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W J Gehring

Publications and source records attributed to W J Gehring.

At least 127 records · Page 7Linked to original sources
PubMed1987

Homeo boxes in the study of development.

The body plan of Drosophila is determined to a large extent by homeotic genes, which specify the identity and spatial arrangement of the body segments. Homeotic genes share a characteristic DNA segment, the homeo box, which encodes a defined domain of the homeotic proteins. The homeo domain seems to mediate the binding to specific DNA sequences, whereby the homeotic proteins exert a gene regulatory function. By isolating the normal Antennapedia gene, fusing its protein-coding sequences to an inducible promoter, and reintroducing this fusion gene into the germline of flies, it has been possible to transform head structures into thoracic structures and to alter the body plan in a predicted way. Sequence homologies suggest that similar genetic mechanisms may control development in higher organisms.

Animals↗
PubMed1987

The white gene as a marker in a new P-element vector for gene transfer in Drosophila.

We describe new vectors suitable for P-element mediated germ line transformation of Drosophila melanogaster using passenger genes whose expression does not result in a readily detectable phenotypic change of the transformed flies. The P-element vectors contain the white gene fused to the heat shock protein 70 (hsp70) gene promoter. Expression of the white gene rescues the white phenotype of recipient flies partly or completely even without heat treatment. Transformed descendents of most founder animals (GO) fall into two classes which are distinguishable by their orange and red eye colours. The different levels of white expression are presumably due to position effects associated with different chromosomal sites of insertion. Doubling of the gene dose in orange eyed fly stocks results in an easily visible darkening of the eye colour. Consequently, the generation of homozygous transformants is easily possible by simple inbreeding due to the phenotypic distinction of homo- and heterozygous transformants. Cloning into these P-element vectors is facilitated by the presence of polylinkers with 8 and 12 unique restriction sites.

Animals↗
PubMed1987

Expression of the caudal gene in the germ line of Drosophila: formation of an RNA and protein gradient during early embryogenesis.

The caudal (cad) gene of Drosophila encodes a maternal and a zygotic transcript which have different promoters. Both mRNAs contain the same open reading frame, including a homeo box. In situ hybridization and antibody staining show that the maternal RNA and protein are localized in an anteroposterior gradient during the syncytial blastoderm stage. The protein is found mainly in the nuclei and is also present in the pole cells. Zygotic RNA and protein are localized in the primordia of the terminal abdominal segment, the hindgut, and in the posterior midgut rudiment. In third instar larvae, cad is expressed in the gut, the gonads, and parts of the genital discs. It is the first homeo box-containing gene expressed in the germ line of Drosophila.

Animals↗
PubMed1987

Molecular analysis of the dominant homeotic Antennapedia phenotype.

Most of the dominant alleles of the homeotic gene Antennapedia (Antp) which show a transformation of antennae into legs are associated with large chromosomal inversions. To determine the molecular mechanisms underlying the dominant phenotype, one of the strongest Antp alleles (Antp) was studied in more detail. The mutant chromosome has been cloned and the structure of the inversion has been identified. The inversion breaks two genes apart: the Antp gene and a previously unidentified gene, tentatively called responsible for dominant phenotype (rfd), located at 84D1-2. The two genes are transcribed in opposite directions and the breakpoints lie within introns of both genes. Through the inversion event, a reciprocal exchange of the first exons including promoters occurred leading to the production of new transcripts. The transcripts containing the entire Antp protein coding region which have been fused to the promoter of the rfd gene are lost in revertants of the dominant phenotype indicating a correlation between this fusion gene and the dominant phenotype. The molecular structure of inversion Antp suggests that the dominant phenotype arises via ectopic expression of the normal Antp protein due to a gene fusion event.

Journal Article↗
PubMed1987

Detection in situ of genomic regulatory elements in Drosophila.

