scholarly journals Functional organization of the Aspergillus nidulans trpC promoter.

1987 ◽  
Vol 7 (7) ◽  
pp. 2352-2359 ◽  
Author(s):  
J E Hamer ◽  
W E Timberlake
Keyword(s):  
Gene Expression ◽  
Aspergillus Nidulans ◽  
Functional Organization ◽  
Fusion Gene ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
S1 Nuclease ◽  
Promoter Mutations ◽  
Beta Galactosidase ◽  
Trpc Gene

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

Functional organization of the Aspergillus nidulans trpC promoter

1987 ◽  
Vol 7 (7) ◽  
pp. 2352-2359
Author(s):  
J E Hamer ◽  
W E Timberlake
Keyword(s):  
Gene Expression ◽  
Aspergillus Nidulans ◽  
Functional Organization ◽  
Fusion Gene ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
S1 Nuclease ◽  
Promoter Mutations ◽  
Beta Galactosidase ◽  
Trpc Gene

We investigated the functional organization of the Aspergillus nidulans trpC promoter by the sequential removal of sequences upstream of the major trpC mRNA cap site (+1). DNA fragments containing promoter mutations were fused to the Escherichia coli lacZ gene, and a novel method was used to select for integration of the fusion gene at the Aspergillus argB locus. beta-Galactosidase assays and S1 nuclease protection experiments demonstrated that the promoter mutations affected gene expression in three ways: (i) 5' deletions up to -82 resulted in variable increases in beta-galactosidase activity, depending on the growth conditions; (ii) a deletion from -67 to -11 did not alter the level of beta-galactosidase activity, but did give rise to mRNAs with aberrant 5' ends; and (iii) a 5' deletion with an endpoint at -11 and an internal deletion from -142 to -11 abolished gene expression. These results indicate that sequences upstream of -82 reduce transcription of the trpC gene and that distinct DNA sequence elements are required for expression versus correct initiation of transcription of the trpC gene. The sequences essential for trpC expression do not include the common eucaryotic promoter elements CCAAT and TATAAA. To our knowledge, this is the first functional analysis of a promoter from a fungus other than Saccharomyces cerevisiae.

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region

1982 ◽  
Vol 152 (2) ◽  
pp. 829-839
Author(s):  
A M Easton ◽  
R H Rownd
Keyword(s):  
Gene Expression ◽  
Target Site ◽  
Plasmid Replication ◽  
Lacz Gene ◽  
Origin Of Replication ◽  
Wild Type ◽  
Base Pairs ◽  
Replication Region ◽  
Lambda Phages ◽  
Beta Galactosidase

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

2000 ◽  
Vol 46 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Takashi Kubo ◽  
Toshiko Aiso ◽  
Reiko Ohki
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Lacz Gene ◽  
Permissive Temperature ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Genes ◽  
Beta Galactosidase ◽  
Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

Intrapericardial administration of adenovirus for gene transfer

1997 ◽  
Vol 272 (1) ◽  
pp. H310-H317 ◽  
Author(s):  
K. G. Lamping ◽  
C. D. Rios ◽  
J. A. Chun ◽  
H. Ooboshi ◽  
B. L. Davidson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Left Atrial ◽  
Histochemical Staining ◽  
Adenoviral Vectors ◽  
Galactosidase Activity ◽  
Parietal Pericardium ◽  
The Right ◽  
Beta Galactosidase ◽  
Epicardial Myocytes

Gene transfer to the heart has been accomplished with intravascular administration of adenoviral vectors into the pericardial sac, by increasing the duration of exposure to the adenovirus, would result in gene expression in the pericardium and perhaps myocardium and therefore might provide an alternative method to intravascular administration for gene transfer. We injected a replication-deficient adenovirus (average 1 x 10(12) particles/ml in 3% sucrose; 1 x 10(10) plaque forming units/ml containing cDNA encoding a nuclear-targeted bacterial beta-galactosidase into the pericardial sac of dogs. Samples of the pericardium and heart were examined for enzymatic activity of beta-galactosidase and after histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. One day after injection of the adenovirus (1-3 ml), beta-galactosidase activity was highest in the parietal pericardium and left atrial tissue and lower in the right and left ventricles. Histochemical expression of the transgene was predominantly in the visceral pericardium of atria and ventricles and occasionally in the epicardial myocytes, arterioles, and venules. Pretreatment with doxycycline (5 mg) before adenovirus administration increased transgene activity in left ventricles. Thus adenovirus injected into the pericardial sac provides an effective method for gene transfer to the visceral and parietal pericardium over atria and ventricles.

