ACE2, an activator of yeast metallothionein expression which is homologous to SWI5

1991 ◽  
Vol 11 (1) ◽  
pp. 476-485
Author(s):  
G Butler ◽  
D J Thiele
Keyword(s):  
Steady State ◽  
Gene Product ◽  
Large Deletion ◽  
Yeast Genome ◽  
Coding Region ◽  
Reading Frame ◽  
Enhanced Expression ◽  
Multiple Copies ◽  
Steady State Levels ◽  
Cup1 Gene

Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper. Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene. In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells. We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain. The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background. A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1. The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus. This protein is 37% identical to the SWI5 gene product, an activator of HO gene transcription in S. cerevisiae, suggesting that ACE2 and SWI5 may have functional similarities.

scholarly journals ACE2, an activator of yeast metallothionein expression which is homologous to SWI5.

1991 ◽  
Vol 11 (1) ◽  
pp. 476-485 ◽  
Author(s):  
G Butler ◽  
D J Thiele
Keyword(s):  
Steady State ◽  
Gene Product ◽  
Large Deletion ◽  
Yeast Genome ◽  
Coding Region ◽  
Reading Frame ◽  
Enhanced Expression ◽  
Multiple Copies ◽  
Steady State Levels ◽  
Cup1 Gene

Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper. Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene. In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells. We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain. The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background. A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1. The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus. This protein is 37% identical to the SWI5 gene product, an activator of HO gene transcription in S. cerevisiae, suggesting that ACE2 and SWI5 may have functional similarities.

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

scholarly journals Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443 ◽  
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

scholarly journals MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis.

1990 ◽  
Vol 10 (5) ◽  
pp. 2379-2389 ◽  
Author(s):  
J Engebrecht ◽  
G S Roeder
Keyword(s):  
Gene Product ◽  
Chromosome Pairing ◽  
Meiotic Recombination ◽  
Genetic Recombination ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Coding Region ◽  
Reading Frame ◽  
Acid Protein ◽  
Genes Expression

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.

Nucleotide sequence of the cDNA encoding the common α subunit of the chicken pituitary glycoprotein hormones

1992 ◽  
Vol 8 (1) ◽  
pp. 21-27 ◽  
Author(s):  
D. N. Foster ◽  
D. Galehouse ◽  
T. Giordano ◽  
B. Min ◽  
I. C. Lamb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Cdna Clones ◽  
Glycoprotein Hormones ◽  
Coding Region ◽  
Α Subunit ◽  
Reading Frame ◽  
Pituitary Glands ◽  
Steady State Levels ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Recombinant cDNA clones that encode the α subunit of the chicken pituitary glycoprotein hormones were isolated from a pituitary library. The longer of the two cDNA clones that were sequenced was 754bp in length. It contained 81 nucleotides of the 5′-untranslated region (UTR), an open-reading frame of 360bp that encoded a 24 amino acid leader polypeptide sequence as well as the 96 amino acid mature α subunit, and 268 nucleotides of the 3′-UTR, followed by a 45 bp poly(A) tract. There was 69–79% homology between the nucleotide sequence of the coding region for the chicken and mammalian α-subunit cDNAs. Northern blot analysis revealed that the steady-state levels of an approximately 800 bp α-subunit specific transcript increased quantitatively when dispersed chicken pituitary glands were treated in culture with chicken gonadotrophin-releasing hormone-I.

scholarly journals IS195, an Insertion Sequence-Like Element Associated with Protease Genes in Porphyromonas gingivalis

1998 ◽  
Vol 66 (7) ◽  
pp. 3035-3042 ◽  
Author(s):  
Janina P. Lewis ◽  
Francis L. Macrina
Keyword(s):  
Porphyromonas Gingivalis ◽  
Cysteine Protease ◽  
Insertion Sequence ◽  
Biochemical Characterization ◽  
Etiologic Agent ◽  
Coding Region ◽  
Reading Frame ◽  
Acid Protein ◽  
Multiple Copies

ABSTRACT Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homolog from P. gingivalis W83 encoding a cysteine protease with Lys-X specificity. However, thisprtP gene was interrupted by an insertion sequence-like element which we designated IS195. Furthermore, IS195 and another element, IS1126, were present downstream of prtP gene homologs (kgp) found inP. gingivalis H66 and 381. IS195, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of P. gingivalis W83, one carrying IS195 within the coding region of theprtP gene and another containing an intact prtPgene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalisW83 variant carrying the disrupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of theprtP gene. An allelic-exchange mutant defective in theprtP gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type andprtP::IS195 mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.

MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis

1990 ◽  
Vol 10 (5) ◽  
pp. 2379-2389 ◽  
Author(s):  
J Engebrecht ◽  
G S Roeder
Keyword(s):  
Gene Product ◽  
Chromosome Pairing ◽  
Meiotic Recombination ◽  
Genetic Recombination ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Coding Region ◽  
Reading Frame ◽  
Acid Protein ◽  
Genes Expression

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.

scholarly journals Identification of sequences in c-myc mRNA that regulate its steady-state levels.

1996 ◽  
Vol 16 (7) ◽  
pp. 3511-3522 ◽  
Author(s):  
N M Yeilding ◽  
M T Rehman ◽  
W M Lee
Keyword(s):  
Steady State ◽  
Reverse Transcription ◽  
Cell Metabolism ◽  
C2c12 Cells ◽  
Mrna Levels ◽  
Beta Globin ◽  
Coding Region ◽  
Globin Mrna ◽  
Protein Coding ◽  
Steady State Levels

The level of cellular myc proto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-myc mRNA, which enables cells to rapidly alter c-myc mRNA levels. Efforts to identify elements in myc mRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-myc mRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-myc mRNA region on the steady-state levels of c-myc mRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of human myc mRNAs transcribed from two similarly constructed myc genes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced into myc genes, and the levels of their mRNAs were compared with that of a near-normal, reference myc mRNA. Deletion of most of the myc 3' untranslated region (UTR) raised myc mRNA levels, while deletion of sequences in the myc 5' UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3' UTR is responsible for keeping myc mRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two beta-globin-myc fusion mRNAs, we showed that fusion of the myc 3' UTR lowers globin mRNA levels by destabilizing beta-globin mRNA. Surprisingly, fusion of the protein-coding region of myc exon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-myc mRNA turnover, we demonstrated that these sequences, but not myc 3' UTR sequences, are necessary for the normal posttranscriptional downregulation of c-myc mRNA during myoblast differentiation. We conclude that, while two elements within c-myc mRNA can act as instability determinants in a heterologous context, only the instability element in the 3' UTR regulates its steady-state levels in proliferating C2C12 cells.

scholarly journals Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

2008 ◽  
Vol 190 (23) ◽  
pp. 7719-7727 ◽  
Author(s):  
Kayo Okumura ◽  
Kaori Ohtani ◽  
Hideo Hayashi ◽  
Tohru Shimizu
Keyword(s):  
Gene Product ◽  
Clostridium Perfringens ◽  
Complete Sequence ◽  
Northern Analysis ◽  
Reading Frame ◽  
Rna Molecules ◽  
Transcription Start Sites ◽  
Negative Effect ◽  
In The Wild ◽  
Steady State Levels

ABSTRACT Analysis of the complete sequence of the genome of Clostridium perfringens strain 13 resulted in identification of five genes, including pfoA (encoding theta toxin) and vrr (encoding VirR/VirS-regulated RNA), with consensus VirR-binding sequences upstream of the open reading frame (ORF), suggesting that expression of these genes may be regulated directly by the two-component VirR/VirS system. To test this possibility, we examined VirR/VirS system-mediated transcriptional regulation of three genes, virT, ccp (encoding alpha-clostripain), and virU, with the novel VirR-binding sequences. Northern analysis revealed that the steady-state levels (increases or decreases in the amounts of RNA expressed) of virT, ccp, and virU mRNAs were lower in a virR mutant strain than in the wild-type strain, as were the levels of the pfoA and vrr transcripts. The consensus VirR-binding sites were located similarly relative to the transcription start sites in the virT, ccp, and virU promoters. Mutation and overexpression analyses with virT and virU revealed that the virT gene product has a negative effect on expression of pfoA and ccp, whereas the virU gene product positively affects expression of pfoA, virT, ccp, and vrr. Nonsense and frameshift mutations in the virT or virU putative ORF did not affect the regulatory functions, suggesting that virT and virU may encode RNA regulators rather than proteins. These results suggest that a complex regulatory network, perhaps involving several regulatory RNA molecules, governs the expression of the VirR/VirS regulon in C. perfringens.

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