Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes

Lauer, Gail D.; Roberts, Thomas M.; Klotz, Lynn C.

doi:10.1016/0022-2836(77)90175-9

Cited by 125 publications

(33 citation statements)

References 42 publications

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“…A genomic expression library has been successfully used to isolate yeast nuclear genes (42) and, although the Chlamydomonas genome is approximately seven times the size of the yeast genome (11,17), it is small enough to make screening a genomic library feasible.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

Williams

Mitchell

Rosenbaum

1986

The Journal of Cell Biology

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Abstract. Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in Lgtll. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26~ (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybridselect mRNA from mutant pf-26~ cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26~ mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar ct-tubulin mRNA.

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

Williams

Mitchell

Rosenbaum

1986

The Journal of Cell Biology

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“…Because C. albicans and related species lack sexual cycles (46), genetic studies have been difficult. Analyses of mitotic recombination of auxotrophic markers (47,48), DNA content (32,42), and complexity (32) and chromosome organization by orthogonal field alternating gel electrophoresis (21) indicate that C. albicans possesses a diploid genome organized into about eight chromosome pairs (16,20), with a DNA content and complexity similar to those of bakers' yeast, Saccharomyces cerevisiae (17).…”

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Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification

et al. 1991

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Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida albicans. These include two families of large dispersed repeat sequences (Ca3, Ca24) and a short (23-bp) tandemly repeated element (Ca7) associated with C. albicans telomeres. In addition, a large subtelomeric repeat (WOL17) has been cloned. DNA fragments containing the telomeric repeats are highly variable among different C. albicans strains. We have shown that the Ca3 repeat is relatively more stable and is suitable for use as a species-specific and strain-specffic probe for C. albicans.Candida albicans and related species are responsible for the majority of oral and vaginal yeast infections (la, 26) and continue to increase in frequency as a major cause of systemic infections in immunocompromised hosts (1,4,5,25,30). Because C. albicans and related species lack sexual cycles (46), genetic studies have been difficult. Analyses of mitotic recombination of auxotrophic markers (47, 48), DNA content (32, 42), and complexity (32) and chromosome organization by orthogonal field alternating gel electrophoresis (21) indicate that C. albicans possesses a diploid genome organized into about eight chromosome pairs (16,20), with a DNA content and complexity similar to those of bakers' yeast, Saccharomyces cerevisiae (17).The lack of a sexual phase in C. albicans and related species presumably prevents opportunities for recombination between strains, leading to a direct clonal relationship in the descendants. This characteristic may be useful in clinical analyses of colonization and epidemiological studies, since it allows direct determination of strain relatedness by employing genomic markers. Distinguishing between species and strains is of paramount importance in such studies, since it has been demonstrated that more than one Candida species and more than one strain of a single species can colonize or infect the same individual (9,23,27,28,44). In addition, multiple "switch phenotypes" (growth forms with distinctive cell types and colony morphologies [40,41]) of the same strain can be found at the same site of infection (43). Sugar assimilation and biotyping methods are normally accurate in distinguishing species and, in many cases, strains (15, 29, 31) but can provide false distinctions between switch phenotypes of the same strain (1). Serological typing methods can discriminate between only two C. albicans subtypes (8). Even electrophoretic karyotyping can be misleading, since subclones of single strains can exhibit significant changes in chromosome size or structure over relatively short periods of culture (36,45 Repetitive DNA sequences provide useful markers for strain identification and also provide keys to understanding chromosome structure and organization. Scherer and Stevens (38) reported the isolation of a dispersed middle repetitive DNA sequence from C. albicans and showed that it could be used for fingerprinting strains of that species. We have also generated species-specific repetitive DNA probes that have been ...

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“…In this paper, we describe a project that is aimed at constructing a restriction map of total nuclear DNA from the yeast Saccharomyces, whose haploid genome size has been estimated to be 1.5 x 104 kb (4). The data collection has involved picking X clones at random and measuring the sizes of the restriction fragments generated by a single digest of each clone.…”

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confidence: 99%

Random-clone strategy for genomic restriction mapping in yeast.

Olson¹,

Dutchik²,

Graham³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

362

192

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An approach to global restriction mapping is described that is applicable to any complex source DNA. By analyzing a single restriction digest for each member of a redundant set of A clones, a data base is constructed that contains fragment-size lists for all the clones. The clones are then grouped into subsets, each member of which is related to at least one other member by a significant overlap. Finally, a tree-searching algorithm seeks restriction maps that are consistent with the fragment-size lists for all the clones in each subset. The feasibility of the approach has been demonstrated by collecting data on 5000 A clones containing random 15-kilobase inserts of yeast DNA. It is shown that these data can be analyzed to produce regional maps of the yeast genome, extending in some cases for over 100 kilobases. In combination with hybridization probes to previously cloned genes, these local maps are already useful for defining the physical arrangement of closely linked genes. They may in the future serve as building blocks for the construction of a continuous global map.Fifteen years after the discovery of site-specific restriction endonucleases, the restriction map has become the standard medium for displaying the functional organization of short segments of DNA. For regions spanning up to 50 kilobases (kb), restriction maps are routinely constructed and have become indispensable tools both for organizing existing data and for planning experiments. In a few favorable cases, maps as long as 600 kb have been constructed at a resolution of a few kb (1-3), but it has proven difficult to extend existing mapping methods beyond this range. Given that the average human chromosome contains 105 kb of DNA, there is a gross disparity between the present limits of restriction-mapping techniques and the sequence complexity of cellular genomes.In this paper, we describe a project that is aimed at constructing a restriction map of total nuclear DNA from the yeast Saccharomyces, whose haploid genome size has been estimated to be 1.5 x 104 kb (4). The data collection has involved picking X clones at random and measuring the sizes of the restriction fragments generated by a single digest of each clone. Because enough clones have been analyzed to provide redundant sampling of most regions of the genome, it is possible to extract mapping information from the unordered fragment-size lists by an algorithm that systematically imposes the requirement that the set of restriction fragments generated from a single clone must be contiguous.At the present stage of the yeast project, we have analyzed 5000 X clones with average insert sizes of 15 kb, thereby achieving a sampling redundancy on the order of five. Using the data, it is now possible to construct maps of from 20 to >100 kb around most arbitrary starting points in the yeast genome. These local maps have already defined the physical arrangement of clusters of genes that previously had only been linked genetically, and they point the way toward the development of a global phy...

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Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes

Cited by 125 publications

References 42 publications

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification

Random-clone strategy for genomic restriction mapping in yeast.

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