The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants.

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

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The Legionella pneumophila iraAB Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence

Infection and Immunity ◽

10.1128/iai.68.3.1069-1079.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1069-1079 ◽

Cited By ~ 52

Author(s):

V. K. Viswanathan ◽

Paul H. Edelstein ◽

C. Dumais Pope ◽

Nicholas P. Cianciotto

Keyword(s):

Amino Acid ◽

Legionella Pneumophila ◽

Open Reading Frame ◽

Host Cells ◽

Reading Frame ◽

Intracellular Infection ◽

Acid Protein ◽

Iron Deficient ◽

Iron Assimilation ◽

Amino Acid Protein

ABSTRACT Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that theiraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionellaspecies. A newly derived mutant containing a targeted disruption ofiraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest thatiraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

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ISCce1 and ISCce2, Two Novel Insertion Sequences in Clostridium cellulolyticum

Journal of Bacteriology ◽

10.1128/jb.185.3.714-725.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 714-725 ◽

Cited By ~ 9

Author(s):

Hédia Maamar ◽

Pascale de Philip ◽

Jean-Pierre Bélaich ◽

Chantal Tardif

Keyword(s):

Amino Acid ◽

Insertion Site ◽

Open Reading Frame ◽

Inverted Repeats ◽

Target Sequence ◽

Insertion Sequences ◽

Reading Frame ◽

Clostridium Cellulolyticum ◽

Acid Protein ◽

Amino Acid Protein

ABSTRACT Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.

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Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1711-1721.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1711-1721

Author(s):

E M McIntosh ◽

R H Haynes

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frame ◽

Northern Analysis ◽

Hindiii Site ◽

Reading Frame ◽

Hindiii Restriction Site ◽

Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

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Identification of the ubiD Gene on theEscherichia coli Chromosome

Journal of Bacteriology ◽

10.1128/jb.182.21.6243-6246.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6243-6246 ◽

Cited By ~ 19

Author(s):

Haitao Zhang ◽

George T. Javor

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Gene Product ◽

Mutant Strain ◽

Amino Acid Residue ◽

Open Reading Frame ◽

Reading Frame

ABSTRACT The open reading frame at 86.7 min on the Escherichia coli chromosome, “yigC,” complemented aubiD mutant strain, AN66, indicating that yigCis the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

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The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5546-5553.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5546-5553 ◽

Cited By ~ 37

Author(s):

Kazuhiro Iwashita ◽

Tatsuya Nagahara ◽

Hitoshi Kimura ◽

Makoto Takano ◽

Hitoshi Shimoi ◽

...

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Enzyme Assays ◽

Partial Amino Acid Sequence ◽

Reading Frame ◽

Acid Protein ◽

Aspergillus Kawachii ◽

Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

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Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽

10.1093/genetics/131.3.531 ◽

1992 ◽

Vol 131 (3) ◽

pp. 531-539 ◽

Cited By ~ 6

Author(s):

C Bornaes ◽

J G Petersen ◽

S Holmberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Sequence ◽

Transcription Initiation ◽

Open Reading Frame ◽

Predicted Amino Acid Sequence ◽

S1 Nuclease ◽

Reading Frame ◽

Threonine Dehydratase ◽

Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

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Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1711 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1711-1721 ◽

Cited By ~ 38

Author(s):

E M McIntosh ◽

R H Haynes

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frame ◽

Northern Analysis ◽

Hindiii Site ◽

Reading Frame ◽

Hindiii Restriction Site ◽

Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

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Cloning of a Mucin-Desulfating Sulfatase Gene fromPrevotella Strain RS2 and Its Expression Using aBacteroides Recombinant System

Journal of Bacteriology ◽

10.1128/jb.182.11.3002-3007.2000 ◽

2000 ◽

Vol 182 (11) ◽

pp. 3002-3007 ◽

Cited By ~ 30

Author(s):

Damian P. Wright ◽

Catriona G. Knight ◽

Shanthi G. Parkar ◽

David L. Christie ◽

Anthony M. Roberton

Keyword(s):

Amino Acid ◽

Sequence Data ◽

Signal Sequence ◽

Active Form ◽

Open Reading Frame ◽

Expression Vectors ◽

Gene Encoding ◽

Reading Frame ◽

Gene Coding ◽

Acid Protein

ABSTRACT A gene encoding the mucin-desulfating sulfatase inPrevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into aBacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

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Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1999.0501 ◽

1999 ◽

Vol 354 (1389) ◽

pp. 1577-1581 ◽

Cited By ~ 35

Author(s):

Y. Ohsumi

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Conjugation System ◽

Yeast Saccharomyces Cerevisiae ◽

Whole Process ◽

Unique Protein ◽

Acid Protein ◽

Major Route ◽

Lytic Compartment ◽

Amino Acid Protein

Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development. This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole. We found that yeast, Saccharomyces cerevisiae , induces autophagy under various starvation conditions. The whole process is essentially the same as macroautophagy in higher eukaryotic cells. However, little is known about the mechanism of autophagy at a molecular level. To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy–defective mutants ( apg ) were isolated. So far, 14 APG genes have been cloned. Among them we recently found a unique protein conjugation system essential for autophagy. The C–terminal glycine residue of a novel modifier protein Apg12p, a 186–amino–acid protein, is conjugated to a lysine residue of Apg5p, a 294–amino–acid protein, via an isopeptide bond. We also found that apg7 and apg10 mutants were unable to form an Apg12p–Apg5p conjugate. The conjugation reaction is mediated via Apg7p, E1–like activating enzyme and Apg10p, indicating that it is a ubiquitination–like system. These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human. Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy.

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