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J N Strathern

Publications and source records attributed to J N Strathern.

49 records · Page 3Linked to original sources

A position-effect control for gene transposition: state of expression of yeast mating-type genes affects their ability to switch.

Mating-type switches of the yeast Saccharomyces cerevisiae occur by unidirectional transposition of copies of unexpressed mating-type genetic information, residing at HML and HMR loci, into the expressed MAT locus. The HML and HMR loci remain unchanged. In contrast, in appropriate strains where the silent loci are also allowed to express, for example in mar mutants, efficient switches of HML and HMR are shown to occur at rates equivalent to those observed for MAT. Thus the position-effect control on the direction of transposition is affected by the state of expression of the locus under study the expressed loci switch regardless of their location.

Crosses, Genetic↗

Evidence for a physical interaction between the transposed and the substituted sequences during mating type gene transposition in yeast.

Mating type switches in the yeast Saccharomyces cerevisiae occur by transposition of a replica of the "source" unexpressed loci HML and HMR to the mating type locus (MAT). The incoming information replaces previously expressed DNA, resulting in an interconversion of MAT alleles. A strain of genotype HML alpha/HML alpha MAT alpha/mata-missense HMR alpha/hmra-nonsense HO/ho generates cells with the genotype HML alpha/HML alpha MAT alpha/MAT a HMR alpha/hmra-nonsense HO/ho; that is, wild-type MATa+ recombinants are produced efficiently by a strain in which the incoming a information and the resident mata allele bear different mutations. Production of the wild-type MATa recombinants requires the homothallism (switching) function, and the incoming a information and the resident mata allele must bear different mutations. This result is consistent with the formation of a heteroduplex between the incoming and the outgoing DNA at MAT. Thus a process of unidirectional gene conversion as a mechanism for mating type gene transposition is favored. A molecular model based on a single-strand transfer is proposed. Results also favor the idea that the direction of switching is controlled by cell's mating phenotype rather than by the genetic content of MAT.

Alleles↗

Structure and organization of transposable mating type cassettes in Saccharomyces yeasts.

Cell type in Saccharomyces yeasts is regulated by two transposable blocks of DNA, the a and alpha cassettes. There are three loci where either cassette can exist. At the HML and HMR loci the cassettes are not expressed. The cassette at the MAT locus is expressed and controls the cell type. Changes of cell type involve transposition-substitution of cassettes from HML or HMR into MAT. We recently reported the molecular cloning of the alpha cassette at the HML locus, HML alpha, and showed that it contained sequences homologous to HMR and MAT. Using HML alpha as a hybridization probe, we have isolated HMLa, HMR alpha, HMRa, MAT alpha, and MATa. Heteroduplex analysis and restriction endonuclease mapping studies indicate that the a and alpha cassettes differ by a substitution corresponding to about 750 base pairs in alpha and about 600 base pairs in a. The HML, HMR, and MAT loci have regions of homology flanking the position of the a versus alpha substitution. We have used specific chromosome rearrangements fusing MAT and HML and MAT with HMR to orient the cloned sequences on the genetic map and have found that all three genes have the same left-to-right polarity on the chromosome.

Alleles↗

Precise mapping of the homothallism genes HML and HMR in Saccharomyces cerevisiae.

The HML and HMR loci carry unexpressed copies of MATa and MAT alpha information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).

Chromosome Mapping↗

Transposable mating type genes in Saccharomyces cerevisiae.

A functional copy of the alpha mating type gene of Saccharomyces cerevisiae has been cloned by transformation in yeast. Using the Southern Blotting procedure it has been shown that three distinct genetic loci implicated in mating type interconversion (HML, HMR and MAT) contain sequences homologous to the clone fragment. The restriction fragment associated with each locus exhibits a characteristic size which can be correlated with the mating type allele present at that locus. The characteristic size difference between the a and alpha genetic elements made it possible to demonstrate that the homothallic interconversion of mating types in this yeast occurs by DNA rearrangement as proposed in the 'cassette hypothesis'.

Chromosome Mapping↗

Isolation of a circular derivative of yeast chromosome III: implications for the mechanism of mating type interconversion.

We describe genetic and physical characterization of rearrangements of chromosome III which result in changes of cell type in S. cerevisiae. Two types of rearrangements were obtained as rare events which caused a change at the locus controlling cell type, MAT, associated with a recessive lethal mutation, in one case from MATalpha to MATa-lethal, and in the other case from MATa to MATalpha-lethal. The MATa-lethal mutation is a deletion on the right arm of chromosome III, which we demonstrate extends to (or near) HMalpha. We suggest this deletion removes MATalpha and activates cryptic MATa information stored in HMalpha as proposed in the cassette model of mating type interconversion. The MATalpha-lethal mutation is the result of the formation of a circular chromosome III, which we interpret to remove MATa and activate the cryptic MATalpha information stored at HMa. Strains carrying the MATalpha-lethal chromosome contain a circular chromosome of length 62.6 plus or minus 5.7 mum, which is absent in related strains. This chromosome was confirmed to be chromosome III by hybridization of specific yeast DNA fragments to supercoiled DNA obtained from MATalpha-lethal strains. The isolation of a large circular derivative of chromosome III allows correlation of genetic and physical distance based on large distances-1 centimorgan corresponds to approximately 2700 base pairs.

Chromosome Aberrations↗

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast.

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement ("cassette" insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as "rules" of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided ("experienced cells"). Spores and buds ("inexperienced cells") rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.

Animals↗

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.

Arginine↗

Healing of mat mutations and control of mating type interconversion by the mating type locus in Saccharomyces cerevisiae.

Homothallic yeasts switch cell types (mating types a and alpha) at high frequency by changing the alleles of the mating type locus, MATa and MATalpha. We have proposed in the cassette model that yeast cells contain silent MATa and MATalpha blocs ("cassettes"), copies of which can be substituted at the mating type locus for the resident information. The existence of silent cassettes was originally proposed to explain efficient switching of a defective MATalpha locus (matalpha) to a functional MATalpha locus. We report here that this "healing" of mat mutations is a general property of the mating type interconversion system and is not specific to the class of matalpha mutations studied earlier: a defective MATa (mata1) switches readily to MATa and various matalpha loci switch readily to MATalpha. These observations satisfy the prediction of the cassette model that all mutations within MATa and MATalpha be healed. These studies also identify MAT functions that control the switching process: the same functions known to promote sporulation and prevent mating in a/alpha cells also inhibit the switching system in a/alpha cells. Finally, we present additional characterization of a natural variant of MATalpha, MATalpha-inc [Takano, I., Kusumi, T. & Oshima, Y. (1973) Mol. Gen. Genet. 126, 19-28] that is insensitive to switching. Our observation that MATalpha-inc acts in cis suggests that it may be altered in a site concerned with excision of MATalpha-inc or its replacement by another cassette.

Journal Article↗

A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci.

A mutation has been identified that suppresses the mating and sporulation defects of all mutations in the mating-type loci of S. cerevisiae. This suppressor, sir1-1, restores mating ability to mat alpha 1 and mat alpha 2 mutants and restores sporulation ability to mat alpha 2 and mata1 mutants. MATa sir1-1 strains exhibit a polar budding pattern and have reduced sensitivity to alpha-factor, both properties of a/alpha diploids. Furthermore, sir1-1 allows MATa/MATa, mat alpha 1/mat alpha/, and MAT alpha/MAT alpha strains to sporulate efficiently. All actions of sir1-1 are recessive to SIR1. The ability of sir1-1 to supply all functions necessary for mating and sporulation and its effects in a cells are explained by proposing that sir1-1 allows expression of mating type loci which are ordinarily not expressed. The ability of sir1-1 to suppress the mat alpha 1-5 mutation is dependent on the HMa gene, previously identified as required for switching of mating types from a to alpha. Thus, as predicted by the cassette model, HMa is functionally equivalent to MAT alpha since it supplies functions of MAT alpha. We propose that sir1-1 is defective in a function. Sir ("Silent-information regulator"), whose role may be to regulate expression of HMa and HM alpha.

Chromosome Mapping↗

Resolution of recombination intermediates generated during yeast mating type switching.

Interchromosomal gene conversion between alleles has been shown in yeast frequently to be associated with the recombination of flanking genetic markers. Although this also holds true for gene conversion between two alleles of the yeast mating-type (MAT) locus, initiated by the homothallic switching system, we find no evidence that crossing-over ever accompanies gene conversion between the non-allelic HMR and MAT genes when initiated by this same homothallic switching system.

Alleles↗