We have developed an approach for the in situ detection of genomic elements that regulate transcription zin Drosophila melanogaster. The approach is analogous to a powerful method of bacterial genetics, the random generation of operon fusions, that enables the isolation and characterization of genes simply by knowing or postulating their pattern of expression; it is not necessary initially to screen for mutant phenotypes. To apply this approach to Drosophila, we have used the expression of the lacZ gene of Escherichia coli from the P-element promoter in germ-line transformant flies to screen for chromosomal elements that can act at a distance to stimulate expression from this apparently weak promoter. Of 49 transformed fly lines obtained, approximately 70% show some type of spatially regulated expression of the lacZ gene in embryos; many of these express lacZ specifically in the nervous system. The P-lacZ fusion gene is, therefore, an efficient tool for the recovery of elements that may regulate gene expression in Drosophila and for the generation of a wide variety of cell-type-specific markers.

Animals↗
PubMed1986

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs.

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to beta-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.

Journal Article↗
PubMed1986

Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22.

Downstream of the transcription start site in the Drosophila heat-shock gene hsp22, we have identified a region that is necessary for efficient transcription, and also for selective translation during heat shock. We assayed the expression of mutated genes after P-factor-mediated insertion into the genome. Deletions within the first 26 nucleotides block the preferential translation of hsp22 mRNA at high temperature. The rate of transcription is also decreased, though transcription is still heat-inducible. Leaving this region intact, up to 86% of the leader can be deleted without affecting translation or transcription. The functional region coincides with a region of sequence homology between the heat-shock genes. Only partial homology is found to a conserved sequence, ATCAGTTCT, found at the very 5' end of other insect genes.

Animals↗
PubMed1986

Germline transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes.

Two Drosophila mutants KM75 and HH5, which are mutated in the act88F actin gene specific for the indirect flight muscles (IFM), synthesize heat shock proteins (hsps) constitutively in a tissue-specific manner. We have introduced cloned mutant act88F genes into a strain containing the wild-type act88F allele by P-element-mediated transformation. Flies transformed with a 4.05 kb KM75 act88F gene fragment encoding the p42 actin variant express both p42 and hsps specifically in the IFM. Using normal/mutant chimeric genes, the mutation sites of KM75 and HH5 were mapped within the sequence encoding the last 72 amino acids of actin. An in vitro mutated gene encoding a protein that lacks the 72 carboxy-terminal amino acids also induces constitutive hsp synthesis.

Actins↗
PubMed1986

Structural organization and sequence of the homeotic gene Antennapedia of Drosophila melanogaster.

The structure of the Drosophila melanogaster Antennapedia (Antp) gene has been investigated by the isolation and sequencing of different cDNAs and genomic clones. Northern analysis, S1 mapping and primer extension experiments reveal a complex and unusual gene structure. The gene is composed of two promoters, eight exons spanning >100 kb, and two termination processing regions. Four major polyadenylated transcripts were found, two of them starting at a second internal promoter in front of exon 3. All four transcripts have extremely long untranslated leader and trailer sequences in the range of 1-2 kb. Despite the complex transcriptional organization, the open reading frame is the same in all transcripts, and starts in exon 5 giving rise to a protein of mol. wt. 42 800. The putative protein is rich in glutamine (18%) and proline (10%). The homeobox, a region which previously has been shown to be highly conserved among homeotic genes, is contained in the open reading frame and located in the last exon. Functional implications of the complex structure with respect to development and its relation to the mutant phenotypes are discussed.

Amino Acid Sequence↗
PubMed1986

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.

Animals↗
PubMed1986

Sequence requirement for expression of the Drosophila melanogaster heat shock protein hsp22 gene during heat shock and normal development.

A 14-base-pair sequence element present in almost all Drosophila melanogaster heat shock genes has been implicated in the heat inducibility of transcription. The D. melanogaster gene encoding the smallest heat shock protein, hsp22, contains within its 5' flanking sequences three such repeats, two close to the transcription start site and a distally located third one 101 base pairs further upstream. Deletion analyses reveal that the 5' flanking sequences required for full expression of the hsp22 gene extend beyond the distal repeat. Deletion of the furthest upstream repeat results in a five to sixfold reduction of gene expression. The small heat shock genes are transiently expressed in the late third instar larval and early pupal stages without external stimulation. A deletion of 5' flanking sequences to position -194, which includes two nucleotides of the distal heat shock element, has no effect on the developmental expression, whereas removal of an additional 18 nucleotides, including 12 nucleotides of the distal heat shock element, severely reduces developmental expression.

