Temperature-dependent dimorphism of the yeast Arxula adeninivorans Ls3

Arxula adeninivorans Ls3 is described as an ascomycetous, arthroconidial, anamorphic, xerotolerant yeast, which was selected from wood hydrolysates in Siberia. By using minimal salt medium or yeast-extract-peptone-medium with glucose or maltose as carbon source it was shown that this yeast is able to grow at up to 48 degrees C. Increasing temperatures induce changes in morphology from the yeast phase to mycelia depending on an altered programme of gene expression. This dimorphism is an environmentally conditioned (reversible) event and the mycelia can be induced at a cultivation temperature of 45 degrees C. Depending on the morphology of strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium changed. The activities of the accumulated extracellular enzymes glucoamylase and invertase were 2 to 3 times higher in cultures grown at 45 degrees C than in those grown at 30 degrees C. While the level of the glucoamylase protein secreted from mycelia between 45 and 70 hours did not change, biochemical activity decreased after a cultivation time of 43 hours. It was shown that this effect depended on both the catabolic repression of the glucoamylase by glucose and the thermal inactivation of this enzyme in media without or with low concentrations of starch or maltose.     

Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

The glucose-dependent transactivation activity of ABF1 on the expression of the TDH3 gene in yeast

We used titanium anchors for the surgical repair of rotator cuff tears in 34 selected patients, all of whom were < 60 years of age, had good bone quality, and had no known metabolic bone diseases. Nine tears were repaired within 6 months, 15 within 6-12 months, and 10 later than 12 months after injury. Tear size was graded as small (10 patients), medium (15 patients), and large (nine patients) during open operation. After 6-24 months of follow-up, 30 patients reported satisfactory pain relief, function, active forward flexion, and muscle strength [18 excellent and 12 good results based on the University of California at Los Angeles rating system (UCLA scores)]; there were no implant failures (p < 0.001). Two patients had unsatisfactory function but good relief of pain, whereas two patients were dissatisfied with their overall result (four poor results based on UCLA scores). Although trans-bone suturing is presently the most common and successful surgical technique for rotator cuff tears, we found that use of titanium anchors shortens operative time and has results comparable with the traditional technique. Titanium anchors should not be used when bone quality is poor or good patient compliance is doubtful. They are also contraindicated, as our four poor results indicate, when the tear is old (> 6 months) and large (diameter > 5 cm with significant tissue degeneration).     

Identification of the orotidine-5'-phosphate decarboxylase gene and development of a transformation system in the yeast Saccharomyces exiguus Yp74L-3

To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5'-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.     

A novel esterase from Saccharomyces carlsbergensis, a possible function for the yeast TIP1 gene

An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16.9 kDa. The optimal pH for activity was in the range of four to five. Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization. Caprylic acid, which is present in beer, competitively inhibited the esterase. The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms. The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed. N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S. cerevisiae TIP1 gene. The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Lack of association between manic-depressive illness and a highly polymorphic marker from GABRA3 gene

Subcutaneous sarcoidosis appears to be rare and is usually associated with hiliar adenopathy. Finger swelling is well recognized in patients with sarcoidosis and usually results from bony involvement or tenosynovitis. We report a patient with subcutaneous sarcoidosis and dactylitis. Biopsy of a finger and of subcutaneous nodules showed similar features with a granulomatous infiltrate. There were no osseus or tendinous lesions.     

Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.     

The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae

To test the hypothesis that the major irrational evaluative beliefs postulated by Rational Emotive Behavior Therapy are related to marital conflict, 15 married couples participated in a thought-listing procedure. During this procedure, three idiosyncratic scenes portraying marital conflict and three control scenes free of conflict were identified for and presented to each member of the dyad. Analysis indicated that the conflict-portraying scenes were associated with significantly more irrational evaluative beliefs and significantly fewer rational cognitions than the control scenes.     

Identification of the ure1+ gene encoding urease in fission yeast

We describe a case of subacute cor pulmonale caused by tumor embolism from a gallbladder carcinoma in a 63-year-old woman. The patient was admitted to hospital with increasing dyspnea. Physical examination and echocardiography showed signs of pulmonary hypertension. She died of circulatory failure. At autopsy microscopic studies revealed tumor embolism in the pulmonary vessels and subsequent lesions causing the lethal pulmonary hypertension. This is the first case report of pulmonary hypertension caused by embolism from a gallbladder carcinoma in the literature worldwide.     

