Structure and expression of ubiquitin genes of Drosophila melanogaster.

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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A Genetically Marked I Element in Drosophila melanogaster Can Be Mobilized When ORF2 Is Provided in trans

Genetics ◽

10.1093/genetics/148.1.267 ◽

1998 ◽

Vol 148 (1) ◽

pp. 267-275

Author(s):

Isabelle Busseau ◽

Sophie Malinsky ◽

Maria Balakireva ◽

Marie-Christine Chaboissier ◽

Danielle Teninges ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Reverse Transcriptase ◽

Germ Line ◽

Ltr Retrotransposons ◽

Low Frequencies ◽

High Frequencies ◽

In Trans ◽

Very High Frequencies ◽

Very High

Abstract I factors in Drosophila melanogaster are non-LTR retrotransposons similar to mammalian LINEs. They transpose at very high frequencies in the germ line of SF females resulting from crosses between reactive females, devoid of active I factors, and inducer males, containing active I factors. The vermilion marked IviP2 element was designed to allow easy phenotypical screening for retrotransposition events. It is deleted in ORF2 and therefore cannot produce reverse transcriptase. IviP2 can be mobilized at very low frequencies by actively transposing I factors in the germ line of SF females. This paper shows that IviP2 can be mobilized more efficiently in the germ line of strongly reactive females in the absence of active I factors, when it is trans-complemented by the product of ORF2 synthesized from the hsp70 heat-shock promoter. This represents a promising step toward the use of marked I elements to study retrotransposition and as tools for mutagenesis.

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P transposons controlled by the heat shock promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1640 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1640-1649 ◽

Cited By ~ 50

Author(s):

H Steller ◽

V Pirrotta

Keyword(s):

Heat Shock ◽

Germ Line ◽

Fusion Gene ◽

P Element ◽

Translational Efficiency ◽

Heat Shock Promoter ◽

P Elements ◽

Heat Shocks ◽

Neomycin Resistance ◽

Heat Shock Induction

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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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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Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1746 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1746-1753 ◽

Cited By ~ 63

Author(s):

H Xiao ◽

J T Lis

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Fusion Gene ◽

Developmental Expression ◽

Regulatory Sequence ◽

Mrna Levels ◽

Hsp70 Gene ◽

High Level ◽

Hsp83 Gene

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.

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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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Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1746-1753.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1746-1753

Author(s):

H Xiao ◽

J T Lis

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Fusion Gene ◽

Developmental Expression ◽

Regulatory Sequence ◽

Mrna Levels ◽

Hsp70 Gene ◽

High Level ◽

Hsp83 Gene

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.

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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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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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Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.522 ◽

1996 ◽

Vol 16 (2) ◽

pp. 522-528 ◽

Cited By ~ 23

Author(s):

K J Keeler ◽

T Dray ◽

J E Penney ◽

G B Gloor

Keyword(s):

Drosophila Melanogaster ◽

Gene Targeting ◽

Germ Line ◽

Drosophila Embryo ◽

Homologous Sequence ◽

Specific Gene ◽

Drosophila Genome ◽

White Locus ◽

Dna Break ◽

Double Strand Dna

We report an efficient and specific gene targeting method for transforming the germ line of Drosophila melanogaster. The targeting occurs during the repair of a double-strand DNA break that is induced at the white locus by the excision of a P transposable element. The break is repaired when homologous sequence is copied from a plasmid injected into the Drosophila embryo. The procedure efficiently integrates DNA into the targeted locus of the Drosophila genome. Heterologous sequence of up to 13 kbp in length can be inserted, permitting the intergration of entire genes into a common genomic site for further study.

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