P transposons controlled by the heat shock promoter.

We have transformed Drosophila melanogaster with modified P-element transposons, which express the transposase function from the heat-inducible hsp70 heat shock promoter. The Icarus transposon, which contains a direct hsp70-P fusion gene, behaved like a very active autonomous P element even before heat shock induction. Although heat shock led to abundant somatic transcription, transposition of the Icarus element was confined to germ line cells. To reduce the constitutive transposase activity observed for the Icarus element, we attenuated the translational efficiency of the transposase RNA by inserting the transposon 5 neomycin resistance gene between the hsp70 promoter and the P-element sequences. The resulting construct, called Icarus-neo, conferred resistance to G418, and its transposition was significantly stimulated by heat shock. Heat shocks applied during the embryonic or third instar larval stage had similar effects, indicating that transposition of P elements is not restricted to a certain developmental stage. Both Icarus and Icarus-neo destabilized snw in a P-cytotype background and thus at least partially overcome the repression of transposition. Our results suggest that the regulation of P-element transposition occurs at both the transcriptional and posttranscriptional levels.

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Structure and expression of ubiquitin genes of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4727 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4727-4735 ◽

Cited By ~ 119

Author(s):

H S Lee ◽

J A Simon ◽

J T Lis

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Germ Line ◽

Fusion Gene ◽

Binding Motif ◽

Drosophila Genome ◽

Nucleic Acid Binding ◽

Positional Identity ◽

Polyubiquitin Gene ◽

A Minor

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.

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Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.663 ◽

1986 ◽

Vol 6 (2) ◽

pp. 663-673 ◽

Cited By ~ 32

Author(s):

E Hoffman ◽

V Corces

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Dna Sequences ◽

Germ Line ◽

Initiation Site ◽

P Element ◽

Heat Shock Gene ◽

Base Pairs ◽

Consensus Sequences ◽

Shock Gene

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.

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Duration and Persistence of Heat Shock Induction of Chilling Tolerance in Cucumber Seedling Roots

HortScience ◽

10.21273/hortsci.32.3.538e ◽

1997 ◽

Vol 32 (3) ◽

pp. 538E-538

Author(s):

Hua Zhang ◽

Paul H. Jennings

Keyword(s):

Heat Shock ◽

Root Growth ◽

Electrolyte Leakage ◽

Chilling Tolerance ◽

Shock Duration ◽

Dependent Manner ◽

Heat Shocks ◽

Shock Proteins ◽

Membrane Peroxidation ◽

Heat Shock Induction

The effects of heat shock duration and persistence on the induction of chilling tolerance in cucumber roots were studied using total root growth, electrolyte leakage, and membrane peroxidation as injury indices after chilling. Heat shock reduced the chilling induced electrolyte leakage, decreased membrane peroxidation as measured by MDA content, and resulted in a greater total root growth after chilling compared to the control. Heat shocks at 40°C, applied to 36 hr germinated seedlings for time periods from 1 to 15 hr, all resulted in an increase in chilling tolerance in a time-dependent manner. The heat shock induction of chilling tolerance is most effective when heat shock was imposed immediately before chilling, but the effect is persistent even 32 hr after heat shock when seedlings are held at 25°C before chilling. The possible mechanism of heat shock effect and its persistence will be discussed in relation to heat shock proteins and antioxidant enzyme systems.

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Germ-line and somatic recombination induced by in vitro modified P elements in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/124.2.331 ◽

1990 ◽

Vol 124 (2) ◽

pp. 331-337 ◽

Cited By ~ 2

Author(s):

J A Sved ◽

W B Eggleston ◽

W R Engels

Keyword(s):

Drosophila Melanogaster ◽

Germ Line ◽

Somatic Cells ◽

Chromosome Breakage ◽

P Element ◽

Male Germ Line ◽

Male Recombination ◽

P Elements ◽

Somatic Recombination

Abstract The P element insertion delta 2-3(99B) has previously been shown to activate incomplete P elements elsewhere in the genome. We show that this element, in conjunction with a second incomplete P element, P[CaSpeR], also induces recombination in the male germ line. The recombination is induced preferentially in the region of the P[CaSpeR] element. Recombinant chromosomes contain the P[CaSpeR] element in more than 50% of cases, and alternative models of transposon replication and preferential chromosome breakage are put forward to explain this finding. As is the case with male recombination induced by P-M dysgenic crosses, recombination appears to be premeiotic in a high proportion of cases. The delta 2-3(99B) element is known to act in somatic cells. Correspondingly, we show that the delta 2-3(99B)-P[CaSpeR] combination elevates the incidence of somatic recombination.

