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FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca 2+ -dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca 2+ /calmodulin-dependent...
In order to characterize individual protein components of the mitochondrial (mt) ribosome for regulatory, functional and evolutionary studies, the yeast nuclear gene MRP-L4 (accession No. Z30582), coding for the mt ribosomal protein (MRP) YmL4, has been cloned using oligodeoxyribonucleotides (oligos) deduced from a partial amino acid (aa) sequence [Graack et al., FEBS Lett. 242 (1988) 4-8] as screening...
A Saccharomyces cerevisiae GAL7 expression vector for the production of protein fusions to glutathione S-transferase (GST) has been constructed. Using this vector, a GST fusion to human papillomavirus type 6 (HPV-6) E7 protein was produced and purified by affinity chromatography in a single step, at a yield of 2 μg/ml of culture. The E7 portion of the fusion protein was phosphorylated, in contrast...
We report the isolation and sequencing of MTJ1, a 1792-bp cDNA from an M27 murine lung carcinoma cell line. The largest ORF within MTJ1 encodes a 63869-Da protein, containing a 73-amino-acid (aa) sequence (the J domain) that is conserved in proteins of the DnaJ family of chaperonins. The J domain of MTJ1 is bracketed by potential transmembrane domains in a similar configuration to the J domain of...
This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus [Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx...
CDC54 is a gene essential for initiation of DNA replication in Saccharomyces cerevisiae, and which is known to genetically interact with other regulators of the S-phase, including CDC46. We describe the isolation and sequencing ofCDC54 and show that it encodes a protein structurally related to Cdc46p, Mcm2p and Mcm3p by the presence of a conserved domain of 145 amino acids which is internal to each...
Candida albicans (Ca) FAS2, the gene encoding the α-subunit of fatty acid synthase (FAS), has been isolated and characterized. Saccharomyces cerevisiae (Sc) FAS2 was used as a probe to screen genomic libraries of Ca, strain 4918. Clones were obtained that contained all but the first 1 kb of the gene. The 5 end, as well as upstream sequences, were subsequently isolated by PCR. The Ca FAS2 gene...
An expression system for Saccharomyces cerevisiae (Sc) has been developed which, depending on the chosen vector, allows the constitutive expression of proteins at different levels over a range of three orders of magnitude and in different genetic backgrounds. The expression system is comprised of cassettes composed of a weak CYC1 promoter, the ADH promoter or the stronger TEF and GPD promoters, flanked...
Using degenerate oligodeoxyribonucleotides based on the N-terminal amino acid (aa) sequence of a yeast glutathione reductase (GR) CNBr-generated peptide fragment and a conserved C-terminal region of known GR aa sequences, the yeast gene encoding GR, GLR1, was isolated using PCR followed by screening of a yeast genomic DNA plasmid library. GLR1 encodes a 467-aa protein with a deduced M r of...
The acidic transcriptional activation domain of the herpes virus activator VP16 requires an accessory protein complex for function, termed an adaptor. Although the activation domain of the yeast activator HAP4 is also highly negatively charged, its function is independent of at least one component of the adaptor complex, ADA2. In this study, we have used an in vitro inhibition assay to determine...
We report the genetic and biochemical properties of a staurosporine (ST)- and temperature-sensitive mutant, sst10, of Saccharomyces cerevisiae. The stt10 mutant shows an osmoremedial phenotype in a medium with 1 M sorbitol. ST sensitivity of the stt10 mutant was suppressed by overexpression of PKC1STT1, showing the genetic interactions ofSTT10 with the PKC1STT1 pathway. The nucleotide sequence of...
A short stretch of strong homology between the Saccharomyces cerevisiae chromosome segregation protein Mif2 and the DNA-binding motifs of the Drosophila D1 and mammalian HMGI(Y) chromosomal proteins suggested that Mif2 may act directly on chromosomes. Because this conserved motif is involved in binding A T DNA, it was proposed that Mif2 may interact with chromosomes at the highly A + T-rich DNA...
Mutations of genes involved in the STT1PKC1 pathway in yeast show staurosporine and temperature sensitivities (stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis...
The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p),...
We describe the construction of a new vector, pWITCH, designed to facilitate the characterisation of proteins encoded by novel cDNAs isolated using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while translation of the expressed cDNAs results in proteins...
ARS2 and ARS3 are two Candida albicans (Ca) DNA fragments with autonomous replicating activity that have been shown to promote non-integrative genetic transformation of both Ca and Saccharomyces cerevisiae (Sc). We have developed several shuttle vectors based on either ARS fragment, or the combination of both, and using the CaURA3 gene as a selection marker. The combination of ARS2 and ARS3 fragments...
The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading α-amylase (αAmy) that liberates reducing groups from glucose polymers containing both α-1,4 and α-1,6 bonds. The LKA1 gene encoding this industrially important αAmy was cloned as a 2261-bp cDNA fragment from a glucose-depressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment...
An 1.7-kb Arabidopsis thaliana (At) cDNA was isolated by complementation of a bap1 mutation affecting the transport of branched-chain amino acids (aa) in the yeast Saccharomyces cerevisiae. The determination of the nucleotide (nt) sequence revealed an open reading frame of 1383 nt which may encode a protein of 461 aa with a predicted molecular mass of 51 038 Da. The deduced aa sequence exhibited...
The yeast PDR1 locus encodes a member of the C6 zinc cluster family of transciptional regulatory proteins. Among the targets of PDR1 is the yeast PDR5 locus. The product of this gene is a member of the ATP-binding cassette (ABC) transmembrane protein family and plays a major role in inhibitor efflux. Mutations in PDR1 affect the relative level ofPDR5 transcript and can therefore result in increased...
The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm). The originally characterized Rm-resistant (Rm R ) Tor1-1 and Tor2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of...
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