Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis

The mammalianCAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5 end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a completeCAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of theCAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.     

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene,XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant

Summary A P. stipitis cDNA library in gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.     

Molecular analysis and characterization of a protein involved in the replication of intracellular Toxoplasma gondii

Previous studies in our laboratory have identified a cytoplasmic protein (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replication in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not expressed on the surface of the tachyzoite. Polyclonal sera raised against the purified native protein was used to isolate a cDNA of 3.3 kb from a library. The product of this gene expresses a protein of approximate M_r 97 kDa that is reactive to the antibody (1B8) raised against the native antigen. The protein sequence of this product suggests that it is within the cytoplasm as suggested by the lack of a signal sequence or hydrophobic trans-membrane domain. This protein fails to dissociate into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrates a homologous gene sequence in two closely related Apicomplexa, Neospora caninum and Besnoitia jellisoni suggesting this protein is conserved among certain species of the Sarcocystidae.     

Isolation of a Trichoderma reesei cDNA encoding GTP: a-d-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation

A cDNA coding for GTP: α-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant. Received: 21 July 1997 / 24 April 1998     

Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae

Summary Orotate phosphoribosyl transferase (OPRTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.     

Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in Saccharomyces cerevisiae

A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-β-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1 _S ). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae. Received: 1 March 1996 / 9 April 1996     

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP ⁺ transformants). Plasmids isolated from the transformants (URA ⁺ TRP ⁺) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.     

Isolation,sequencing, and characterization of crp-5, a gene encoding a Neurospora ribosomal protein

A Neurospora crassa cytoplasmic ribosomal protein gene, crp-5, has been isolated and characterized. The cDNA was isolated by a differential screening of a cDNA library for glucose-inducible genes. The cDNA was subsequently used to identify and isolate crp-5 genomic sequences. Computer analysis of the DNA sequences showed that they contain an open reading frame which encodes a protein homologous to the rat ribosomal protein S26. The crp-5 mRNA levels are regulated in a carbon-source-dependent manner. The organization of the gene and the region upstream of the coding sequences are discussed.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Molecular genetic properties of the yeast Torulaspora pretoriensis: Characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids

Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogenous-electric-field gel electrophoresis. Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed. By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae. A uracil auxotrophic mutant derived from T. pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S. cerevisiae by the lithium acetate method.     

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.     

Cloning and functional characterization of a fatty acid synthase component FAS2 gene from Saccharomyces kluyveri

A gene coding the α subunit of fatty acid synthase (FAS2) was isolated from the budding yeast Saccharomyces kluyveri. Nucleotide sequence analysis indicated that this gene, termed Sk-FAS2, coded a protein having an amino acid sequence 83% identical to the FAS2 protein of S. cerevisiae (Sc-FAS2). The Sk-FAS2 gene was able to functionally complement an S. cerevisiae fas2 disruptant. This Sk-FAS2-expressing strain was found to produce larger amounts of C18 than C16, in contrast to the Sc-FAS2-expressing fas2 strain. In addition, fusion genes of Sk-FAS2 and Sc-FAS2 were transformed into a fas2-disrupted strain of S. cerevisiae, and fatty acid analysis of these transformants suggested that the region containing the acyl carrier protein and β-ketoacyl reductase domains of yeast FAS2 protein play an important role in determining carbon chain length of fatty acids.GenBank Submission: The nucleotide sequences reported in this paper have been submitted to GenBank Nucleotide Sequence Database under the accession number AB115969.     

Characterization of the nuclear gene encoding chloroplast ribosomal protein S13 from Arabidopsis thaliana

We have characterised a cDNA clone and a nuclear gene encoding the chloroplast 30 s ribosomal protein S13 from Arabidopsis thaliana. The identification is based on the high similarity of the predicted amino-acid sequence with eubacterial S13 protein sequences, and immunodetection of a 14.5-kDa chloroplast ribosomal polypeptide using antibodies raised against the polypeptide produced from part of the cDNA expressed in bacteria. The predicted amino-acid sequence contains an N-terminal extension which has several features characteristic of chloroplast transit peptides. Experiments suggest there is a single copy of this gene in A. thaliana and multiple copies in Brassica species. The origin of the mitochondrial S13 polypeptide in crucifers is also discussed.     

The cloning and sequencing of thealcB gene,coding for alcohol dehydrogenase II,inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in gt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5 region of the gene is discussed in relation to the regulation of the gene.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.     

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia

We isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1–q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder. Received: September 17, 1997 / Accepted: January 9, 1998     

Effect of ochre nonsense mutations on yeast URA1 mRNA stability

Summary The genetic map of 27 mutants of the URA1 yeast gene has been established by meiotic recombination and 16 nonsense mutations characterized. The half life of URA1 mRNA was determined by two independent methods in the wild-type and in two ochre mutants localized at each extremity of the genetic map. A halflife of 15 min was found for the wild-type and for one of the ochre mutants. This half-life was radically reduced in the other ochre mutant whereas the instantaneous rate of its mRNA synthesis remained constant. After subcloning various endonucleolytic fragments the coding sequence of the URA1 gene was restricted to a 1.65 kb fragment within a 5.7 kb yeast DNA segment. Direct visualization of the URA1 mRNA by Northern hybridization of denatured RNA with a URA1 specific DNA probe revealed a length of 1.5 kb.