Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.     

Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae.

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.     

Genetic mapping of the integrin alpha 1 gene (Vla1) to mouse chromosome 13.

The integrin alpha 1 chain (Vla1) associates with the beta 1 chain to form a heterodimer that functions as a dual laminin/collagen receptor in neural cells and hematopoietic cells. We have used an interspecies backcross gene-mapping technique to map the Vla1 gene to the distal end of chromosome 13 in the mouse genome. The Vla1 locus is located 3.5 cM distal to Ctla-3 and 7.8 cM distal to Htrla. We have further characterized this locus in recombinant inbred (RI) mice by examining the strain distribution patterns of nine genomic DNA restriction fragment length variants detected with alpha 1 cDNA probes. The RI gene mapping did not show linkage to previously mapped genes or mutants in the AXB, BXA, or AKXD RI sets and therefore defines a new genetic marker for the distal end of chromosome 13 in these RI sets.     

Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker

Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40–80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40–80 copies of its chromosome per cell, we have developed an in vivo chromosome counting technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this In vivo chromosome counting technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.     

荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立

建立一种精确定量人胚胎干细胞线粒体DNA拷贝数的方法。构建包含线粒体DNANDl和核单拷贝基β-globin基因序列的重组质粒作为标准品;收集无饲养层培养体系下人胚胎干细胞DNA样本,结合2个单独的Taqman探针实时荧光定量PCR对待测样本中线粒体NDl和核β-globin基因分别进行定量,从而对人胚胎干细胞线粒体DNA的含量进行了精确定量。结果提示,人胚胎干细胞线粒体DNA的平均拷贝数／细胞为1321±228。研究表明,该技术可对人胚胎干细胞线粒体DNA拷贝数进行准确的测定,为研究培养条件对人胚胎干细胞线粒体DNA拷贝数的影响及优化体外培养条件奠定了基础。     

Duplications and deletions of Vh genes in inbred strains of mice

evolution of variable region (Vh) gene family copy number and polymorphism was investigated by the analysis of the immunoglobulin heavy chain variable region (Igh-V) locus in 74 inbred strains and substrains of mice. Several strains were found to have slight differences from Igh-V haplotypes previously identified, usually of a single Vh gene family. These results indicate that the evolution of copy number in the mouse Igh-V locus proceeds largely by the accumulation of incremental changes, reflecting the clustered organization of the mouse Igh-V locus. We have found no evidence of very large or frequent duplication or deletion events indicative of rapid expansion or contraction processes. The existence of one or more particularly large Vh gene families most likely reflects random copy number variation, rather than selection for the amplification of their members. The identification of strains with recombinant Vh gene arrays demonstrates that recombination, both within and between haplotypes, appears to be the predominant mechanism generating the high restriction fragment length polymorphism in the Igh-V locus.Abbreviations used in this paper Igh-V immunoglobulin heavy chain variable region locus - Vh heavy chain variable region gene - Dh heavy chain diversity region gene - V immunoglobulin kappa light chain variable region gene - V T-cell receptor beta chain variable region gene     

Adaptor complex-independent clathrin function in yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.     

Yeast clathrin has a distinctive light chain that is important for cell growth

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.     

Sensitivity to the Yeast Plasmid 2mu DNA is conferred by the nuclear allele nibl.

Two strains of Saccharomyces carlsbergensis that lacked the plasmid 2mu DNA responded differently when the plasmid was introduced into them. In one strain, cells lacking 2mu DNA ("cir0") produced the normal "smooth" colony morphology, but cells bearing 2mu DNA ("cir+") produced heterogeneous "nibbled" colonies. In the second strain, both cir+ and cir0 strains exhibited a smooth colony morphology. Crosses between these strains revealed that a single recessive nuclear gene, called nibl, conferred the nibbled colony morphology in the presence of 2mu DNA. By a series of backcrosses, nibl was introduced into a Saccharomyces cerevisiae background. nibl caused a nibbled colony morphology in this background just as it did in S. carlsbergensis. nibl was mapped to the left arm of chromosome XVI. Twelve independent smooth revertants were isolated from two nibl [cir+] strains. Seven were analyzed, and all were found to be chromosome VII disomes. Chromosome VII disomy and suppression of the nibbled phenotype cosegregated in crosses. Thus, chromosome VII disomy can suppress the nibbled phenotype. The results of other experiments (C. Holm, Cell 29:585-594, 1982) indicate that the nibbled colony morphology is the result of lethal sectoring and that the lethality is caused by a high copy number of 2mu DNA. I suggest, therefore, that the product of the nibl gene may play a role in controlling the copy number of 2mu DNA. Possible models for the suppression of the nibbled phenotype by chromosome VII disomy are discussed.     

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 ± 920, significantly higher than that in skeletal muscle, 3650 ± 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.     

Chromosome 1 trisomy compromises the virulence of Candida albicans

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.     

Satellite DNA and cytogenetic evolution

The genusDipodomys (kangaroo rats) exhibits major interspecies variations in the proportions of highly reiterated satellite DNA sequences in the genome as well as in the chromosome number and the proportions of uni-armed and bi-armed chromosomes. For nearly all of the approximately 22 species of the genus and several subspecies, liver DNA was distributed in neutral CsCl buoyant density gradients into four fractions: principal DNA (1.698 g/ml), intermediate-density DNA (1.702 g/ml), MS satellite (1.707 g/ml) and HS (heavy) satellites (1.713 g/ml). The total nuclear DNA content of diploid liver cells measured in eleven species by quantitative cytophotometry, ranged from 6.9 to 10.9 pg. These data were correlated with known features of the karyotypes of individual species. The salient findings were: (1) that interspecies variations in diploid chromosome number cluster at 52–54, 60–64 and 70–72 (2) that high total nuclear DNA was associated with high chromosome number, and with relatively large amounts of satellite DNA (3) that a high ratio of HS satellites to intermediate-density DNA was generally correlated with a predominance of metacentric and submetacentric chromosomes (high fundamental number). The relationships of satellite DNA to karyotype structure reveal a new level of hierarchy in the genome that appears capable of exerting global control over environmental adaptation and the evolution of new species. This mechanism is consistent with recent hypotheses that changes in the macro-structure of the genome are more important than point mutations in facilitating the rapid phases of animal evolution.     

Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.     

A high-resolution map of segmental DNA copy number variation in the mouse genome

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.