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Biomedical subjects

V Pirrotta

Publications and source records attributed to V Pirrotta.

At least 19 recordsLinked to original sources
PubMed1992

Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication.

We have analyzed the eye and testis enhancers located 1 kb upstream of the transcription start site of the white gene. Both enhancers confer the corresponding tissue-specific expression on a heterologous promoter as well as on the white promoter. The eye determinant consists of multiple elements, each able to stimulate eye-specific expression. It also contains five binding sites for the zeste protein while the immediately adjacent testis element contains none. Site-directed mutation of these zeste binding sites abolishes the zeste-white interaction but does not significantly affect the eye enhancer activity, indicating that they are not important for the eye enhancer activity per se. Other zeste binding sites just upstream of the promoter are not necessary for the zeste-white interaction. We conclude that the overlap of the eye enhancer with the zeste binding sites is responsible for the zeste-white interaction and explains why this interaction affects eye but not testis expression. Sequence deletion or substitution experiments suggested that the white promoter is internal to the transcription start site; the zeste protein is not required for distant enhancer action but a 95-bp promoter-proximal sequence is essential for distant enhancer-promoter interaction. This element may serve as an anchor to stabilize formation of a loop that brings the enhancer to the vicinity of the promoter.

Animals↗
PubMed1992

Conserved DNA binding and self-association domains of the Drosophila zeste protein.

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

Amino Acid Sequence↗
PubMed1992

The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes.

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.

Amino Acid Sequence↗
PubMed1991

The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes.

The Drosophila homeotic gene Ultrabithorax (Ubx) is regulated by complex mechanisms that specify the spatial domain, the timing and the activity of the gene in individual tissues and in individual cells. In early embryonic development, Ubx expression is controlled by segmentation genes turned on earlier in the developmental hierarchy. Correct Ubx expression depends on multiple regulatory sequences located outside the basal promoter. Here we report that a 500 bp DNA fragment from the bx region of the Ubx unit, approximately 30 kb away from the promoter, contains one of the distant regulatory elements (bx region enhancer, BRE). During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). We show that the hb protein binds to the BRE element and that such binding is essential for hb repression in vivo, hb protein also binds to DNA fragments from abx and bxd, two other regulatory regions of the Ubx gene. We conclude that hb represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps.

Animals↗
PubMed1991

Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes.

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.

Animals↗
PubMed1979

Operators and promoters in the OR region of phage 434.

The OR operator region of phage 434 contains three 14 bp blocks with sequence ACAAGA-A--TTGT which are presumed to be the 434 repressor recognition sites. Operator constitutive mutations are located in two of these blocks, while a mutation affecting repressor levels in the lysogenic state is located in the third. Two transcripts obtained in vitro, one leftwards and one rightwards, are tentatively identified as the Prm and PR transcription starts. the arrangement of the 434 operator region appears to be very similar to that of the OR of phage lambda.

Base Sequence↗
PubMed1978

Transcription of cloned tRNA gene fragments and subfragments injected into the oocyte nucleus of Xenopus laevis.

Cloned 3.18 kilobase fragments of Xenopus laevis DNA containing genes for tRNAMet1 and for at least one other 4S RNA species are transcribed rapidly after their injection into the nucleus of X. laevis oocytes. The newly synthesized RNA can be resolved by gel electrophoresis into a few predominant 4S RNA species and a series of slower migrating components. One of the 4S RNA species appears to be identical, by fingerprint analysis, to the tRNAMet1 isolated by hybridization of somatic cell RNA to this cloned tRNA gene fragment (tDNA). Thus, the tRNAMet1 produced after injection can be both fully processed and modified. Its rate of synthesis is estimated to be about 6 x 10(9) molecules/hr in each oocyte injected with 2 ng of tDNA. When the tDNA fragment is cleaved into two halves with restriction endonuclease Sst I, each injected half gives rise to a subset of the RNAs produced after injection of the intact fragment. This experiment thus suggests the presence of at least two transcriptional units on this cloned tDNA. This simple way of biologically testing defined restriction fragments may be of value for analyzing the functional organization of other cloned eukaryotic DNA units.

Animals↗
PubMed1978

Characterization of a mouse DNA clone containing an immunoglobulin variable region gene.

A 4.8 kilobase mouse embryo DNA fragment was inserted into a phage lambda genome and was subsequently characterized by electron microscopy, restriction enzyme mapping and partial DNA sequencing. This fragment contains a 400 base sequence which is homologous to that of an immunoglobulin light lambda chain mRNA which spans 3.3 to 3.7 kilobases from one end of the fragment. Restriction enzyme mapping as well as partial nucleotide sequencing of the 3' terminal of the homology region confirm the previous conclusion [Tonegawa, Brack, Hozumi and Schuller, Proc. Natl. Acad. Sci. USA. 74, 3518-3522 (1977)] that the cloned DNA fragment contains a Vlambda gene sequence which is separate from any Clambda sequence.

Animals↗
PubMed1978

Organization of immunoglobulin genes.

The nucleotide-sequence determination of a cloned, embryonic Vlambda gene directly demonstrated that V genes are separate from a corresponding C gene in embryonic cells. Analysis by restriction enzymes of total cellular DNA from various sources strongly suggested that the two separate immunoglobulin genes become continuous during differentiation of B lymphocytes. There seems to be a strict correlation between the joining event and activation of the joined genes. Cloning of more immunoglobulin genes from embryo and plasma cells will not only provide direct demonstration of such a gene-joining event but also help in the elucidation of a possible relationship of the event to gene activation mechanisms.

Animals↗
PubMed1977

The 5S genes of Drosophila melanogaster.

We have cloned embryonic Drosophila DNA using the poly (dA-DT) connector method (Lobban and Kaiser, 1973) and the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975) as a cloning vehicle. Two clones, containing hybrid plasmids with sequences complementary to a 5S RNA probe isolated from Drosophila tissue culture cells, were identified by the Grunstein and Hogness (1975) colony hybridization procedure. One hybrid plasmid has a Drosophila insert which is comprised solely of tandem repeats of the 5S gene plus spacer sequences. The other plasmid contains an insert which has about 20 tandem 5S repeat units plus an additional 4 kilobases of adjacent sequences. The size of the 5S repeat unit was determined by gel electrophoresis and was found to be approximately 375 base pairs. We present a restriction map of both plasmids, and a detailed map of of the5S repeat unit. The 5S repat unit shows slight length and sequence heterogeneity. We present evidence suggesting that the 5S genes in Drosophila melanogaster may be arranged in a single continuous cluster.

Base Sequence↗
PubMed1977

A simple, general procedure for purifying restriction endonucleases.

A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography.

Bacillus↗
PubMed1976

Lambda repressor regulates the switch between PR and Prm promoters.

The DNA region containing the Or operator, Pr and Prm promoters and their transcription initiations is sequenced. By binding to Or, repressor turns off Pr, turns on Prm and at higher concentrations turns off Prm, regulating its own synthesis. Prm mRNA is unique in beginning immediately with the initiation of translation, without a preceding leader sequence.

Base Sequence↗
PubMed1976

Two restriction endonucleases from Bacillus globiggi.

The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped. This enzyme recognises the sequence 5' ...AGATCT...3' and makes staggered cuts producing sticky ends. In lambda DNA, the second A in this sequence is methylated about 50% of the time by a bacterial methylase absent in E. coli dam. In contrast to Bgl II, Bgl I makes many cuts in lambda DNA and produces 5' terminals which are not substrates for polynucleotide kinase.

Bacillus↗