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation

The aim was to describe the appearance of the calcified cartilage zone (CCZ) and to determine its dimensional relationship to the articular cartilage thickness in the normal human temporomandibular joint. An autopsy material comprising 21 joints from 12 elderly individuals was examined microscopically. The appearance of the CCZ was examined, and the thickness of the CCZ and of the total articular cartilage was measured in 18 different positions in each joint. The CCZ was outlined by a flat or gently undulating tidemark and an irregular osteochondral junction. The cellularity of the CCZ varied extensively. The cells were numerous in the CCZ when the overlying articular cartilage displayed high cellularity. Statistical analysis of the measurements demonstrated a relationship (p < 0.001) between the thickness of the CCZ and of the articular cartilage. Our findings, both qualitative and quantitative, indicate a close relationship between the physiology of the CCZ and of the overlying articular cartilage.     

The yeast HSM3 gene acts in one of the mismatch repair pathways

Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae. One bearing the hsm3-1 mutation was used for this study. This mutation does not increase sensitivity to the lethal action of different mutagens. The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7. This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene. The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain. All these phenotypes are similar to ones caused by pms1, mlhl and msh2 mutations. In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light. Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems. The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene. Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant. The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene). The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs.     

RNA binding analysis of yeast REF2 and its two-hybrid interaction with a new gene product, FIR1

The product of the REF2 gene is required for optimal levels of endonucleolytic cleavage at the 3' ends of yeast mRNA, prior to the addition of a poly(A) tail. To test the role of the previously demonstrated nonspecific affinity of REF2 for RNA in this process, we have identified RNA binding mutants in vitro and tested them for function within the cell. One REF2 variant, with an internal deletion of 82 amino acids (269-350), displays a 10-fold reduction in RNA binding, yet still retains full levels of processing activity in vivo. Conversely, a series of carboxyl-terminal deletions that maintain full RNA binding capability have progressively decreasing activity. These results rule out a major role for the central RNA binding domain of REF2 in mRNA 3' end processing and demonstrate the importance of the carboxyl-terminal region. To ask if the stimulatory role of REF2 depends on interactions with other proteins, we used a two-hybrid screen to identify a new protein termed FIR1 (Factor Interacting with REF) encoded on chromosome V. FIR1 interacts with two independent regions of REF2, one of which (amino acids 268-345) overlaps the RNA binding domain and is dispensible for REF2 function, whereas the other (amino acids 391-533) is located within the critical carboxyl-terminus. As with REF2, FIR1 has a small but detectable role in influencing the efficiency of poly(A) site use. Yeast strains containing a disrupted FIR1 gene are slightly less efficient in the use of cryptic poly(A) sites located within the lacZ portion of an ACT1-lacZ reporter construct. Likewise, a double delta ref2, delta fir1 mutant is more defective in processing of a reporter CYC1 poly(A) site than delta ref2 alone. This synergistic response provides additional support for the interaction of FIR1 with REF2 in vivo, and suggests that a number of gene products may be involved in regulating the cleavage reaction in yeast.     

A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.     

Leishmania major: histone H1 gene expression from the sw3 locus

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.     

Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human

LAG1 is a longevity gene, the first such gene to be identified and cloned from the yeast Saccharomyces cerevisiae. A close homolog of this gene, which we call LAC1, has been found in the yeast genome. We have cloned the human homolog of LAG1 with the ultimate goal of examining its possible function in human aging. In the process, we have also cloned a homolog from the nematode worm Caenorhabditis elegans. Both of these homologs, LAG1Hs and LAG1Ce-1, functionally complemented the lethality of a lag1delta lac1delta double deletion, despite low overall sequence similarity to the yeast proteins. The proteins shared a short sequence, the Lag1 motif, and a similar transmembrane domain profile. Another, more distant human homolog, TRAM, which lacks this motif, did not complement. LAG1Hs also restored the life span of the double deletion, demonstrating that it functions in establishing the longevity phenotype in yeast. LAG1Hs mapped to 19p12, and it was expressed in only three tissues: brain, skeletal muscle, and testis. This gene possesses a trinucleotide (CTG) repeat within exon 1. This and its expression profile raise the possibility that it may be involved in neurodegenerative disease. This possibility suggests at least one way in which LAG1Hs might be involved in human aging.