Biomedical subjects

V Pirrotta

Publications and source records attributed to V Pirrotta.

At least 37 records · Page 2Linked to original sources

PubMed1996

Hunchback-independent silencing of late Ubx enhancers by a Polycomb Group Response Element.

Drosophila homeotic genes are kept silent outside of their appropriate expression domains by a repressive chromatin complex formed by the Polycomb Group proteins. In the case of the Ubx gene, it has been proposed that the early repressor HB, binding at enhancers, recruits the Polycomb complex and specifies the domain of repression. We show that some Ubx enhancers are activated after blastoderm. If a Polycomb Response Element (PRE) is combined with such late enhancers, repression of a reporter gene can be established everywhere in the embryo, irrespective of the presence or absence of hunchback protein. If, however, these late enhancers are combined with a Ubx early enhancer, as well as a PRE, repression is established only where the reporter gene was inactive at early stages. These results imply that the Polycomb complex is not dependent on hunchback and suggest that the pattern of silencing reflects rather the state of activity of the gene at the time the Polycomb complex is formed.

Animals↗

PubMed1995

Chromatin complexes regulating gene expression in Drosophila.

Polycomb-group proteins form chromatin complexes at target genes such as Ubx, providing a cellular memory of gene activity in early development and determining the later activity of the gene. The complexes, whose constituents vary depending on site and genomic context, initiate at specific sites, but can extend to involve larger chromatin domains. How they persist through cell proliferation and how they silence gene activity are still open issues.

Animals↗

PubMed1995

Dosage compensation of the Drosophila white gene requires both the X chromosome environment and multiple intragenic elements.

The X-linked white gene when transposed to autosomes retains only partial dosage compensation. One copy of the gene in males expresses more than one copy but less than two copies in females. When inserted in ectopic X chromosome sites, the mini-white gene of the CaspeR vector can be fully dosage compensated and can even achieve hyperdosage compensation, meaning that one copy in males gives more expression than two copies in females. As sequences are removed gradually from the 5' end of the gene, we observe a progressive transition from hyperdosage compensation to full dosage compensation to partial dosage compensation. When the deletion reaches -17, the gene can no longer dosage compensate fully even on the X chromosome. A deletion reaching +173, 4 bp preceding the AUG initiation codon, further reduces dosage compensation both on the X chromosome and on autosomes. This truncated gene can still partially dosage compensate on autosomes, indicating the presence of dosage compensation determinants in the protein coding region. We conclude that full dosage compensation requires an X chromosome environment and that the white gene contains multiple dosage-compensation determinants, some near the promoter and some in the coding region.

ATP-Binding Cassette Transporters↗

PubMed1995

Distinct parasegmental and imaginal enhancers and the establishment of the expression pattern of the Ubx gene.

The expression domain of the Ubx gene in Drosophila embryos is bounded by the product of the hb gene, acting as a repressor. We show that all Ubx fragments that bind Hb protein in vitro contain parasegmental enhancers active in the embryo in specific parasegmental patterns. We have found three new embryonic enhancer elements in the upstream region, in addition to the two previously identified. Each produces a pattern initially bounded at PS6 by Hb but sooner or later breaks down this boundary and begins to express in the anterior region. These enhancers do not respond to the long-term maintenance mediated by the Polycomb group of genes. They also cease functioning after germ band extension. Expression in imaginal tissues is due to a set of entirely separate and independent imaginal disc enhancers. These do not contain Hb binding sites and by themselves have no anterior/posterior positional information, although some distinguish between ventral and dorsal discs. A third kind of element, the Polycomb Response Element (PRE), has no enhancer activity but causes long-term maintenance of the expression domain of other enhancers present in the vicinity. The interaction of these elements results in the correct expression of Ubx in imaginal tissues.

Animals↗

PubMed1994

A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression.

Segmentation genes provide the signals for the activation and regulation of homeotic genes in Drosophila but cannot maintain the resulting pattern of expression because their activity ceases halfway through embryogenesis. Maintenance of the pattern is due to the Polycomb group of genes (Pc-G) and the trithorax group of genes (trx-G), responsible for the persistence of the active or repressed state of homeotic genes. We have identified a regulatory element in the Ubx gene that responds to Pc-G and trx-G genes. Transposons carrying this element create new binding sites for Pc-G products in the polytene chromosomes. This Pc-G maintenance element (PRE), establishes a repressive complex that keeps enhancers repressed in cells in which they were originally repressed and maintains this state through many cell divisions. The trx-G products stimulate the expression of enhancers in cells in which they were originally active. This mechanism is responsible for the correct regulation of imaginal disc enhancers, which lack themselves antero-posterior positional information. The PRE also causes severe variegation of the mini-white gene present in the transposon, a phenomenon very similar to heterochromatic position-effect variegation. The significance of this mechanism for homeotic gene regulation is discussed.

Animals↗

PubMed1994

White gene expression, repressive chromatin domains and homeotic gene regulation in Drosophila.

The use of Drosophila chromosomal rearrangements and transposon constructs involving the white gene reveals the existence of repressive chromatin domains that can spread over considerable genomic distances. One such type of domain is found in heterochromatin and is responsible for classical position-effect variegation. Another type of repressive domain is established, beginning at specific sequences, by complexes of Polycomb Group proteins. Such complexes, which normally regulate the expression of many genes, including the homeotic loci, are responsible for silencing, white gene variegation, pairing-dependent effects and insertional targeting.

Animals↗

PubMed1994

Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells.

Double-minute (dm) chromosomes are cytogenetically resolvable DNA amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remained inconclusive, dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, we report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a "transcript selection" strategy, we cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, we constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Molecular and cytogenetical characterization of the 10A1-2 band and adjoining region in the Drosophila melanogaster polytene X chromosome.

Some 300 kb of DNA from the 9F12-10A7 X chromosome region (seven bands) uncovered by Df(1)vL3 were cloned and 31 break points of chromosome rearrangements within the region were mapped. Positions of 12 genes found earlier in genetic saturation experiments, transcripts and P element-induced mutations were located on the physical map using either chromosome rearrangements or Southern blot hybridizations. Data on the position of the break points, genes and polytene chromosome bands allow the following conclusions to be made. (1) The size of the bands in the region varies between 4 kb (10A6 and 7) and 183-195 kb (10A1-2). The compaction ratio of DNA in bands varies from 8-36 (10A6 + 7) to 151-161 (10A1-2). Therefore, fine and thick bands appear to have different kinds of DNP packaging. (2) The bands differ in genetic content. Fine bands contain from one to three genes. In contrast, the 10A1-2 band contains three genes and at least six transcribed DNA fragments. (3) Comparison of genetic and physical maps shows that in this region 0.01 centiMorgan corresponds to 3.3 kb of DNA.

Animals↗

PubMed1993

The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects.

Mutations in the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of a number of tissue-specific mutations caused by insertion of a gypsy retrotransposon. The su(Hw) gene encodes a zinc finger protein which binds to a 430 bp region of gypsy shown to be both necessary and sufficient for its mutagenic effects. su(Hw) protein causes mutations by inactivation of enhancer elements only when a su(Hw) binding region is located between these regulatory sequences and a promoter. To understand the molecular basis of enhancer inactivation, we tested the effects of su(Hw) protein on expression of the mini-white gene. We find that su(Hw) protein stabilizes mini-white gene expression from chromosomal position-effects in euchromatic locations by inactivating negative and positive regulatory elements present in flanking DNA. Furthermore, the su(Hw) protein partially protects transposon insertions from the negative effects of heterochromatin. To explain our current results, we propose that su(Hw) protein alters the organization of chromatin by creating a new boundary in a pre-existing domain of higher order chromatin structure. This separates enhancers and silencers distal to the su(Hw) binding region into an independent unit of gene activity, thereby causing their inactivation.

ATP-Binding Cassette Transporters↗

PubMed1993

Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.

Polycomb group genes are necessary for maintaining homeotic genes repressed in appropriate parts of the body plan. Some of these genes, e.g. Psc, Su(z)2 and E(z), are also modifiers of the zeste-white interaction. The products of Psc and Su(z)2 were immunohistochemically detected at 80-90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of zeste protein and two other Polycomb group proteins, Polycomb and polyhomeotic. The five proteins co-localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in another Polycomb group gene, E(z), the Su(z)2 and Psc products become dissociated from chromatin at non-permissive temperatures from most but not all sites, while the binding of the zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions. These results suggest that the binding of at least some Polycomb group proteins requires interactions with other members of the group and, although zeste can bind independently, its repressive effect on white involves the presence of at least some of the Polycomb group proteins.

Animals↗

PubMed1993

Stepwise assembly of hyperaggregated forms of Drosophila zeste mutant protein suppresses white gene expression in vivo.

The zeste gene is involved in two chromosome pairing-dependent phenomena: transvection and the suppression of white gene expression. Both require the ability of zeste protein to multimerize, dependent on three interlaced hydrophobic heptad repeats in the C-terminal domain. The first step is dimerization through a leucine zipper. Two other heptad repeats are then required to form higher multimers. The zeta(1) mutation, which causes the pairing-dependent suppression of white, creates a new hydrophobic nucleus that allows the formation of a new and larger aggregate. The zeta(op6) mutation, which suppresses even unpaired copies of white, makes even larger aggregates. The phenotypic suppression of white by a series of mutants is strictly correlated with hyperaggregation and the larger the hyperaggregates, the weaker the requirement for the pairing of white. Hyperaggregation of the Z1 protein in vitro is suppressed by co-translation with the C-terminal peptide of wild-type protein, lacking the DNA-binding domain. This C-Z+ peptide also complements the zeta(1) allele in vivo and restores normal color, demonstrating that zeste product also exists in a multimeric complex in the cell. Complementation in vivo is strictly correlated with the prevention of hyperaggregation of the zeste mutant products in vitro, supporting the interpretation that excessive association of zeta(1) and zeta(op6) proteins is responsible for their repression of white gene expression.

ATP-Binding Cassette Transporters↗

PubMed1993

Multimerization of the Drosophila zeste protein is required for efficient DNA binding.

The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.

ATP-Binding Cassette Transporters↗

PubMed1993

Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene.

The core activity of the Ubx gene enhancer BRE (bx region enhancer) is encoded within a 500 bp module. bx DNA outside this active module increases the level of expression, expands the expression into ventro-lateral ectoderm and partially stabilizes the late expression pattern. The products of the gap genes hb and tll and of the pair-rule gene ftz bind to the 500 bp BRE module and control directly its initial pattern of expression. ftz enhances expression in even-numbered parasegments within the correct spatial domain whose boundaries are set by hb and tll. In addition, en and twi products activate the enhancer, probably directly. en broadens the parasegmental stripe while twi cooperates with ftz to enhance expression in the mesoderm. Binding sites for the five regulators are closely clustered, often overlapping extensively with one another. In vitro, hb blocks the binding of ftz and can also displace ftz protein pre-bound to an overlapping site, suggesting that competitive binding and/or interference by hb sets the initial boundaries of the domain of expression. Our results also suggest that this interaction is short-range and the long distance interactions among different enhancers may depend on each enhancer's ability to complex with the promoter.

Animals↗

PubMed1993

Flow cytometric study of the expression of neutral endopeptidase (CD10/CALLA) on the surface of newborn granulocytes.

The susceptibility of newborn infants to bacterial infections is well documented. Neutrophils play an important role in defense against bacterial infection, the most common kind of infection in the newborn period. Many studies of lymphocyte surface characteristics during that period of life are available, but there are no reports on the surface immunophenotype of the granulocytes at birth. Because some of their membrane associated antigens have been identified as enzymes (CALLA/CD10), neutral endopeptidase, and (CD13) amino peptidase that could play a role in the biological functions of neutrophils, a study of the membrane phenotype appeared potentially important. Using flow cytometry, we studied the expression of a panel of the antigens expressed on mature neutrophils including CD10, CD13, and CD33 in 28 full-term babies and 19 adults. A significantly (p < 0.001) lower expression of CD10, CD13, and CD33 was found in full-term babies compared with 19 adults. These data raise two points: first, that because CD10 is detected only on segmented granulocytes, the low level of CD10 observed in neonates is consistent with a degree of immaturity of the neutrophil membrane, and second, that the deficiency of endopeptidase may impair neutrophil interactions with peptide effectors and thus play a role in the increased susceptibility to bacterial infections exhibited in newborns.

Adult↗

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↗