Identification of cis-acting regulatory sequences by P element-mediated germline transformation of Drosophila embryos

Author(s):  
Eric Hoffman ◽  
Victor Corces
Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 565-578 ◽  
Author(s):  
Drzislav Mismer ◽  
Gerald M Rubin

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.


Genetics ◽  
1989 ◽  
Vol 121 (1) ◽  
pp. 77-87 ◽  
Author(s):  
D Mismer ◽  
G M Rubin

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.


Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1245-1254 ◽  
Author(s):  
W. Sullivan ◽  
P. Fogarty ◽  
W. Theurkauf

Cytoplasmic organization, nuclear migration, and nuclear division in the early syncytial Drosophila embryo are all modulated by the cytoskeleton. In an attempt to identify genes involved in cytoskeletal functions, we have examined a collection of maternal-effect lethal mutations induced by single P-element transposition for those that cause defects in nuclear movement, organization, or morphology during the syncytial embryonic divisions. We describe three mutations, grapes, scrambled, and nuclear-fallout, which define three previously uncharacterized genes. Females homozygous for these mutations produce embryos that exhibit extensive mitotic division errors only after the nuclei migrate to the surface. Analysis of the microfilament and microtubule organization in embryos derived from these newly identified mutations reveal disruptions in the cortical cytoskeleton. Each of the three mutations disrupts the actin-based pseudocleavage furrows and the cellularization furrows in a distinct fashion. In addition to identifying new genes involved in cytoskeletal organization, these mutations provide insights into cytoskeletal function during early Drosophila embryogenesis.


1989 ◽  
Vol 9 (8) ◽  
pp. 3517-3523
Author(s):  
D P McDonnell ◽  
J W Pike ◽  
D J Drutz ◽  
T R Butt ◽  
B W O'Malley

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.


1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick

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.


Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 659-669 ◽  
Author(s):  
S.E. Bergsten ◽  
E.R. Gavis

Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3′ untranslated region (3′UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3′UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3′UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.


Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 189-200 ◽  
Author(s):  
U. Grossniklaus ◽  
H.J. Bellen ◽  
C. Wilson ◽  
W.J. Gehring

We have stained the ovaries of nearly 600 different Drosophila strains carrying single copies of a P-element enhancer detector. This transposon detects neighbouring genomic transcriptional regulatory sequences by means of a beta-galactosidase reporter gene. Numerous strains are stained in specific cells and at specific stages of oogenesis and provide useful ovarian markers for cell types that in some cases have not previously been recognized by morphological criteria. Since recent data have suggested that a substantial number of the regulatory elements detected by enhancer detection control neighbouring genes, we discuss the implications of our results concerning ovarian gene expression patterns in Drosophila. We have also identified a small number of insertion-linked recessive mutants that are sterile or lead to ovarian defects. We observe a strong correlation with specific germ line staining patterns in these strains, suggesting that certain patterns are more likely to be associated with female sterile genes than others. On the basis of our results, we suggest new strategies, which are not primarily based on the generation of mutants, to screen for and isolated female sterile genes.


Development ◽  
1994 ◽  
Vol 120 (10) ◽  
pp. 2945-2955 ◽  
Author(s):  
J.A. Langeland ◽  
S.F. Attai ◽  
K. Vorwerk ◽  
S.B. Carroll

We present a genetic and molecular analysis of two hairy (h) pair-rule stripes in order to determine how gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. We have delimited regulatory sequences critical for the expression of h stripes 5 and 6 to 302 bp and 526 bp fragments, respectively, and assayed the expression of stripe-specific reporter constructs in several gap mutant backgrounds. We demonstrate that posterior stripe boundaries are established by gap protein repressors unique to each stripe: h stripe 5 is repressed by the giant (gt) protein on its posterior border and h stripe 6 is repressed by the hunchback (hb) protein on its posterior border. Interestingly, Kruppel (Kr) limits the anterior expression limits of both stripes and is the only gap gene to do so, indicating that stripes 5 and 6 may be coordinately positioned by the Kr repressor. In contrast to these very similar cases of spatial repression, stripes 5 and 6 appear to be activated by different mechanisms. Stripe 6 is critically dependent upon knirps (kni) for activation, while stripe 5 likely requires a combination of activating proteins (gap and non-gap). To begin a mechanistic understanding of stripe formation, we locate binding sites for the Kr protein in both stripe enhancers. The stripe 6 enhancer contains higher affinity Kr-binding sites than the stripe 5 enhancer, which may allow for the two stripes to be repressed at different Kr protein concentration thresholds. We also demonstrate that the kni activator binds to the stripe 6 enhancer and present evidence for a competitive mechanism of Kr repression of stripe 6.


Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 437-449 ◽  
Author(s):  
E. Sanchez-Herrero

The abdominal-A (abd-A) and Abdominal-B (Abd-B) genes of the bithorax complex (BX-C) specify the identity of most of the Drosophila abdomen. Six different classes of infraabdominal (iab) mutations within the BX-C transform a subset of the parasegments affected by the lack of these two genes. It is thought that these mutations define parasegmental cis-regulatory regions that control the expression of abd-A and Abd-B. By staining embryos mutant for different iab mutations with anti-abd-A and anti-Abd-B antibodies I show here that the expression of Abd-B (and probably also abd-A) exhibit a parasegmental regulation. I have also studied the significance of the chromosomal order of parasegmental iab regulatory sequences, and the possible presence of chromosomal ‘boundaries’ between them, by looking at the expression of abd-A and Abd-B in embryos carrying the Uab and Mcp mutations. These data are discussed in the light of models of parasegmental-specific regulatory regions within the BX-C.


1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.


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