The use of an inhibitor of protein synthesis to investigate the roles of ecdysteroids and sex-determination genes on the expression of the genes encoding the Drosophila yolk proteins

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 931-941
Author(s):  
M. Bownes ◽  
A. Scott ◽  
M. Blair
Keyword(s):  
Protein Synthesis ◽  
Sex Determination ◽  
Fat Body ◽  
Yolk Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Yolk Protein Genes ◽  
Fat Bodies ◽  
Inhibitor Of Protein Synthesis

The three yolk-protein genes of Drosophila are normally expressed only in adult female fat bodies and ovaries. 20-hydroxyecdysone can affect the transcription of these genes in males and females, as can mutations in the sex-determining genes tra, tra-2, ix and dsx. We have asked a number of basic questions about how these genes are regulated, using an inhibitor of protein synthesis (cycloheximide), labelling RNA in vivo, a temperature-sensitive sex-determination mutant (tra-2ts1), and 20-hydroxyecdysone. We have found that the yolk-protein genes are continuously transcribed in the fat bodies of adult females and that maintenance of this transcription requires protein synthesis. Hormone induction in males is also inhibited by cycloheximide, suggesting that the products of other genes are essential both for 20-hydroxyecdysone to be able to switch on the genes, and for their continuous transcription in the female fat body. The products of the tra-2 gene are also required for continuous transcription of the yolk-protein genes, suggesting that the pathway inhibited by the cycloheximide is that of the sex-determination hierarchy. 20-hydroxyecdysone can override the sex-determination system and induce yolk protein synthesis in normal males and tra-2ts reared and maintained at the restrictive temperature.

scholarly journals Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor.

1995 ◽  
Vol 15 (12) ◽  
pp. 6943-6952 ◽  
Author(s):  
M Lossky ◽  
P C Wensink
Keyword(s):  
Fat Body ◽  
Regulatory Element ◽  
Ovarian Follicle ◽  
In Vitro Transcription ◽  
Yolk Protein ◽  
Follicle Cells ◽  
Gata Factor ◽  
Yolk Protein Genes

The divergently transcribed yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed only in adult females, in fat body tissue and in ovarian follicle cells. Using an in vitro transcription assay, we have identified a single 12-bp DNA element that activates transcription from the promoters of both Yp genes. In vivo, this regulatory element is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. We show that among the Drosophila genes known to encode GATA factors, only dGATAb is expressed in ovaries. The single transcript that we detect in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed dGATAb binds to the 12-bp element; a similar binding activity is also present in the Kc0 nuclear extracts used for in vitro transcription assays. These in vitro and in vivo results lead us to propose that dGATAb makes several developmentally regulated products, one of which is a follicle cell-specific protein activating transcription of Yp1 and Yp2 from a known regulatory element.

Expression of the yolk-protein genes in the mutant doublesex dominant (dsxD) of Drosophila melanogaster

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 241-257
Author(s):  
Mary Bownes ◽  
Maureen Dempster ◽  
Mairearad Blair
Keyword(s):  
Fat Body ◽  
Adult Life ◽  
Yolk Protein ◽  
Heterozygous State ◽  
X Chromosomes ◽  
Yolk Proteins ◽  
Transcript Levels ◽  
Yolk Protein Genes ◽  
Males And Females ◽  
Fat Bodies

Adult flies mutant for doublesex dominant (dsxD) are intermediate in phenotype between males and females. The dsxD mutation acts in the heterozygous state to transform only flies with two X chromosomes into intersexes, XY flies are unaffected by the mutation. Yolkprotein synthesis, which normally occurs in the ovaries and fat bodies of females, but not in males unless stimulated with 20-hydroxy-ecdysone, is reduced. The dsxD fat body synthesizes less yolk proteins throughout adult life, and the gonads rarely make yolk proteins. Using cloned yolk-protein genes as probes for measuring transcript levels we have shown that expression of these genes in dsxD is regulated both transcriptionally and post-transcriptionally. We suggest that the dsxD locus regulates the expression of the yolk-protein genes from within the fat body cells and does not operate by modulating ecdysteroid titres in the adults.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Disparity between in vivo and in vitro synthesis of yolk protein by fat bodies of vitellogenic Locusta migratoria after allatectomy

1980 ◽  
Vol 41 (3) ◽  
pp. 417-420 ◽  
Author(s):  
Mark Pines ◽  
Esther Lubzens ◽  
Paula Harry ◽  
Shalom W. Applebaum
Keyword(s):  
Locusta Migratoria ◽  
Yolk Protein ◽  
In Vitro Synthesis ◽  
Fat Bodies

Hymenolepis diminuta: influence of metacestodes on synthesis and secretion of fat body protein and its ovarian sequestration in the intermediate host, Tenebrio molitor

Parasitology ◽  
1986 ◽  
Vol 93 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Hilary Hurd ◽  
C. Arme
Keyword(s):  
Intermediate Host ◽  
Post Infection ◽  
Fat Body ◽  
Tenebrio Molitor ◽  
Hymenolepis Diminuta ◽  
Body Protein ◽  
Concomitant Reduction ◽  
Fat Bodies

SUMMARYFemale Tenebrio molitor infected with metacestodes of Hymenolepis diminuta exhibit elevated concentrations of female-specific proteins in their haemolymph and the origin of these has been investigated. Following a 4 h in vitro incubation with [14C]leucine, fat bodies from non-infected females secreted 13 times more protein than those from females 12 days post-infection. A comparison of the uptake in vivo of radio-isotope labelled amino acids by ovaries from non-infected and infected beetles of various ages revealed no differences; however, a 51·5% decrease in protein sequestration was detected in females 12 days post-infection. Electrophoresis of homogenates of radio-isotope labelled ovaries demonstrated that the majority of label was associated with vitellin sub-units. It is suggested that the decrease in vitellogenin sequestration associated with infection results in an increase in the haemolymph concentration of these proteins despite a concomitant reduction in their secretion by fat bodies. Both fat body synthesis and ovarian sequestration are under juvenile hormone control and it is proposed that metacestodes of H. diminuta may cause a reduction in the concentration of this hormone in the intermediate host.

Autonomous yolk protein synthesis in ovaries ofDrosophila cultured in vivo

1979 ◽  
Vol 187 (3) ◽  
pp. 255-266 ◽  
Author(s):  
Ž. Srdić ◽  
C. Reinhardt ◽  
H. Beck ◽  
H. Gloor
Keyword(s):  
Protein Synthesis ◽  
Yolk Protein

scholarly journals A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

1996 ◽  
Vol 16 (3) ◽  
pp. 1017-1026 ◽  
Author(s):  
M M Smith ◽  
P Yang ◽  
M S Santisteban ◽  
P W Boone ◽  
A T Goldstein ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Nuclear Division ◽  
Dna Recombination ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Histone H4 ◽  
Eukaryotic Chromosome ◽  
Chromosome Transmission

The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation.

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