Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

Download Full-text

Repression and activation of the Drosophila dopa decarboxylase gene in glia

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5659-5666.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5659-5666

Author(s):

G S Mastick ◽

S B Scholnick

Keyword(s):

Germ Line ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Regulatory Elements ◽

Dopa Decarboxylase ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Cell Activator ◽

Germ Line Transformation

Download Full-text

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4548-4557.1986 ◽

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.

Download Full-text

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

Canadian Journal of Genetics and Cytology ◽

10.1139/g84-060 ◽

1984 ◽

Vol 26 (3) ◽

pp. 386-389 ◽

Cited By ~ 1

Author(s):

Linda J. Reha-Krantz ◽

Sükran Parmaksizoglu

Keyword(s):

Dna Polymerase ◽

Low Temperatures ◽

Bacteriophage T4 ◽

Effect Of Temperature ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

Download Full-text

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4087-4097.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4087-4097

Author(s):

J Wang ◽

N Suzuki ◽

Y Nishida ◽

T Kataoka

Keyword(s):

Heat Shock ◽

Adenylyl Cyclase ◽

Gene Replacement ◽

Yeast Cells ◽

Cyclase Activity ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Download Full-text

Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2913-2917.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2913-2917

Author(s):

F Maschat ◽

M L Dubertret ◽

J A Lepesant

Keyword(s):

Drosophila Melanogaster ◽

Hormonal Regulation ◽

Germ Line ◽

Regulatory Elements ◽

Fat Bodies ◽

Germ Line Transformation ◽

Third Instar Larvae

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.

Download Full-text

Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region

Journal of Cell Science ◽

10.1242/jcs.108.4.1779 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1779-1789 ◽

Cited By ~ 2

Author(s):

K.C. Chang ◽

K. Fernandes ◽

M.J. Dauncey

Keyword(s):

Myosin Heavy Chain ◽

Heavy Chain ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Chain Gene ◽

Specific Expression ◽

Slow Fibre ◽

Skeletal Myosin

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.

Download Full-text

Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

Journal of Bacteriology ◽

10.1128/jb.184.12.3242-3252.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3242-3252 ◽

Cited By ~ 11

Author(s):

Françoise Blain ◽

A. Lydia Tkalec ◽

Zhongqi Shao ◽

Catherine Poulin ◽

Marc Pedneault ◽

...

Keyword(s):

Transcriptional Start Site ◽

Expression System ◽

Regulatory Region ◽

Start Codon ◽

Upstream Region ◽

Wild Type ◽

Heparinase I ◽

High Level ◽

Flavobacterium Heparinum ◽

Transcriptional Start Sites

ABSTRACT A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

Download Full-text

Dual Negative Control of spx Transcription Initiation from the P3 Promoter by Repressors PerR and YodB in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.01520-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1736-1744 ◽

Cited By ~ 43

Author(s):

Montira Leelakriangsak ◽

Kazuo Kobayashi ◽

Peter Zuber

Keyword(s):

Oxidative Stress ◽

Bacillus Subtilis ◽

Transcription Initiation ◽

Transcriptional Start Site ◽

Regulatory Region ◽

Point Mutations ◽

Negative Control ◽

Response To Treatment ◽

Additive Increase

ABSTRACT The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P3 promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P3 promoter. Expression of an spx-bgaB fusion carrying the P3 promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P3 transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P3 promoter DNA was used as a template. Point mutations at positions within the P3 promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P3 −10 and −35 regions, while PerR binds to a region downstream of the P3 transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.

Download Full-text

Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

Infection and Immunity ◽

10.1128/iai.00052-07 ◽

2007 ◽

Vol 75 (7) ◽

pp. 3315-3324 ◽

Cited By ~ 47

Author(s):

Eric J. Gauger ◽

Mary P. Leatham ◽

Regino Mercado-Lubo ◽

David C. Laux ◽

Tyrrell Conway ◽

...

Keyword(s):

Escherichia Coli ◽

Regulatory Region ◽

Flagellar Motor ◽

Wild Type ◽

E Coli ◽

Mouse Intestine ◽

Flagellum Biosynthesis ◽

Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

Download Full-text

A Novel ENU-Induced Intronic Mutation of Ank1 Causing Quantitative Defect of Ankyrin-1 Models Hemolytic Hereditary Spherocytosis in Mouse

Blood ◽

10.1182/blood.v120.21.989.989 ◽

2012 ◽

Vol 120 (21) ◽

pp. 989-989

Author(s):

Xin Du ◽

Hua Huang ◽

Lara Krieg

Keyword(s):

Cell Biology ◽

Hereditary Spherocytosis ◽

Conflicts Of Interest ◽

Membrane Surface ◽

Regulatory Region ◽

Wild Type Mouse ◽

Wild Type ◽

Intronic Mutation ◽

Vertical Linkages

Abstract Abstract 989 The ability of red blood cells (RBCs) to maintain the surface area and deformability is vital for their survival. The maintenance of membrane surface is dependent upon the strong cohesion between the lipid bilayer and the skeletal network, achieved by vertical linkages between transmembrane proteins and spectrin tetramers. Mutations causing functional deficiencies in these proteins have been identified in various hemolytic anemias. Here we reported a mild hereditary spherocytosis (HS) and hemolytic anemia phenotype in mouse, named hema6, induced by N-ethyl-N-nitrosourea (ENU) mutagenesis. Hema6 phenotype is transmitted as a semidominant trait as heterozygous mice are less severely affected than homozygotes. The causal mutation was traced to a single nucleotide transition in the deep intronic region of intron 13 of gene Ank1, encoding the anchorage protein ankyrin-1 in RBC. In vitro minigene assay revealed two abnormally spliced transcripts in addition to wild-type mRNA. The wild-type Ank1 transcript was detected in the homozygous mutant mouse with 30% reduction in expression level compared to that in wild type mouse. The aberrant transcripts presumably encoded a 509 amino acids protein, which lacks beta-spectrin binding domain and C-terminal regulatory region. The truncated protein was not detected by western blotting using currently available antibodies against full-length ankyrin-1 in homozygous hema6 erythrocyte ghosts, whereas the wild-type Ankyrin-1 are present with reduced quantity. Employing biochemical and cell biology assays, we characterized the mechanism by which Ank1hema6 mutation causes hemolytic hereditary spherocytosis in mouse. Hema6 strain provides a novel tool to study ankyrin-1 and its pathogenesis role in HS. Disclosures: No relevant conflicts of interest to declare.

Download Full-text