Analysis of transcriptional regulation of the s38 chorion gene of Drosophila by P element-mediated transformation

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 137-146
Author(s):  
Laura Kalfayan ◽  
Barbara Wakimoto ◽  
Allan Spradling
Keyword(s):  
Transcriptional Regulation ◽  
P Element ◽  
Developmental Expression ◽  
Developmental Profile ◽  
E Coli ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
P Element Transformation ◽  
Lac Z

Transcriptional regulation of the s38 chorion gene was studied using P element-mediated germline transformation. A 5·27 kb DNA fragment containing the s38 gene and 5′- and 3′-flanking sequences, was tested for its ability to be transcribed with correct developmental specificity. Five single-insert transformed lines were generated by microinjection of this DNA fragment cloned into a marked P element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native s38 gene. The 1·3 kb at the 5′ end of this tested fragment was fused to the E. coli lac z gene. This fragment was also capable of initiating transcription of E. coli lac z RNA with the developmental profile of the native s38 gene. In vitro deletion studies are underway to determine which sequences in the 1·3 kb fragment are necessary for regulating the developmental expression of the gene.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2298-2304 ◽  
Author(s):  
L W Bergman
Keyword(s):  
Transcriptional Regulation ◽  
Nucleosome Positioning ◽  
Vector System ◽  
Growth Media ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
Acid Phosphatase Gene ◽  
Nucleosomal Structure ◽  
Relationship Of ◽  
Pho5 Gene

The functional relationship of nucleosome positioning and gene expression is not known. Using high-copy plasmids, containing the yeast phosphate-repressible acid phosphatase gene (PHO5) and the TRP1/ARS1 vector system, I have determined the nucleosomal structure of the 5' region of the PHO5 gene and demonstrated that the nucleosomal positioning of this region is independent of orientation or position in the various plasmid constructions utilized. However, deletion of a 278-base pair BamHI-ClaI fragment from the 5'-flanking sequences of the PHO5 gene causes the nucleosome positioning to become dependent on orientation or position in the plasmids tested. Use of PHO5-CYC1-lACZ fusions have demonstrated that this DNA fragment contains the sequences responsible for the transcriptional regulation of the PHO5 gene in response to the level of phosphate in the growth media. The nucleosome positioning in the 5' region of PHO5 may be determined by an interaction with the sequences or machinery responsible for transcriptional regulation of the gene.

scholarly journals The promoter of DNA puff gene II/9-1 of Sciara coprophila is inducible by ecdysone in late prepupal salivary glands of Drosophila melanogaster.

1991 ◽  
Vol 2 (11) ◽  
pp. 875-888 ◽  
Author(s):  
B Bienz-Tadmor ◽  
H S Smith ◽  
S A Gerbi
Keyword(s):  
Drosophila Melanogaster ◽  
Salivary Glands ◽  
Dna Amplification ◽  
P Element ◽  
Control Of Gene Expression ◽  
Evolutionarily Conserved ◽  
Dna Puffs ◽  
P Element Transformation ◽  
Transgenic Drosophila

DNA puffs occur in Sciarid salivary gland chromosomes; they are sites of DNA amplification and intense transcription and they appear to encode secreted structural proteins needed for pupation. In this report we have used P-element transformation of Drosophila to study regulation of a Sciara DNA puff gene. We found that a 718-bp promoter fragment of DNA puff gene II/9-1 from Sciara coprophila directs expression of the bacterial reporter gene CAT in late prepupal salivary glands of transgenic Drosophila melanogaster. The identical tissue and analogous stage specificity indicate that some aspects of the ecdysone response are evolutionarily conserved between Drosophila and Sciara. When transgenic salivary glands are cultured in vitro, CAT activity is rapidly induced by ecdysone, suggesting direct control of gene expression by the ecdysone receptor. Putative stage-specific factors limit expression of the chimeric Sciara-CAT gene in transgenic Drosophila to late prepupae but not to third instar larvae when ecdysone titers are also high.

scholarly journals Gene Cluster Responsible for Validamycin Biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

2005 ◽  
Vol 71 (9) ◽  
pp. 5066-5076 ◽  
Author(s):  
Yi Yu ◽  
Linquan Bai ◽  
Kazuyuki Minagawa ◽  
Xiaohong Jian ◽  
Lei Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Control Agent ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
E Coli ◽  
Sheath Blight Disease ◽  
Dna Fragment ◽  
Blight Disease

