Molecular analysis of UAS(E), a cis element containing stress response elements responsible for ethanol induction of the KlADH4 gene of Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we report the molecular analysis of UAS(E), an element of the KlADH4 promoter which is essential for the induction of KlADH4 in the presence of ethanol. UAS(E) contains five stress response elements (STREs), which have been found in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress. Whereas KlADH4 is not responsive to stress conditions, the STREs present in UAS(E) seem to play a key role in the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae. Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) under non-inducing conditions. Moreover, we observed that the RAP1 binding site present in UAS(E) binds KlRap1p.     

Isolation,heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.     

Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans

In a previous study, we reported the isolation and characterization of the two-component response regulator SSK1 gene of Candida albicans. This gene is a structural but not a functional homolog of the SSK1 and mcs4(+) genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. In the present study, we have constructed and phenotypically characterized Deltassk1 mutants of C. albicans. The results confirmed our previous observation that CaSSK1, unlike SSK1 or mcs4(+), does not regulate cellular responses to either osmotic or oxidative stress. Instead, Deltassk1 null strains showed severely reduced hyphal formation on serum agar and were totally defective in hyphal development on other solid media, such as medium 199 (pH 7.5) and Spider medium. In contrast, under conditions of low nitrogen availability on solid media, Deltassk1 null strains dramatically hyperinvaded the agar. However, while forming germ tubes and hyphae in liquid media similar to those of the wild type, Deltassk1 null strains flocculated in a manner similar to that of Deltachk1 two-component histidine kinase mutants, which we have previously described. Finally, virulence studies indicated that SSK1 is essential for the pathogenesis of C. albicans, suggesting that the Ssk1p response regulator could be a good target for antifungal therapy.     

A potential hexose transporter gene expressed predominantly in the bloodstream form of Trypanosoma brucei.

A cDNA cloned from Trypanosoma brucei brucei codes for a putative membrane protein which is homologous to the erythrocyte glucose transporter and several other sugar transporters from Escherichia coli, yeast, algae and Leishmania. This cDNA hybridizes to a 2.3-kb mRNA that accumulates to a much higher degree in the bloodstream mammalian form than in the procyclic insect form of the parasite. The correlation between the expression of this gene and the hexose metabolism of Leishmania enriettii and T. brucei suggest that these 2 related genes probably encode hexose transporters. The gene encoding this mRNA is a member of a multigene family. The putative hexose transporter gene is highly conserved among Kinetoplastidae, indicating an important role for this protein in the parasite life cycle.     

Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment

Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.     

Mechanism of interferon action. Expression of reovirus S3 gene in transfected COS cells and subsequent inhibition at the level of protein synthesis by type I but not by type II interferon

The effect of interferon on the expression of the reovirus serotype 1 Lang strain S3 gene was examined in simian COS cells transfected with the expression vector pSVS3 containing the S3 gene under the control of the SV40 late promoter. When COS cells were treated with type I interferon-alpha 24 hr after transfection, the synthesis of the reovirus S3-encoded sigma NS polypeptide was inhibited about 10-fold as compared to that in untreated control cells. By contrast, under the same conditions, neither the plasmid DNA copy number nor the S3 gene mRNA levels were significantly affected by interferon treatment. Type II interferon-gamma, unlike the type I interferons-alpha, did not affect the rate of synthesis of polypeptide sigma NS in pSVS3-transfected cells.     

Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe

The Cks1 protein is a component of the Cdc28 protein kinase in the budding yeast Saccharomyces cerevisiae. This paper reports the cloning of two homologs of the S. cerevisiae CKS1 gene from human cells. These homologs, CKShs1 and CKShs2, both encode proteins of 79 amino acids that share considerable homology at the amino acid level with the products of CKS1 from S. cerevisiae and suc1+ from the fission yeast Schizosaccharomyces pombe. Both human homologs are capable of rescuing a null mutation of the S. cerevisiae CKS1 gene when expressed from the S. cerevisiae GAL1 promoter. S. pombe suc1+ expressed from the GAL1 promoter is also capable of rescuing a S. cerevisiae cks1 null mutation. Ckshs1 or Ckshs2 protein linked to Sepharose beads can bind the Cdc28/Cdc2 protein kinase from both S. cerevisiae and human cells. The CKShs1 and CKShs2 mRNAs are expressed in different patterns through the cell cycle in HeLa cells, which may reflect specialized roles for the encoded proteins.