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PubMed2005

Genome-scale profiling of histone H3.3 replacement patterns.

Histones of multicellular organisms are assembled into chromatin primarily during DNA replication. When chromatin assembly occurs at other times, the histone H3.3 variant replaces canonical H3. Here we introduce a new strategy for profiling epigenetic patterns on the basis of H3.3 replacement, using microarrays covering roughly one-third of the Drosophila melanogaster genome at 100-bp resolution. We identified patterns of H3.3 replacement over active genes and transposons. H3.3 replacement occurred prominently at sites of abundant RNA polymerase II and methylated H3 Lys4 throughout the genome and was enhanced on the dosage-compensated male X chromosome. Active genes were depleted of histones at promoters and were enriched in H3.3 from upstream to downstream of transcription units. We propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin.

Animals↗
PubMed1996

Selection on X-linked genes during speciation in the Drosophila athabasca complex.

We present the results of a restriction site survey of variation at five loci in Drosophila athabasca, complimenting a previous study of the period locus. There is considerably greater differentiation between the three semispecies of D. athabasca at the period locus and two other X-linked genes (no-on-transient-A and E74A) than at three autosomal genes (Xdh, Adh and RC98). Using a modification of the HKA test, which uses fixed differences between the semispecies and a test based on differences in Fst among loci, we show that the greater differentiation of the X-linked loci compared with the autosomal loci is inconsistent with a neutral model of molecular evolution. We explore several evolutionary scenarios by computer simulation, including differential migration of X and autosomal genes, very low levels of migration among the semispecies, selective-sweeps, and background selection, and conclude that X-linked selective sweeps in at least two of the semispecies are the best explanation for the data. This evidence that natural selection acted on the X-chromosome suggests that another X-linked trait, mating song differences among the semispecies, may have been the target of selection.

Alcohol Dehydrogenase↗
PubMed2002

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos.

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.

Anaphase↗
PubMed1998

CYP12A1, a mitochondrial cytochrome P450 from the house fly.

Eukaryotic P450 proteins are membrane proteins found predominantly in the endoplasmic reticulum. In vertebrates, several biosynthetic P450s are found in mitochondria as well. We cloned three putative insect mitochondrial P450s from larval house fly cDNA. These P450s are members of a new P450 family, CYP12. The CYP12 proteins are most closely related to the mammalian mitochondrial P450 of the CYP11, CYP24, and CYP27 families. The most abundant cDNA, CYP12A1, was expressed in Escherichia coli and purified. NADPH-dependent reduction of CYP12A1 was rapid and efficient with the bovine mitochondrial proteins adrenodoxin reductase and adrenodoxin as electron transfer partners. In contrast, house fly microsomal NADPH cytochrome P450 reductase reduced CYP12A1 only poorly. In a reconstituted system with the bovine mitochondrial electron donors, CYP12A1 metabolized a variety of insecticides and other xenobiotics, but did not metabolize ecdysteroids, juvenoids, or fatty acids. Subcellular localization of CYP12A1 by immunogold histochemistry established the mitochondrial nature of this protein. CYP12A1 mRNA levels are constitutively higher in an insecticide-resistant strain than in a susceptible strain, and this trait maps to chromosome II in the house fly, where the constitutive overexpression of the pesticide-metabolizing microsomal CYP6A1 also maps. Multiple mitochondrial P450s have evolved in insects and may play a role in the metabolism of xenobiotics in addition to their possibly ancestral functions in steroidogenesis.

Amino Acid Sequence↗
PubMed2000

courtless, the Drosophila UBC7 homolog, is involved in male courtship behavior and spermatogenesis.

The courtless (col) mutation disrupts early steps of courtship behavior in Drosophila males, as well as the development of their sperm. Most of the homozygous col/col males (78%) do not court at all. Only 5% perform the entire ritual and copulate, yet these matings produce no progeny. The col gene maps to polytene chromosome band 47D. It encodes two proteins that differ in their carboxy termini and are the Drosophila homologs of the yeast ubiquitin-conjugating enzyme UBC7. The col mutation is caused by an insertion of a P element into the 3' UTR of the gene, which probably disrupts translational regulatory elements. As a consequence, the homozygous mutants exhibit a six- to sevenfold increase in the level of the COL protein. The col product is essential, and deletions that remove the col gene are lethal. During embryonic development col is expressed primarily in the CNS. Our results implicate the ubiquitin-mediated system in the development and function of the nervous system and in meiosis during spermatogenesis.

Alleles↗
PubMed1993

Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.

R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.

Animals↗
PubMed2001

Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections.