Hsp104 expression and morphological changes associated with disinfectants in saccharomyces cerevisiae: environmental bioassay using stress response

1998 ◽  
Vol 38 (7) ◽  
pp. 237-243 ◽  
Author(s):  
K. Fujita ◽  
H. Iwahashi ◽  
R. Kawai ◽  
Y. Komatsu
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Stress Proteins ◽  
Morphological Changes ◽  
Chemical Stress ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Hybrid Gene ◽  
Sublethal Concentrations

On exposure of Saccharomyces cerevisiae to sublethal concentrations of disinfectants such as TPN, Thiuram, Captan and Oxine-copper; 70-, 90-kDa proteins and heat-shock protein Hsp104 were induced without morphological changes. Considering these stress proteins as critical signs, we can determine how cells are damaged by pesticides under sublethal conditions. Furthermore, Hsp104-lacZ hybrid gene (a lacZ gene put under control of Hsp104 promoter) in S. cerevisiae was sensitively expressed in the presence of sublethal concentrations of these disinfectants by measuring the relative β-galactosidase activity. It follows that not only monitoring the growth phase or the induction of synthesized proteins but also detecting the level of gene expression shows the chemical stress response rapidly, conveniently and reproducibly. We conclude that the use of a yeast strain with a stress reporter gene is a novel and simple bioassay relative to human health and to the ecosystem in general.

Negative regulation of STE6 gene expression by the alpha 2 product of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2420-2427
Author(s):  
K L Wilson ◽  
I Herskowitz
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Insertion Mutation ◽  
Lacz Gene ◽  
Alpha Cells ◽  
Type Locus ◽  
A Cells ◽  
Product Expression ◽  
Beta Galactosidase

The alpha 2 product of the alpha mating type locus of Saccharomyces cerevisiae is proposed to be a negative regulator of a set of dispersed genes concerned with specialized properties of a cells. This set of genes includes those, termed a-specific STE genes (STE2, STE6, and STE14), which are required for mating by a cells but not by alpha cells. We cloned the STE6 gene to determine whether its expression is limited to a cells and, if so, whether its expression is inhibited in alpha cells by the alpha 2 product. Expression of STE6 was assayed in two ways: by blot hybridization, RNA and by beta-galactosidase activity in strains carrying a STE6-lacZ hybrid gene. We found that STE6 expression was limited to a cells and was negatively regulated by the alpha 2 product. STE6 RNA was not detectable in strains containing the wild-type alpha 2 gene product. Expression of STE6 was at least 150-fold lower in alpha cells than in a cells, based on beta-galactosidase activities in a and alpha cells carrying the STE6-lacZ gene. These results confirmed that the alpha 2 product is a negative regulator of gene expression and showed that it acts at the level of RNA production. We also examined the phenotype of a mutant carrying an insertion mutation of the STE6 gene, the ste6::lacZ allele. In addition, an a-specific defect in mating, this mutant was greatly reduced (but not completely deficient) in a-factor production. Other phenotypes characteristic of a cells--Barrier activity, agglutination, and response to alpha-factor--were normal. STE6 thus appears to be necessary for biosynthesis of a-factor.

scholarly journals Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (11) ◽  
pp. 4882-4888 ◽  
Author(s):  
D M Kinney ◽  
C J Lusty
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Mrna Translation ◽  
Single Amino Acid ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Beta Galactosidase

delta-N-(Phosphonacetyl)-L-ornithine (PALO), a transition state analog inhibitor of ornithine transcarbamylase, induced arginine limitation in vivo in Saccharomyces cerevisiae. Arginine restriction caused increased expression of HIS3 and TRP5, measured by the beta-galactosidase activity in strains carrying chromosomally integrated fusions of the promoter regions of each gene with the lacZ gene of Escherichia coli. The increase in beta-galactosidase activity induced by PALO was reversed by the addition of arginine and was dependent on GCN4 protein. These results indicate that PALO, like 3-amino-1,2,4-triazole DL-5-methyltryptophan, can be used to study the effect of limitation of a single amino acid, arginine, on the expression of genes under the general amino acid control regulatory system. Arginine deprivation imposed by PALO also caused increased expression of CPA1 and CPA2, coding respectively for the small and large subunits of arginine-specific carbamyl-phosphate synthetase. The observed increase was GCN4 dependent and was genetically separable from arginine-specific repression of CPA1 mRNA translation. The 5'-flanking regions of CPA1 (reported previously) and CPA2 determined in this study each contained at least two copies of the sequence TGACTC, shown to bind GCN4 protein. The beta-galactosidase activities expressed from CPA1- and CPA2-lacZ fusions integrated into the nuclear DNA of gcn4 mutant strains were five to six times less than in the wild type, when both strains were grown under depressed conditions. The gcn4 mutation reduced basal expression of both CPA1 and CPA2. The addition of arginine to strains containing the CPA1-lacZ fusion further reduced beta-galactosidase activity of the gcn4 mutant, indicating independent regulation of the CPA1 gene by the general amino acid control and by arginine-specific repression. In strains overproducing GCN4 protein, the translational control completely overrode transcriptional activation of CPA1 by general amino acid control.

scholarly journals Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806 ◽  
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

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