Animals↗
PubMed1985

Cloning of the homeotic Sex combs reduced gene in Drosophila and in situ localization of its transcripts.

We have extended our ;chromosomal walk' in the Antennapedia-complex (ANT-C) by isolating overlapping DNA sequences spanning the chromosomal segment between Antennapedia (Antp) and Deformed (Dfd). The transcription units, homeoboxes and M-repeats were mapped within this region. Four transcription units Antp, fushi tarazu (ftz), Sex combs reduced (Scr) and Dfd contain both a homeobox and an M repeat, whereas at least two additional transcription units, x and z, were found to lack these elements. The Scr locus was identified by deletion mapping. It consists of at least two exonic regions separated by a large intron. The homeobox is located in the 3' exon and is 82% homologous to the one in Antp. Scr encodes a major 3.9-kb RNA. A corresponding cDNA clone was used as a probe for in situ hybridization to sections of various embryonic stages. At gastrula stages Scr transcripts accumulate in the posterior head and the anterior thoracic region of the germ band. At later stages a strong accumulation of transcripts is observed in the suboesophageal and the prothoracic ganglion of the ventral nervous system.

Journal Article↗
PubMed1985

Molecular cloning of lethal(2)giant larvae, a recessive oncogene of Drosophila melanogaster.

Recessive mutations at the lethal(2)giant larvae (l(2)gl) locus of Drosophila melanogaster cause a complex syndrome, which has as its most striking features the development of malignant neuroblastomas in the larval brain and tumors of the imaginal discs. A chromosomal segment containing the l(2)gl gene has been cloned. Within this segment a transcription unit has been localized which is structurally changed in all l(2)gl alleles examined. The developmental profile of expression of the two RNAs (6 and 4.5 kb) made by this transcription unit coincides with the two major terminal phases of cell proliferation in the developing fly, namely, early embryogenesis and late third instar larvae. Tumors are produced when both normal l(2)gl alleles are inactivated by deletion or insertional mutation. The normal function of the l(2)gl presumably controls the normal cell proliferation of the optic centers of the brain and the imaginal discs, as well as their post-mitotic differentiation.

Alleles↗
PubMed1985

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.

Alcohol Dehydrogenase↗
PubMed1985

Isolation of caudal, a Drosophila homeo box-containing gene with maternal expression, whose transcripts form a concentration gradient at the pre-blastoderm stage.

We report the isolation and characterization of caudal (cad), a previously unknown Drosophila homeo box-containing gene from the 38E region on the left arm of the second chromosome. This homeo box has diverged from the prototype sequence in Antennapedia, but contains subregions which are highly homologous. By Northern analysis and in situ hybridization experiments two transcripts of 2.4 kb were found to accumulate in nurse cells and in the oocyte during oogenesis. These transcripts generate a transient concentration gradient along the antero-posterior axis at the syncytial blastoderm stage. At the cellular blastoderm stage transcripts accumulate in a single band from 13-19% egg length at the posterior end. One zygotic transcript of 2.6 kb is detected. At later stages this transcript is localized in ectodermally and endodermally derived tissues such as the proctodeum, the Malpighian tubules and the posterior midgut. The 2.6-kb transcript is detectable until the onset of metamorphosis.

Journal Article↗
PubMed1985

Excision of copia element in a revertant of the white-apricot mutation of Drosophila melanogaster leaves behind one long-terminal repeat.

The spontaneous mutation white-apricot (wa) in Drosophila melanogaster has a considerably lighter eye colour than the wild-type, and is caused by the insertion of a copia transposable element into a small intron of the white gene. We have analyzed an X-ray induced wa revertant (waR59K1), whose eye pigmentation is incompletely restored, by in situ hybridization, Southern blotting and sequencing analysis. At the site where copia had originally inserted, we found one long terminal repeat of copia, flanked by a 5 bp duplication with the same polarity as the direction of transcription of the white locus. These results suggest that the wa reversion is due to homologous recombination between the two long terminal repeats of copia.

Animals↗