Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

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PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3843 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3843-3856 ◽

Cited By ~ 20

Author(s):

J P O'Connor ◽

C L Peebles

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Genomic Library ◽

Essential Gene ◽

Growth Defect ◽

Temperature Sensitive ◽

Putative Open Reading Frame ◽

Reading Frame ◽

Trna Processing ◽

Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

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The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2214 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2214-2223 ◽

Cited By ~ 79

Author(s):

Y Wada ◽

K Kitamoto ◽

T Kanbe ◽

K Tanaka ◽

Y Anraku

Keyword(s):

Saccharomyces Cerevisiae ◽

Genomic Library ◽

Carboxypeptidase Y ◽

Amino Acid Residues ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Lethal Event ◽

Vacuolar Protein ◽

And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

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The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2214-2223.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2214-2223

Author(s):

Y Wada ◽

K Kitamoto ◽

T Kanbe ◽

K Tanaka ◽

Y Anraku

Keyword(s):

Saccharomyces Cerevisiae ◽

Genomic Library ◽

Carboxypeptidase Y ◽

Amino Acid Residues ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Lethal Event ◽

Vacuolar Protein ◽

And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

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PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3843-3856.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3843-3856

Author(s):

J P O'Connor ◽

C L Peebles

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Genomic Library ◽

Essential Gene ◽

Growth Defect ◽

Temperature Sensitive ◽

Putative Open Reading Frame ◽

Reading Frame ◽

Trna Processing ◽

Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

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Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/125.4.739 ◽

1990 ◽

Vol 125 (4) ◽

pp. 739-752 ◽

Cited By ~ 6

Author(s):

C A Woolford ◽

C K Dixon ◽

M F Manolson ◽

R Wright ◽

E W Jones

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mass ◽

Open Reading Frame ◽

Relative Molecular Mass ◽

Cell Fractionation ◽

Calculated Molecular Mass ◽

Reading Frame ◽

Dna Library ◽

Isolation And Characterization ◽

Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

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Characterization of the gene celD and its encoded product 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa

Biochemical Journal ◽

10.1042/bj2850947 ◽

1992 ◽

Vol 285 (3) ◽

pp. 947-955 ◽

Cited By ~ 30

Author(s):

J E Rixon ◽

L M A Ferreira ◽

A J Durrant ◽

J I Laurie ◽

G P Hazlewood ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Plant Cell Wall ◽

Genomic Library ◽

Cell Envelope ◽

Sequence Similarity ◽

Significant Proportion ◽

Structural Similarity ◽

Reading Frame ◽

E Coli ◽

Beta Glucosidase

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.

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HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.)

Genome ◽

10.1139/g09-064 ◽

2009 ◽

Vol 52 (11) ◽

pp. 904-911 ◽

Cited By ~ 12

Author(s):

M. Buti ◽

T. Giordani ◽

M. Vukich ◽

L. Gentzbittel ◽

L. Pistelli ◽

...

Keyword(s):

Helianthus Annuus ◽

Sequence Similarity ◽

Protein Domain ◽

Long Terminal Repeats ◽

High Sequence Identity ◽

Nonsense Mutations ◽

Reading Frame ◽

Sequence Identity ◽

Isolation And Characterization ◽

Copia Retrotransposon

In this paper we report on the isolation and characterization, for the first time, of a complete 6511 bp retrotransposon of sunflower. Considering its protein domain order and sequence similarity to other copia elements of dicotyledons, this retrotransposon was assigned to the copia retrotransposon superfamily and named HACRE1 ( Helianthus annuus copia-like retroelement 1). HACRE1 carries 5′ and 3′ long terminal repeats (LTRs) flanking an internal region of 4661 bp. The LTRs are identical in their sequence except for two deletions of 7 and 5 nucleotides in the 5′ LTR. Based on the sequence identity of the LTRs, HACRE1 was estimated to have inserted within the last ∼84 000 years. The isolated sequence contains a complete open reading frame with only one complete reading frame. The absence of nonsense mutations agrees with the very high sequence identity between LTRs, confirming that HACRE1 insertion is recent. The haploid genome of sunflower (inbred line HCM) contains about 160 copies of HACRE1. This retrotransposon is expressed in leaflets from 7-day-old plantlets under different light conditions, probably in relation to the occurrence of many putative light-related regulatory cis-elements in the LTRs. However, sequenced cDNAs show less variability than HACRE1 genomic sequences, indicating that only a subset of this family is expressed under these conditions.

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Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.264 ◽

1995 ◽

Vol 15 (1) ◽

pp. 264-271 ◽

Cited By ~ 63

Author(s):

M Wu ◽

B Repetto ◽

D M Glerum ◽

A Tzagoloff

Keyword(s):

Saccharomyces Cerevisiae ◽

Flavin Adenine Dinucleotide ◽

Genomic Library ◽

Sequence Similarity ◽

Complementation Group ◽

Flavin Mononucleotide ◽

Synthetase Activity ◽

Multicopy Plasmid ◽

Multiple Copies ◽

Extragenic Suppression

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

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Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/124.4.797 ◽

1990 ◽

Vol 124 (4) ◽

pp. 797-806 ◽

Cited By ~ 2

Author(s):

A Petitjean ◽

F Hilger ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Critical Role ◽

Missense Mutations ◽

Nucleotide Exchange ◽

Carboxy Terminus ◽

Reading Frame ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Carboxy Terminal

Abstract The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate cyclase pathway. Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor. We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene. All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene. Three of the four are missense mutations in the same amino acid codon. A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTE1, a gene required for growth at low temperature and SCD25, a suppressor of cdc25. Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTE1 or SCD25, may have related activities.

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