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Transcription regulation in the cell occurs in the context of chromatin. It follows that a thorough investigation of the mechanism of transcription regulation must take into account the role of chromatin structure. Through classical and molecular genetic experiments in yeast, great strides have been made in understanding the role of chromatin in eukaryotic gene regulation. To achieve a more detailed understanding of the biochemical mechanism of transcription regulation, a yeast chromatin reconstitution system is needed. This need drove us to develop a yeast core histone purification procedure for the reconstitution of these histones into chromatin templates using components wholly derived from yeast. We have purified native yeast core histones in milligram quantities and we have shown these histones to be competent for reconstitution of chromatin templates using yeast nucleosome assembly protein-1. This accomplishment sets the stage for studies using the full power of yeast as an experimental organism to investigate the role of chromatin in transcription regulation.
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The content of fraction 1 and VW-antigens in Y. pestis cultures grown in different media (yeast-casein medium, yeast medium with Hottinger digest, and yeast medium with sunflower-seed protein) was studied over the course of their growth by means of the antibody neutralization and microprecipitation in agar tests. The media under study were not inferior to the casein sulfuric hydrolysate-based medium used for control in their capacity for ensuring the synthesis of VW-antigens. The maximum accumulation of fraction 1 was observed in yeast medium with sunflower-seed protein. In all media the maximum content of fraction 1 was registered on day 3 of cultivation, and the maximum accumulation of VW-antigens on days 8-9 of incubation at 37 degrees C. The data obtained in this study make it possible to regard fraction 1 and VW-antigens as the secondary metabolites of Y. pestis.
The biosynthesis of glycogen involves multiple proteins that associate with each other and the glycogen macromolecule. In efforts to understand the nature of these proteins, a two-hybrid screen was undertaken to detect proteins able to interact with Gsy2p, a major form of glycogen synthase in Saccharomyces cerevisiae. Two positives expressed proteins derived from genes designated PIG1 and PIG2, on chromosomes XIIR and IXL respectively. PIG1 codes for a protein with 38% identity over a 230 residue segment to Gac1p, a protein thought to be a type 1 protein phosphatase targeting subunit whose loss impairs glycogen synthesis. Pig2p has 30% identify to the protein corresponding to an open reading frame, YER054, on chromosome V. Deletion of PIG1 on its own had little effect on glycogen storage but, in combination with loss of GAC1, caused a more severe glycogen-deficient phenotype than seen in gac1 mutants. This result is consistent with Pig1p being functionally related to Gac1p and we propose that Pig1p may be a type 1 phosphatase regulatory subunit. Delection of PIG2, YER054, or both genes together caused no detectable change in glycogen metabolism under the conditions tested. Gac1p, Pig1p, Pig2p and the YER054p are the only four proteins coded by the yeast genome that share a conserved segment of approximately 25 residues, designated the GVNK motif, that is identifiable also in RGI, the mammalian type 1 phosphatase targeting subunit.
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The yeast Saccharomyces cerevisiae (baker's yeast or budding yeast) is an excellent eukaryotic model system for cellular biology with a well-explored, completely sequenced genome. Yeast cells possess robust systems for osmotic adaptation. Central to the response to high osmolarity is the HOG pathway, one of the best-explored MAP kinase pathways. This pathway controls via different transcription factors the expression of more than 150 genes. In addition, osmotic responses are also controlled by protein kinase A via a general stress response pathway and by presently unknown signaling systems. The HOG pathway partially controls expression of genes encoding enzymes in glycerol production. Glycerol is the main yeast osmolyte, and its production is essential for growth in a high osmolarity medium. Upon hypo-osmotic shock, yeast cells transiently stimulate another MAP kinase pathway, the so-called PKC pathway, which appears to orchestrate the assembly of the cell surface and the cell wall. In addition, yeast cells show signs of a regulated volume decrease by rapidly exporting glycerol through Fps1p. This unusual MIP channel is gated by osmotic changes and thereby plays a key role in controlling the intracellular osmolyte content. Yeast cells also possess two aquaporins, Aqy1p and Aqy2p. The production of both proteins is strictly regulated, suggesting that these water channels play very specific roles in yeast physiology. Aqy1p appears to be developmentally regulated. Given the strong yeast research community and the excellent tools of genetics and functional genomics available, we expect yeast to be the best-explored cellular organism for several years ahead, and osmotic responses are a focus of interest for numerous yeast researchers.
Blastomycosis is a medically important systemic fungal infection of dogs and humans. Phagocytic cells are the first line of cellular defence against B. dermatitidis, and are a prominent feature in the lesions and exudate of canine blastomycosis. The adherence of B. dermatitidis yeast to canine phagocytes, and the effects of such adherence on the growth of B. dermatitidis yeast, has not been previously reported. The results of this study demonstrate that canine complement enhances the adherence of B. dermatitidis yeast to canine macrophages. Initiation of the canine complement cascade by B. dermatitidis yeast appeared to occur predominantly by the classical pathway. Adherence of B. dermatitidis yeast to canine macrophages enhanced the growth of the yeast. In the absence of macrophages, this effect could be duplicated by incubating yeast in conditioned medium from co-cultures of macrophages and yeast. This observation suggests that a soluble factor is involved in the growth enhancement of the yeast, These findings provide new insights into the adherence of B. dermatitidis yeast to canine macrophages, and how adherence influences the proliferation of B. dermatitidis yeast.