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The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.973-981.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 973-981

Author(s):

E P Hoffman ◽

S L Gerring ◽

V G Corces

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Dna Sequence ◽

Germ Line ◽

P Element ◽

Position Effects ◽

Consensus Sequences ◽

Basal Expression ◽

Upstream Promoter ◽

Germ Line Transformation

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

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Genetic and molecular characterization of a variegating hsp70-lacZ fusion gene in the euchromatic 31B region of Drosophila melanogaster

Genome ◽

10.1139/g01-038 ◽

2001 ◽

Vol 44 (4) ◽

pp. 698-707

Author(s):

Patrick Morcillo ◽

Ross J MacIntyre

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Fusion Gene ◽

Position Effect ◽

P Element ◽

Lacz Fusion ◽

Position Effect Variegation ◽

Salivary Gland Cells ◽

P Element Transformation

A hsp70lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by P-element transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.Key words: Drosophila, euchromatic position effect, heat shock construct.

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Induced expression of a Drosophila hsp70 promoter-fusion transgene is reduced after repeated heat shocks

Genetics Research ◽

10.1017/s0016672300030469 ◽

1992 ◽

Vol 59 (3) ◽

pp. 183-188 ◽

Cited By ~ 4

Author(s):

S. M. N. Hunt ◽

M. R. Wilkins ◽

H. W. Stokes ◽

G. E. Daggard ◽

R. Frankham

Keyword(s):

Heat Shock ◽

Antisense Rna ◽

Heat Shock Protein 70 ◽

Fusion Gene ◽

Mrna Levels ◽

Hsp70 Promoter ◽

Expression Studies ◽

Promoter Fusion ◽

Heat Shocks

SummaryLevels of transcripts produced by a heat shock protein 70 (hsp70)-antisense white transgene in Drosophila were measured after single and multiple heat shocks to determine whether the hsp70 promoter could produce sustained high levels of transgene transcripts. A single heat shock resulted in typical highly inducible levels of RNA, but the amount of antisense RNA was substantially reduced after multiple heat shocks. Endogenous hsp70 mRNA levels were also less abundant after multiple heat shocks as compared to a single heat shock. The hsp70 promoter is unsuitable for use in fusion gene constructs for long term expression studies where repeated heat shocks are required.

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Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2188 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2188-2195 ◽

Cited By ~ 26

Author(s):

D E Rancourt ◽

V K Walker ◽

P L Davies

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Germ Line ◽

Signal Sequence ◽

Antifreeze Protein ◽

P Element ◽

Western Blots ◽

Gene Coding ◽

Gene Transcripts ◽

Transgenic Drosophila

The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp70 promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.

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Repression of Hybrid Dysgenesis in Drosophila melanogaster by Heat-Shock-Inducible Sense and Antisense P-Element Constructs

Genetics ◽

10.1093/genetics/144.4.1529 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1529-1544

Author(s):

Michael J Simmons ◽

John D Raymond ◽

Craig D Grimes ◽

Carina Belinco ◽

Bret C Haake ◽

...

Keyword(s):

Heat Shock ◽

Gonadal Dysgenesis ◽

Germ Line ◽

Inducible Promoter ◽

P Element ◽

Position Effects ◽

Antisense Constructs ◽

Female Germ Line ◽

Element Activity

Sets of sense and antisense P-element constructs controlled by a heat-shock-inducible promoter were tested for their ability to repress manifestations of P-element activity in vivo. As a group, the antisense constructs repressed pupal lethality, a somatic manifestation of P activity, and this repression was significantly enhanced by heat shock. Three of the 11 antisense constructs also repressed gonadal dysgenesis, a manifestation of P activity in the female germ line; however, none had any effect on P-element-mediated mutability in the male germ line. Among the 13 different heat-shock-inducible sense constructs that were tested, those containing the KP and DP elements were strong repressors of pupal lethality, gonadal dysgenesis and P-element-mediated mutability; however, individual lines carrying these constructs varied in their ability to repress each of these traits, presumably because of genomic position effects. With the exception of the sense construct that contained a complete P element, none of the sense or antisense constructs repressed a lacZ reporter gene driven by the P-element promoter. Overall, the experimental results suggest that in nature, P-element activity could be regulated by P-encoded polypeptides and by antisense P RNAs.

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