We report here the identification and functional characterization of DmMyD88, a gene encoding the Drosophila homolog of mammalian MyD88. DmMyD88 combines a Toll-IL-1R homology (TIR) domain and a death domain. Overexpression of DmMyD88 was sufficient to induce expression of the antifungal peptide Drosomycin, and induction of Drosomycin was markedly reduced in DmMyD88-mutant flies. DmMyD88 interacted with Toll through its TIR domain and required the death domain proteins Tube and Pelle to activate expression of Drs, which encodes Drosomycin. DmMyD88-mutant flies were highly susceptible to infection by fungi and Gram-positive bacteria, but resisted Gram-negative bacterial infection much as did wild-type flies. Phenotypic comparison of DmMyD88-mutant flies and MyD88-deficient mice showed essential differences in the control of Gram-negative infection in insects and mammals.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Y-chromosome effects on Drosophila geotaxis interact with genetic or cytoplasmic background.

Previously, all of the major fruit fly, Drosophila melanogaster, chromosomes (I, II and III) have been shown to be associated with geotaxis, but the Y chromosome has not. Using two methods (back-crossing and chromosome substitution), Y chromosomes from lines that have evolved stable, extreme expressions of geotaxis were placed into different genetic and cytoplasmic backgrounds to test the resulting males for geotaxis. The results of the back-crossing do not support the interpretation of Y-chromosome effects on geotaxis. These tests do not have sufficient statistical power, however, to detect small genetic effects. In the chromosome substitution experiment, the geotaxis-line Y chromosomes were placed into high- and low-selected lines, Canton-S and Champaign wild-type backgrounds. The results of the chromosome substitution experiment provide evidence for a Y-chromosome effect on geotaxis in selected geotaxis lines, but not in wild-type stock, backgrounds. These results suggest that the Y chromosome has a small effect on geotaxis, whose detection depends on genetic and/or cytoplasmic background. The implications of these results are discussed for behaviour genetic analysis of D. melanogaster and for issues of statistical power in detecting small genetic effects.

Animals↗
PubMed2006

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

Animals↗
PubMed1994

Transcription of repetitive DNA sequences in the lampbrush loop pair Nooses formed by sterile alleles of fertility gene Q on the Y chromosome of Drosophila hydei.

The Y chromosomal lampbrush loop-forming male fertility genes of Drosophila consist mainly of repetitive DNA sequences that do not code for proteins. We investigated whether differences in the transcription of these sequences can be detected in male-sterile alleles of male fertility gene Q, which forms the loop pair Nooses. The loop consists, for approximately two-thirds, of repeats of the Y-specific ay1 family of repetitive DNA sequences. Of the remaining one-third, at least one-half is represented by defective retrotransposons of the gypsy family. Both sequence types are interspersed throughout the loop. Using both ay1 and gypsy sequences as probes for transcript in situ hybridization, we show that, at the level of the light microscope, transcription of neither sequence is detectably affected in the loops formed by a male-sterile allele of gene Q. We conclude that the transcription of ay1 and gypsy is required, but not sufficient for the function of gene Q.

Alleles↗
PubMed1997

Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found.

The nucleotide sequence of 37,639 bp of the right arm of chromosome XII has been determined. Twenty-five open reading frames (ORFs) longer than 300 bp were detected, two of which extend into the flanking cosmids. Only two (L2931 and L2961) of the 25 ORFs correspond to previously sequenced genes (HOG1 and YAP3, respectively). Another ORF is distinct from YAP3 but shows pronounced similarity to it. About half of the remaining ORFs show similarity to other genes or display characteristic protein signatures. In particular, ORF L2952 has striking homology with the probable cell cycle control protein crn of Drosophila melanogaster. L2949 has significant similarity to the human ZFM1 (related to a potential suppressor oncogene) and mouse CW17R genes, though it lacks the carboxy-terminal oligoproline and oligoglutamine stretches encoded by these mammalian genes. The small ORF L2922 is similar to part of the much larger yeast flocculation gene FLO1. Other sequences found in the 37639 bp fragment are one delta and one solo-sigma element, the tRNA-Arg3 gene, the small nuclear RNA gene SNR6 and three ARS consensus sequences.

Amino Acid Sequence↗
PubMed1999

Mouse Dac, a novel nuclear factor with homology to Drosophila dachshund shows a dynamic expression in the neural crest, the eye, the neocortex, and the limb bud.

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto-oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild-type limb buds. In contrast, a significant expansion of Dac expression are observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3-23 and further fine-mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities.

Amino Acid Sequence↗
PubMed1997

Nurse cell polytene chromosomes of Drosophila melanogaster otu mutants: morphological changes accompanying interallelic complementation and position effect variegation.