The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.
Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes.
Human peripheral blood mononuclear cells were cultured with either replicating yeast phase Histoplasma capsulatum, intact dead yeasts of soluble yeast phase autolysate antigen for induction of lymphocyte blastogenesis. H. capsulatum replicated to the same extent in cultures of lymphocytes from histoplasmin skin test-positive and negative cell donors, but stimulated far greater blastogenesis in the former. Dead H. capsulatum yeasts, which were more easily quantified, caused nearly as much specific blastogenesis as live yeasts. Blastogenesis was induced by as few as 1 dead yeast per 10,000 lymphocytes, indicating the antigenic potency of Histoplasma yeasts. Specific blastogenesis was also caused by soluble yeast autolysate. Thus, replicating H. capsulatum yeasts, dead yeasts and autolytically released soluble yeast phase antigen each stimulated specific blastogenesis of lymphocytes from persons sensitized to H. capsulatum in vivo.
The killer phenomenon of yeasts was investigated in naturally occurring yeast communities. Yeast species from communities associated with the decaying stems and fruits of cactus and the slime fluxes of trees were studied for production of killer toxins and sensitivity to killer toxins produced by other yeasts. Yeasts found in decaying fruits showed the highest incidence of killing activity (30/112), while yeasts isolated from cactus necroses and tree fluxes showed lower activity (70/699 and 11/140, respectively). Cross-reaction studies indicated that few killer-sensitive interactions occur within the same habitat at a particular time and locality, but that killer-sensitive reactions occur more frequently among yeasts from different localities and habitats. The conditions that should be optimal for killer activity were found in fruits and young rots of Opuntia cladodes where the pH is low. The fruit habitat appears to favor the establishment of killer species. Killer toxin may affect the natural distribution of the killer yeast Pichia kluyveri and the sensitive yeast Cryptococcus cereanus. Their distributions indicate that the toxin produced by P. kluyveri limits the occurrence of Cr. cereanus in fruit and Opuntia pads. In general most communities have only one killer species. Sensitive strains are more widespread than killer strains and few species appear to be immune to all toxins. Genetic study of the killer yeast P. kluyveri indicates that the mode of inheritance of killer toxin production is nuclear and not cytoplasmic as is found in Saccharomyces cerevisiae and Kluyveromyces lactis.
Eukaryotic cells possess two high-molecular-mass proteases, the 700 kDa, 20S proteasome, as well as the even larger 1,400 kDa, 26S proteasome. It has been demonstrated that ornithine decarboxylase is degraded, in vitro, by the 26S proteasome that contains the 20S protease as its catalytic core, but not by the free 20S proteasome. Recently, by demonstrating severe inhibition of mouse and yeast ODC degradation in a mutant yeast cell line, defective in the chymotripsin-like activity of the yeast 20S proteasome, we implicated the 20S proteasome in the degradation of ODC, in vivo, in yeast cells. Here we show that the degradation of ODC is also severely inhibited in the mutant yeast cell lines, cim3-1 and cim5-1, containing a specific lesion in subunits that are unique to the yeast 26S proteasome. We therefore, conclude, that as illustrated in vitro, also in intact cells, it is the 26S proteasome, not the free 20S proteasome, that degrades ODC. We also demonstrate, that while deficiency in the proteasome chymotrypsine-like activity (in the yeast pre1-1 mutant) inhibits the degradation of both yeast and mouse ODCs, deficiency in the peptidyl-glutamyl-peptide-hydrolyzing (PGPH) activity inhibits only yeast ODC degradation. Similarly, we have noted that whereas the putative ATPase activity of both the CIM3 and CIM5 subunits is essential for the degradation of mouse ODC, only that of the CIM3 subunit is required for the degradation of yeast ODC. These results suggest differential utilization of individual proteasomal subunits in the recognition and degradation of individual short-lived proteins.
Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4(+) with a frequency of 10(-4) (about 10(3) transformants per mug of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli "shuttle" remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.
The Quantum II Yeast Identification System (Abbott Laboratories) is a microprocessor-based spectrophotometric system for identification of clinical yeast isolates within 24 h. We compared the Quantum II system with the API Yeast Ident (Analytab Products) and the AutoMicrobic System Yeast Biochemical Card (AMS-YBC; Vitek Systems, Inc.) for the identification of 221 clinical yeast isolates, including 120 common clinical isolates (Candida albicans, C. tropicalis, C. parapsilosis, Torulopsis glabrata, and Cryptococcus neoformans) and 101 relatively uncommon clinical isolates. The API 20C (Analytab) was used as the reference system. The Quantum II and AMS-YBC systems correctly identified 181 (82%) and 184 (83%) isolates, respectively, whereas the Yeast Ident system correctly identified 132 (60%) isolates. Of the 120 common clinical isolates, 113 (94%) were correctly identified by Quantum II, 103 (86%) were correctly identified by AMS-YBC, and 83 (69%) were correctly identified by Yeast Ident. Of the 101 uncommon clinical isolates tested, 68 (67%) were correctly identified by Quantum II, 81 (80%) were correctly identified by AMS-YBC, and 49 (49%) were correctly identified by Yeast Ident. The overall accuracy of the Quantum II, AMS-YBC, and API Yeast Ident was not sufficient to recommend any of these systems for routine use in the clinical microbiology laboratory without substantial expansion of the respective data bases.