Functional analysis of the genes of yeast chromosome V by genetic footprinting

Science

Volumn 274, Issue 5295, 1996, Pages 2069-2074

  Smith, Victoria ^a   Chou, Karen N ^a   Lashkari, Deval ^a   Botstein, David ^a   Brown, Patrick O ^a  

^a STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ARTICLE; BASE PAIRING; CHROMOSOME 5; DNA FOOTPRINTING; GENE DISRUPTION; GENE INDUCTION; GENE INSERTION; GENETIC ANALYSIS; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; TELOMERE; TRANSPOSON;

CHROMOSOMES, FUNGAL; CLONING, MOLECULAR; CULTURE MEDIA; DNA FOOTPRINTING; DNA TRANSPOSABLE ELEMENTS; DNA, FUNGAL; GENE LIBRARY; GENES, FUNGAL; MUTAGENESIS, INSERTIONAL; PHENOTYPE; POLYMERASE CHAIN REACTION; SACCHAROMYCES CEREVISIAE;

ANIMALIA; SACCHAROMYCES CEREVISIAE;

EID: 0030450059     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2069     Document Type: Article

References (45)

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4. F. Dietrich et al., Nature, in press. In subsequent releases, additional telomeric sequences were added, making the full length of chromosome V 574,860 bp [updated in the Martinsried Institute for Protein Sequences (MIPS) Yeast Genome Database]. The number of predicted genes is derived from Dietrich et al. and from the MIPS database. Most of the possible additional and alternative open reading frames noted in the MIPS database were also covered by the primers used in this study. One to three independent gene-specific primers were used to analyze each gene. Primers were designed with the use of the program PRIMER (Whitehead Institute for Biomedical Research, Cambridge, MA) with a specified melting temperature of 69° to 73°C. Primers were synthesized by means of a 96-well array synthesizer [D. A. Lashkari, S. P. Hunicke-Smith, R. Norgren, R. W. Davis, T. Brennan, Proc. Natl. Acad. Sci. U.S.A. 92, 7912 (1995)] and labeled at their 5′ end with 5-carboxyfluorescein (Applied Biosystems or Pharmacia). Approximately 85% of primers produced usable data. Alternative primers were synthesized for any gene for which the first primer failed to produce usable data. Each labeled gene-specific primer was used in a PCR with an unlabeled Ty1 -specific primer, as described (2).
5. F. Dietrich et al., Nature, in press. In subsequent releases, additional telomeric sequences were added, making the full length of chromosome V 574,860 bp [updated in the Martinsried Institute for Protein Sequences (MIPS) Yeast Genome Database]. The number of predicted genes is derived from Dietrich et al. and from the MIPS database. Most of the possible additional and alternative open reading frames noted in the MIPS database were also covered by the primers used in this study. One to three independent gene-specific primers were used to analyze each gene. Primers were designed with the use of the program PRIMER (Whitehead Institute for Biomedical Research, Cambridge, MA) with a specified melting temperature of 69° to 73°C. Primers were synthesized by means of a 96-well array synthesizer [D. A. Lashkari, S. P. Hunicke-Smith, R. Norgren, R. W. Davis, T. Brennan, Proc. Natl. Acad. Sci. U.S.A. 92, 7912 (1995)] and labeled at their 5′ end with 5-carboxyfluorescein (Applied Biosystems or Pharmacia). Approximately 85% of primers produced usable data. Alternative primers were synthesized for any gene for which the first primer failed to produce usable data. Each labeled gene-specific primer was used in a PCR with an unlabeled Ty1 -specific primer, as described (2).
6. Although genetic footprinting analysis of chromosome V had a high success rate, there were some failures. For seven genes (RIP1, PMP2, SWI4, CLK3, GDI1, BRR2, and YER182w), PCR reactions that used two different gene-specific primers failed to produce enough amplified products to allow meaningful data interpretation, in some cases, products corresponding to insertions upstream of the start ATG codon were readily detected, but products representing insertions in the coding sequence were not reproducibly detected. This may reflect a low frequency of Ty1 insertion in the coding sequences of these genes. It is also possible that Ty1 insertions in essential genes may not be detected, even in the time-zero sample, because of selection during the mutagenesis (10). However, at least some of the genes for which no insertions could be detected are known from previous work not to be essential for vegetative growth, so the latter hypothetical explanation cannot account for all failures resulting from insufficient signal. In almost all cases, when two independent primers for any particular gene both produced Ty1 -dependent signal upon PCR analysis, a very similar distribution of PCR products was obtained. In three cases, however, two independent primers for the same gene produced apparently credible data yet gave different results by genetic footprinting analysis (YEL044W, AFG3, and RSP5; Fig. 1). On the basis of the relative frequency of these cases of discordant data produced by two gene-specific primers relative to the frequency of concordant result, we estimate that each analysis using a single primer has a 2 to 3% chance of producing unreliable data.
7. 1 these genes were generally located in the immediate vicinity of tRNA genes. Preferred upstream sites for Ty1 transposition frequently generated a cluster of PCR products with 100 to 1000 times the signal typically observed. This raises the possibility of exhaustion of PCR reagents at a stage earlier than 30 cycles, because of the increased number of initial template molecules. It is thus possible that some of the less abundant, smaller DNA products observed in these cases, which would ordinarily be assumed to represent insertions in the coding sequence of the gene, could be artifactual. In cases of this kind, PCR was repeated with only 23 cycles in an attempt to minimize this possible artifact. In addition, new primers that did not encompass the preferred site were also used; typically, these primers were located in the first 100 bp of the gene and were directed down-stream, toward the stop codon. These "reverse" primers typically allowed a more reliable survey of products corresponding to insertions in coding sequences of genes with very strong upstream site preferences for Ty1 insertion.
8. 8 cells were stored in 25% glycerol at -80°C. After one mutagenesis, cells were immediately transferred to rich medium for growth, without intervening storage. The DNA isolated from these cells was analyzed with primers specific to every gene for which a quantitative growth defect was detected. No cases were found in which storage in glycerol, freezing and resuscitation, or both accounted for the apparent growth defect, although possible contributions of these procedures to some phenotypes are noted in Fig. 1.
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45. We thank R. Norgren for assistance with data analysis and the generation of Fig. 1, and L. McAllister and K. Davis for discussions. Supported by NIH grant 1PO1 HG00205-05 (to R. Davis, P.O.B., and D.B.) and by the Howard Hughes Medical Institute (P.O.B.). Supported in part by grant LT-141/93 from the Human Frontier Science Program Organization to V.S. P.O.B. is an assistant investigator of the Howard Hughes Medical Institute.