ABSTRACT A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2298-2304
Author(s):  
L W Bergman
Keyword(s):  
Transcriptional Regulation ◽  
Nucleosome Positioning ◽  
Vector System ◽  
Growth Media ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
Acid Phosphatase Gene ◽  
Nucleosomal Structure ◽  
Relationship Of ◽  
Pho5 Gene

The functional relationship of nucleosome positioning and gene expression is not known. Using high-copy plasmids, containing the yeast phosphate-repressible acid phosphatase gene (PHO5) and the TRP1/ARS1 vector system, I have determined the nucleosomal structure of the 5' region of the PHO5 gene and demonstrated that the nucleosomal positioning of this region is independent of orientation or position in the various plasmid constructions utilized. However, deletion of a 278-base pair BamHI-ClaI fragment from the 5'-flanking sequences of the PHO5 gene causes the nucleosome positioning to become dependent on orientation or position in the plasmids tested. Use of PHO5-CYC1-lACZ fusions have demonstrated that this DNA fragment contains the sequences responsible for the transcriptional regulation of the PHO5 gene in response to the level of phosphate in the growth media. The nucleosome positioning in the 5' region of PHO5 may be determined by an interaction with the sequences or machinery responsible for transcriptional regulation of the gene.

scholarly journals Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

2005 ◽  
Vol 187 (9) ◽  
pp. 3171-3179 ◽  
Author(s):  
Annik Perrenoud ◽  
Uwe Sauer
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Redox Regulation ◽  
Tca Cycle ◽  
Batch Cultures ◽  
E Coli ◽  
Glucose Catabolism ◽  
The Tca Cycle

ABSTRACT Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

scholarly journals Genetic-Biochemical Analysis and Distribution of the Ambler Class A β-Lactamase CME-2, Responsible for Extended-Spectrum Cephalosporin Resistance inChryseobacterium (Flavobacterium)meningosepticum

2000 ◽  
Vol 44 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Samuel Bellais ◽  
Laurent Poirel ◽  
Thierry Naas ◽  
Delphine Girlich ◽  
Patrice Nordmann
Keyword(s):  
Clavulanic Acid ◽  
Biochemical Analysis ◽  
Agar Plate ◽  
Reading Frame ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Cephalosporin Resistance ◽  
Dna Fragment

ABSTRACT In vitro synergy between extended-spectrum cephalosporins and either clavulanic acid or cefoxitin was found forChryseobacterium meningosepticum isolates during a double-disk assay on an agar plate. An extended-spectrum β-lactamase (ESBL) gene from a C. meningosepticum clinical isolate was cloned and expressed in Escherichia coli DH10B. Its protein conferred resistance to most β-lactams including extended-spectrum cephalosporins but not to cephamycins or to imipenem. Its activity was strongly inhibited by clavulanic acid, sulbactam, and tazobactam, as well as by cephamycins and imipenem. Sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) of 891 bp with a G+C content of 33.9%, which lies close to the expected range of G+C contents of members of the Chryseobacterium genus. The ORF encoded a precursor protein of 297 amino acids, giving a mature protein with a molecular mass of 31 kDa and a pI value of 9.2 in E. coli. This gene was very likely chromosomally located. Amino acid sequence comparison showed that this β-lactamase, named CME-2 (C. meningosepticum ESBL), is a novel ESBL of the Ambler class A group (Bush functional group 2be), being weakly related to other class A β-lactamases. It shares only 39 and 35% identities with the ESBLs VEB-1 from E. coli MG-1 and CBL-A fromBacteroides uniformis, respectively. The distribution ofbla CME-2 among unrelated C. meningosepticum species isolates showed thatbla CME-2-like genes were found in the C. meningosepticum strains studied but were absent from strains of other C. meningosepticum-related species. Each C. meningosepticum strain produced at least two β-lactamases, with one of them being a noninducible serine ESBL with variable pIs ranging from 7.0 to 8.5.

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication

1979 ◽  
Vol 169 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Volker Nüsslein-Crystalla ◽  
Ursula Scheefers-Borchel
Keyword(s):  
Dna Replication ◽  
E Coli ◽  
In Vitro Replication ◽  
Dna Fragment
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