The genetic basis of variation in susceptibility to infection with Histoplasma capsulatum in the mouse.

The pathogenic fungus Histoplasma capsulatum causes disease ranging from mild to fatal in healthy and immunocompromised humans. Infection rates reach 80% in endemic areas, including the Midwestern United States. We used inbred mice to identify a 300-fold difference in fungal burden. A/J mice showed lower fungal burden and morbidity than C57BL/6J mice, a reversal of the trend observed for many bacterial pathogens. We mapped the differences in fungal burden to discrete locations on chromosomes 1, 6, 15 and 17 with high significance. Substitution of a single resistant chromosome 17 onto the susceptible background was sufficient to lower fungal burden. These loci will allow dissection of the fungal-specific immune program.

Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cerevisiae.

Sterol levels affect the expression of many genes in yeast and humans. We found that the paralogous transcription factors Upc2p and Ecm22p of yeast were sterol regulatory element (SRE) binding proteins (SREBPs) responsible for regulating transcription of the sterol biosynthetic genes ERG2 and ERG3. We defined a 7-bp SRE common to these and other genes, including many genes involved in sterol biosynthesis. Upc2p and Ecm22p activated ERG2 expression by binding directly to this element in the ERG2 promoter. Upc2p and Ecm22p may thereby coordinately regulate genes involved in sterol homeostasis in yeast. Ecm22p and Upc2p are members of the fungus-specific Zn[2]-Cys[6] binuclear cluster family of transcription factors and share no homology to the analogous proteins, SREBPs, that are responsible for transcriptional regulation by sterols in humans. These results suggest that Saccharomyces cerevisiae and human cells regulate sterol synthesis by different mechanisms.

Conversion of a gene-specific repressor to a regional silencer.

In Saccharomyces cerevisiae, gene silencing at the HMR and HML loci is normally dependent on Sir2p, Sir3p, and Sir4p, which are structural components of silenced chromatin. Sir2p is a NAD+-dependent histone deacetylase required for silencing. Silencing can be restored in cells lacking Sir proteins by a dominant mutation in SUM1, which normally acts as a mitotic repressor of meiotic genes. This study found that mutant Sum1-1p, but not wild-type Sum1p, associated directly with HM loci. The origin recognition complex (ORC) was required for Sum1-1p-mediated silencing, and mutations in ORC genes reduced association of Sum1-1p with the HM loci. Sum1-1p-mediated silencing also depended on HST1, a paralog of SIR2. Both Sum1-1p and wild-type Sum1p interacted with Hst1p in coimmunoprecipitation experiments. Therefore, the SUM1-1 mutation did not change the affinity of Sum1p for Hst1p, but rather relocalized Sum1p to the HM loci. Sum1-1-Hst1p action led to hypoacetylation of the nucleosomes at HM loci. Thus, Sum1-1p and Hst1p could substitute for Sir proteins to achieve silencing through formation of a compositionally distinct type of heterochromatin.

Overlapping functions of the yeast oxysterol-binding protein homologues.

The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.

DNA replication-independent silencing in S. cerevisiae.

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.

The rye mutants identify a role for Ssn/Srb proteins of the RNA polymerase II holoenzyme during stationary phase entry in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

Membrane biology: membrane-regulated transcription.

The activation of membrane-bound transcription factors involves release from the membrane by proteolysis. Recent studies show that, for some proteins, cleavage is performed by the proteasome, whereas others require specific proteases.

Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+).

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Exclusion of EDNRB and KIT as the basis for white spotting in Border Collies.

BACKGROUND: White spotting patterns in mammals can be caused by mutations in the genes for the endothelin B receptor and c-Kit, whose protein products are necessary for proper migration, differentiation or survival of the melanoblast population of cells. Although there are many different dog breeds that segregate white spotting patterns, no genes have been identified that are linked to these phenotypes. RESULTS: An intercross was generated from a female Newfoundland and a male Border Collie and the white spotting phenotypes of the intercross progeny were evaluated by measuring percentage surface area of white in the puppies. The Border Collie markings segregated as a simple autosomal recessive (7/25 intercross progeny had the phenotype). Two candidate genes, for the endothelin B receptor (EDNRB) and c-Kit (KIT), were evaluated for segregation with the white spotting pattern. Polymorphisms between the Border Collie and Newfoundland were identified for EDNRB using Southern analysis after a portion of the canine gene had been cloned. Polymorphisms for KIT were identified using a microsatellite developed from a bacterial artificial chromosome containing the canine gene. CONCLUSIONS: Both EDNRB and KIT were excluded as a cause of the white spotting pattern in at least two of the intercross progeny. Although these genes have been implicated in white spotting in other mammals, including horses, pigs, cows, mice and rats, they do not appear to be responsible for the white spotting pattern found in the Border Collie breed of dog.

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed.

Identification of SAS4 and SAS5, two genes that regulate silencing in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the alpha-mating phenotype to MATalpha HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration.

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.

Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the Genome Reporter Matrix.

Gene expression profiling is rapidly becoming a mainstay of functional genomic studies. However, there have been relatively few studies of how the data from expression profiles integrate with more classic approaches to examine gene expression. This study used gene expression profiling of a portion of the genome of Saccharomyces cerevisiae to explore the impact of blocks in the isoprenoid biosynthetic pathway on the expression of genes and the regulation of this pathway. Approximately 50% of the genes whose expression was altered by blocks in isoprenoid biosynthesis were genes previously known to participate in the pathway. In contrast to this simple correspondence, the regulatory patterns revealed by different blocks, and in particular by antifungal azoles, was complex in a manner not anticipated by earlier studies.

The boundaries of the silenced HMR domain in Saccharomyces cerevisiae.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains that provide a mechanism to compact the DNA as well as delineate independent units of gene activity. It is believed that insulator/boundary elements separate these domains. Here we report the identification and characterization of boundary elements that flank the transcriptionally repressed HMR locus in the yeast Saccharomyces cerevisiae. Deletion of these boundary elements led to the spread of silenced chromatin, whereas the ectopic insertion of these elements between a silencer and a promoter blocked the repressive effects of the silencer on that promoter at HMR and at telomeres. Sequence analysis indicated that the boundary element contained a TY1 LTR, and a tRNA gene and mutational analysis has implicated the Smc proteins, which encode structural components of chromosomes, in boundary element function.

HMR-I is an origin of replication and a silencer in Saccharomyces cerevisiae.

There appear to be fundamental differences between the properties of the silencers at HML and HMR, with some being origins of replication and others not. Moreover, past studies have suggested that HMR-I's role in silencing may be restricted to plasmid contexts. This study established that HMR-I, like HMR-E and unlike either HML silencer, is an origin of replication. Moreover, both HMR-E and HMR-I contribute to silencing of a chromosomal HMR locus. In addition, we found that Abf1p plays no unique role in silencer function.

A second-generation genetic linkage map of the domestic dog, Canis familiaris.

Purebred strains, pronounced phenotypic variation, and a high incidence of heritable disease make the domestic dog uniquely suited to complement genetic analyses in humans and mice. A comprehensive genetic linkage map would afford many opportunities in dogs, ranging from the positional cloning of disease genes to the dissection of quantitative differences in size, shape, and behavior. Here we report a canine linkage map with the number of mapped loci expanded to 276 and 10-cM coverage extended to 75-90% of the genome. Most of the 38 canine autosomes are likely represented in the collection of 39 autosomal linkage groups. Eight markers were sufficiently informative to detect linkage at distances of 10-13 cM, yet remained unlinked to any other marker. Taken together, the results suggested a genome size of about 27 M. As in other species, the genetic length varied between sexes, with the female autosomal distance being approximately 1.4-fold greater than that of male meioses. Fifteen markers anchored well-described genes on the map, thereby serving as landmarks for comparative mapping in dogs. We discuss the utility of the current map and outline steps necessary for future map improvement.