403–420. doi: 10.1093/nar/11.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Last R. L., Stavenhagen J. B., Woolford J. L., Jr Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2396–2405. doi: 10.1128/mcb.4.11.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lauer G. D., Roberts T. M., Klotz L. C. Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes. J Mol Biol. 1977 Aug 25;114(4):507–526. doi: 10.1016/0022-2836(77)90175-9. [DOI] [PubMed] [Google Scholar]
  33. Levy L. S., Manning J. E. Messenger RNA sequence complexity and homology in developmental stages of Drosophila. Dev Biol. 1981 Jul 15;85(1):141–149. doi: 10.1016/0012-1606(81)90243-8. [DOI] [PubMed] [Google Scholar]
  34. Lillie S. H., Pringle J. R. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol. 1980 Sep;143(3):1384–1394. doi: 10.1128/jb.143.3.1384-1394.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lowry C. V., Weiss J. L., Walthall D. A., Zitomer R. S. Modulator sequences mediate oxygen regulation of CYC1 and a neighboring gene in yeast. Proc Natl Acad Sci U S A. 1983 Jan;80(1):151–155. doi: 10.1073/pnas.80.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Maccecchini M. L., Rudin Y., Blobel G., Schatz G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl Acad Sci U S A. 1979 Jan;76(1):343–347. doi: 10.1073/pnas.76.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae, edition 9. Microbiol Rev. 1985 Sep;49(3):181–213. doi: 10.1128/mr.49.3.181-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae. Microbiol Rev. 1980 Dec;44(4):519–571. doi: 10.1128/mr.44.4.519-571.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nagamine Y., Sentenac A., Fromageot P. Selective blotting of restriction DNA fragments on nitrocellulose membranes at low salt concentrations. Nucleic Acids Res. 1980 Jun 11;8(11):2453–2460. doi: 10.1093/nar/8.11.2453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nasmyth K. A., Reed S. I. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nasmyth K. A., Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. doi: 10.1016/s0092-8674(80)80051-1. [DOI] [PubMed] [Google Scholar]
  42. Ohya Y., Miyamoto S., Ohsumi Y., Anraku Y. Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation. J Bacteriol. 1986 Jan;165(1):28–33. doi: 10.1128/jb.165.1.28-33.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Orr-Weaver T. L., Szostak J. W. Fungal recombination. Microbiol Rev. 1985 Mar;49(1):33–58. doi: 10.1128/mr.49.1.33-58.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. doi: 10.1016/0076-6879(83)01017-4. [DOI] [PubMed] [Google Scholar]
  45. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Percival-Smith A., Segall J. Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):142–150. doi: 10.1128/mcb.4.1.142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Philippsen P., Thomas M., Kramer R. A., Davis R. W. Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis. J Mol Biol. 1978 Aug 15;123(3):387–404. doi: 10.1016/0022-2836(78)90086-4. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Rimm D. L., Horness D., Kucera J., Blattner F. R. Construction of coliphage lambda Charon vectors with BamHI cloning sites. Gene. 1980 Dec;12(3-4):301–309. doi: 10.1016/0378-1119(80)90113-4. [DOI] [PubMed] [Google Scholar]
  50. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  51. Rothstein R. J., Sherman F. Genes affecting the expression of cytochrome c in yeast: genetic mapping and genetic interactions. Genetics. 1980 Apr;94(4):871–889. doi: 10.1093/genetics/94.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sandmeyer S. B., Olson M. V. Insertion of a repetitive element at the same position in the 5'-flanking regions of two dissimilar yeast tRNA genes. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7674–7678. doi: 10.1073/pnas.79.24.7674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  54. Shalit P., Loughney K., Olson M. V., Hall B. D. Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae. Mol Cell Biol. 1981 Mar;1(3):228–236. doi: 10.1128/mcb.1.3.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Shore D., Squire M., Nasmyth K. A. Characterization of two genes required for the position-effect control of yeast mating-type genes. EMBO J. 1984 Dec 1;3(12):2817–2823. doi: 10.1002/j.1460-2075.1984.tb02214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  58. Sprague G. F., Jr Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity. J Bacteriol. 1977 Apr;130(1):232–241. doi: 10.1128/jb.130.1.232-241.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. St John T. P., Davis R. W. The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol. 1981 Oct 25;152(2):285–315. doi: 10.1016/0022-2836(81)90244-8. [DOI] [PubMed] [Google Scholar]
  60. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  61. Strathern J. N., Newlon C. S., Herskowitz I., Hicks J. B. Isolation of a circular derivative of yeast chromosome III: implications for the mechanism of mating type interconversion. Cell. 1979 Oct;18(2):309–319. doi: 10.1016/0092-8674(79)90050-3. [DOI] [PubMed] [Google Scholar]
  62. Struhl K., Davis R. W. Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol. 1981 Nov 5;152(3):535–552. doi: 10.1016/0022-2836(81)90267-9. [DOI] [PubMed] [Google Scholar]
  63. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wilkinson L. E., Pringle J. R. Transient G1 arrest of S. cerevisiae cells of mating type alpha by a factor produced by cells of mating type a. Exp Cell Res. 1974 Nov;89(1):175–187. doi: 10.1016/0014-4827(74)90200-6. [DOI] [PubMed] [Google Scholar]
  67. Wilson K. L., Herskowitz I. Negative regulation of STE6 gene expression by the alpha 2 product of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2420–2427. doi: 10.1128/mcb.4.11.2420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Woolford J. L., Jr, Hereford L. M., Rosbash M. Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae. Cell. 1979 Dec;18(4):1247–1259. doi: 10.1016/0092-8674(79)90236-8. [DOI] [PubMed] [Google Scholar]
  69. Yeh E., Carbon J., Bloom K. Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jan;6(1):158–167. doi: 10.1128/mcb.6.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yoo H. S., Genbauffe F. S., Cooper T. G. Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae. Mol Cell Biol. 1985 Sep;5(9):2279–2288. doi: 10.1128/mcb.5.9.2279. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES