Chapter 12 Genetic Mapping in Yeast

Mortimer, Robert; Hawthorne, Donald C.

doi:10.1016/s0091-679x(08)60325-8

Cited by 169 publications

(77 citation statements)

References 47 publications

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“…Techniques described by Mortimer and Hawthorne [26] Replacement at the chromosomal locus of the wild type FUR4 gene by the in vitro Xbal-Xbal-deleted allele…”

Section: Measurement Of Galactokinase Activitymentioning

confidence: 99%

Primary structure of the uracil transport protein of Saccharomyces cerevisiae

Jund

Weber

Chevallier

1988

European Journal of Biochemistry

111

104

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We present in this paper the nucleotidic sequence of the FUR4 gene encoding the uracil permease in the yeast Saccharomyces cerevisiae. The deduced amino acid sequence of the permease has 633 residues; it consists of many hydrophobic stretches, only the N-terminal and C-terminal ends of the protein (about 100 and 50 amino acids respectively) being mostly hydrophilic. No N-terminal hydrophobic signal peptide is present, although it is shown in this work that the biosynthesis of the uracil permease goes through the secretion/glycosylation pathway. Using the results of three different methods, allowing the prediction of transmembrane a helices in proteic sequences, we drew a model of folding of the permease in the membrane.In yeast many specific permeases have been characterized both by genetic and physiological analysis [I, 21. The lunetic properties of this kind of protein, analysed in microorganisms (where the lactose permease of Escherichia coli deserves special mention [3,4]) and in mammalian cells, led to the formulation of phenomenological models of functioning where the concept of a 'carrier' was developed, but the mechanisms of transport at molecular level are poorly understood. This requires a knowledge of structure and organization of the carrier protein. Unfortunately in microorganisms where the genetic approach is feasible, the biochemical and structural analysis of such proteins lags much behind the physiological and genetic analysis owing to both their low concentration in the plasma membrane and their physicochemical properties.Molecular cloning of the gene, allowing genetic amplification and DNA sequencing, is one way to gain insight into the protein circumventing the isolation of the protein as a first stage. Indeed molecular cloning of the lactose permease of E. coli allowed both determination of the primary structure by DNA sequencing and, thanks to overexpression of the cloned gene, purification of the lactose permease. Also the DNAs coding for the human glucose transporter [5] and murine erythrocyte anion-exchange protein [6] were sequenced leading to the complete elucidation of the primary structure of the proteins. In Saccharomyes cerevisiae the DNA sequences of two amino acid permeases were recently published [7-91. In our laboratory two permeases specific for pyrimidine uptake, namely the uracil permease and the purine-cytosine permease, have been previously described [lo, 111. The genes coding for these permeases were cloned [12, 131 and the nucleotide sequence of these genes determined. This paper presents the sequence of the uracil permease translated from the unique reading frame found for the gene. Features of the primary structure of the protein are shown. Use was made of proteinstructure prediction programs and this led to the proposal of a working model of the protein structure, which will be discussed. MATERIALS AND METHODS Strains and mediaYeast strains are isogenic derivatives of the wild-type strain FLIOO (F. Lacroute), except for GRF18 (a gift from G. R. Fink, M. I. T., Cambr...

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“…Techniques described by Mortimer and Hawthorne [26] Replacement at the chromosomal locus of the wild type FUR4 gene by the in vitro Xbal-Xbal-deleted allele…”

Section: Measurement Of Galactokinase Activitymentioning

confidence: 99%

Primary structure of the uracil transport protein of Saccharomyces cerevisiae

Jund

Weber

Chevallier

1988

European Journal of Biochemistry

111

104

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“…Breakage was monitored microscopically and was always more than 95%. The homogenate was centrifuged successively for 10 min at 20,000 x g and for 10 min at 10,000 x g. The resulting supernatant was dialyzed overnight against 30 mM Tris hydrochloride buffer (pH 7.5) and used for the enzyme assay. Glucoamylase activity was assayed at 50°C using soluble starch as a substrate as described previoulsy (24).…”

Section: Strainsmentioning

confidence: 99%

“…However, we could not obtain any evidence for the significance of SGA in sporulation; on the contrary, we observed that diploid cells homozygous for an insertion mutation at SGA did not degrade glycogen but were able to undergo sporulation. (10). The procedure used for the transformation of yeast has been described previously (20).…”

mentioning

confidence: 99%

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

Yamashita¹,

Fukui²

1985

Mol. Cell. Biol.

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In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

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“…ItsS was mapped near the centromere of chromosome XVI (30), and we show here that it is the same as mak6. Mutations in 11 other MAK genes (mak3, 4,7,8,10,11,12,16,18,19, and 27) do not cause cold sensitivity for growth (R. B. Wickner, unpublished data), and among the low temperature-sensitive mutants of Singh and Manney, only ltsS (mak6) shows the mak-phenotype (30). The ski mutants were first discovered based on their increased production of the K1 toxin (the superkiller phenotype) (18).…”

mentioning

confidence: 99%

Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.

Ridley¹,

Sommer²,

Wickner³

1984

Mol. Cell. Biol.

145

158

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In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30°C but not at 20°C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mksl and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30°C and prevent the cells from growing at 8°C. Elimination of M double-stranded RNA from a coldsensitive ski-strain results in the loss of cold sensitivity. ski-[KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-kl] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski-strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI' products.The killer system of Saccharomyces cerevisiae is unique among eucaryotic virus and plasmid systems in the detail in which interactions of viral and host components have been explored and in the large number of host components shown to be involved. This probably reflects the ease with which these aspects can be investigated in S. cerevisiae rather than any difference in the degree to which viral and host functions are intertwined. Numerous examples are known of host restriction of viral growth in mammalian systems, but analysis of the host genes involved is generally not practical. We show here that overproduction of a S. cerevisiae virus, due to a host mutation, results in host pathology, apparently due to over-utilization by the virus of a specific, essential host component.

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Chapter 12 Genetic Mapping in Yeast

Cited by 169 publications

References 47 publications

Primary structure of the uracil transport protein of Saccharomyces cerevisiae

Primary structure of the uracil transport protein of Saccharomyces cerevisiae

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.

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