scholarly journals Definition of cis-acting elements regulating expression of the Drosophila melanogaster ninaE opsin gene by oligonucleotide-directed mutagenesis.

Genetics ◽  
1989 ◽  
Vol 121 (1) ◽  
pp. 77-87 ◽  
Author(s):  
D Mismer ◽  
G M Rubin
Keyword(s):  
Drosophila Melanogaster ◽  
The Body ◽  
Drosophila Virilis ◽  
P Element ◽  
Opsin Gene ◽  
Directed Mutagenesis ◽  
Homologous Gene ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Cis Acting

Abstract We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter.

Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster.

Genetics ◽  
1988 ◽  
Vol 120 (1) ◽  
pp. 173-180
Author(s):  
D Mismer ◽  
W M Michael ◽  
T R Laverty ◽  
G M Rubin
Keyword(s):  
Drosophila Melanogaster ◽  
Fusion Gene ◽  
Photoreceptor Cells ◽  
Histochemical Staining ◽  
P Element ◽  
Opsin Gene ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Beta Galactosidase ◽  
Cat Expression

Abstract We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells. DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression. Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression. Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs. We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112. We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC). By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli.

scholarly journals Analysis of the Promoter of the ninaE Opsin Gene in Drosophila melanogaster

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 565-578 ◽  
Author(s):  
Drzislav Mismer ◽  
Gerald M Rubin
Keyword(s):  
Control Region ◽  
Photoreceptor Cells ◽  
P Element ◽  
Opsin Gene ◽  
Regulatory Sequences ◽  
Enhancer Element ◽  
Cis Acting ◽  
E Coli ◽  
Promoter Function ◽  
Control Regions

ABSTRACT We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogasteropsin, the protein component of the primary chromophore of photoreceptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (β-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing β-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

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.

scholarly journals Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.

1984 ◽  
Vol 4 (7) ◽  
pp. 1343-1353 ◽  
Author(s):  
W W Mattox ◽  
N Davidson
Keyword(s):  
Drosophila Melanogaster ◽  
Base Pair ◽  
Regulatory Element ◽  
P Element ◽  
Functional Domain ◽  
Lambda Phage ◽  
Base Pairs ◽  
Negative Regulatory Element ◽  
Cis Acting ◽  
Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

scholarly journals Two independent cis-acting elements regulate the sex- and tissue-specific expression ofyp3inDrosophila melanogaster

1995 ◽  
Vol 66 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Elaine Ronaldson ◽  
Mary Bownes
Keyword(s):  
Fat Body ◽  
Germ Line ◽  
Ovarian Follicle ◽  
Yolk Protein ◽  
P Element ◽  
Follicle Cells ◽  
Cell Type Specificity ◽  
Specific Expression ◽  
Cis Acting ◽  
Ideal System

SummaryInDrosophila, the threeyolk protein(yp) genes are transcribed in a sex-, tissue- and developmentally specific manner, providing an ideal system in which to investigate the factors involved in their regulation. The yolk proteins are synthesized in the fat body of adult females, and in the ovarian follicle cells surrounding the developing oocyte during stages 8–10 of oogenesis. We report here an analysis of theyolk protein 3(yp3) gene and its flanking sequences by means of P-element mediated germ-line transformation and demonstrate that a 747 bp promoter region is sufficient to direct sex-specific expression in the female fat body and both the temporal- and cell-type-specificity of expression during oogenesis. Two elements that independently governyp3transcription in these tissues have been separated and no other sequences in the upstream, downstream or coding regions have been identified that are autonomously involved inyp3expression.

scholarly journals Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene

1984 ◽  
Vol 4 (7) ◽  
pp. 1343-1353
Author(s):  
W W Mattox ◽  
N Davidson
Keyword(s):  
Drosophila Melanogaster ◽  
Base Pair ◽  
Regulatory Element ◽  
P Element ◽  
Functional Domain ◽  
Lambda Phage ◽  
Base Pairs ◽  
Negative Regulatory Element ◽  
Cis Acting ◽  
Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

scholarly journals A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn

1987 ◽  
Vol 7 (5) ◽  
pp. 1710-1715
Author(s):  
E Otto ◽  
J M Allen ◽  
J E Young ◽  
R D Palmiter ◽  
G Maroni
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Germ Line ◽  
Initiation Site ◽  
P Element ◽  
Specific Expression ◽  
Metallothionein Gene ◽  
Regulated Expression ◽  
Metallothionein Promoter ◽  
Simplex Virus

Cloned fragments of DNA including the Drosophila melanogaster metallothionein gene Mtn and different amounts of 5' flanking sequences were introduced into flies by P-element-mediated germ line transformation. Comparison of RNA levels in different transformants revealed that metal-regulated and tissue-specific expression of Mtn requires no more than 373 base pairs upstream of the initiation site of transcription. Transformants having an additional, transcribed copy of Mtn could tolerate increased concentrations of cadmium, indicating that Mtn expression is directly related to this phenotype. In separate experiments, these D. melanogaster promoter sequences were fused to the coding sequences of the herpes simplex virus thymidine kinase (TK) gene. After transfection of this fusion into baby hamster kidney cells, increases in TK activity and accumulation of TK RNA were inducible by metals. A series of 5' and 3' deletions showed that D. melanogaster sequences from -130 to -6 were sufficient to confer metal-regulated expression to the TK gene. The function of the D. melanogaster metallothionein promoter in mammalian cells indicates that the mechanism controlling metal regulation is evolutionarily conserved.

Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence.

Genetics ◽  
1992 ◽  
Vol 131 (1) ◽  
pp. 113-128 ◽  
Author(s):  
M T O'Neil ◽  
J M Belote
Keyword(s):  
Alternative Splicing ◽  
Drosophila Virilis ◽  
Evolutionary Divergence ◽  
Sequence Comparisons ◽  
Specific Expression ◽  
Cis Acting ◽  
Conserved Sequence ◽  
Homologous Genes ◽  
Specific Alternative ◽  
High Degree

Abstract The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.

Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron

1991 ◽  
Vol 11 (3) ◽  
pp. 1538-1546
Author(s):  
A C Chain ◽  
S Zollman ◽  
J C Tseng ◽  
F A Laski
Keyword(s):  
Drosophila Melanogaster ◽  
Splice Site ◽  
Germ Line ◽  
Somatic Cells ◽  
Regulatory Region ◽  
Systematic Search ◽  
P Element ◽  
Regulatory Sequence ◽  
Cis Acting ◽  
The Third

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

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.

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