Combinations of certain mutant alleles of the ovarian tumor gene permit the production of viable eggs. Two alleles that behave in this way are otu7 and otu11. Females homozygous for either allele are sterile, and their ovarian nurse cells (NC) contain giant polytene chromosomes of various morphologies. Fertile flies (otu+/otu+, otu+/otu7, otu+/otu11) have endopolyploid nurse cells with typical dispersed chromosomes. Fertile hybrids (otu7/otu11) produce large numbers of polytene chromosomes comparable to, and often larger than, classic salivary gland (SG) chromosomes. Therefore, these otu hybrids provide a unique system for studying, at the chromosomal level, the activation and expression of genes functioning during oogenesis. The otu gene encodes a long and a short isoform. The normal long isoform appears to be responsible for the dispersion of chromosomes during the endomitolic DNA replications occurring in ovarian NCs. The genetic inactivation of euchromatic genes placed next to pericentric heterochromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding chromosome regions. A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SG cells and NCs was made using the Dp(1;1)pn 2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp(1;1)pn 2b rearrangement were 92.6% vs. 15.8%, respectively; for Dp(1;f) 1337, these values were 56.8% vs. 9.7%. Therefore heterochromatin belonging to germ line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterochromatin from somatic chromosomes.

Alleles↗
PubMed1998

Multigeneration exposure test of Drosophila melanogaster to ELF magnetic fields.

Mutations, other than dominant lethals, were accumulated on wild type second chromosomes (+) of Drosophila melanogaster during exposure to 50 Hz sinusoidal alternating magnetic fields of 0.5 or 5 mT (rms) for 40 generations by the Curly/Plum(Cy/Pm) accumulation method. We maintained, for 40 generations under continuous exposure, each (+) chromosome as a heterozygote with (Cy) chromosome. Viability of the (+) chromosome was tested by sib-mating of (Cy/+) male and (Cy/+) female in a culture every 10th generation to obtain the homozygote. Viability indices, defined as twice the ratio of number of (+/+) flies to that of (Cy/+) flies plus 1 in the progeny of the test mating, also were calculated, which equaled 1.00 at the starting point. For the control and 0.5 and 5 mT exposed groups, percent frequencies of recessive lethal lines, defined as a line with (+/+) flies less than 0.3% in the test mating, were, respectively, 1.9, 0.9, and 2.9% (10th), 9.0, 4.9, and 9.5% (20th), 30.3, 22.9, and 30.4% (30th), and 39.9, 32.4, and 43.3% (40th generation). For the control and 0.5 and 5 mT groups, average viability indices, excluding lethals and markedly deleterious, were, respectively, 0.778, 0.796, and 0.752 (20th), 0.704, 0.698, and 0.694 (30th), and 0.669, 0.678, and 0.595 (40th generation). Their decreasing rates were 0.0054, 0.0059, and 0.0078 per generation. No significant difference was detected among the exposure levels in either the recessive lethal mutation frequency or the viability index.

Animals↗
PubMed2001

Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family.

Mutations in cortex and grauzone cause abnormal arrest in Drosophila female meiosis. cortex was mapped to a 14 kb interval in 26F-27A by the male recombination mapping method. While these experiments mapped the gene accurately, they also illustrated some complexities of this method. Rescue results showed that a 2.8 kb genomic fragment from this interval was able to fully rescue the cortex phenotype. The 2.8 kb rescuing fragment contains a single open reading frame. The predicted amino acid sequence indicates that cortex encodes a WD-repeat protein and is a distant member of the Cdc20 protein family. Results from a developmental Northern analysis showed that the cortex transcript is expressed at high levels during oogenesis and early embryogenesis. Interestingly, the meiotic metaphase-anaphase II arrest defect in embryos laid by cortex homozygous females resembles the mitotic metaphase-anaphase defects observed in yeast cdc20 mutants. The predicted nature of the Cortex protein, together with the observed meiotic phenotype in cortex mutants, suggest that a similar pathway to the cdc20 dependent APC-mediated proteolysis pathway, which governs the metaphase-anaphase transition in mitosis, is also important in regulating oocyte meiosis.

Alleles↗
PubMed1990

The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain.

In Drosophila the Abdominal-B (Abd-B) domain of the bithorax complex (BX-C) spans over 100 kb and is responsible for specifying the identities of adult abdominal segments five (A5) to nine (A9), inclusive, and correspondingly, neuromeres 10-14 of the embryonic central nervous system. The domain consists of a region coding for two proteins, ABD-BI (54 kd) and ABD-BII (36 kd) and cis-regulatory regions extending from infra-abdominal-5 (iab-5) to iab-9, inclusive. We have used a monoclonal anti-ABD-B antibody to infer that mutants in iab-8 eliminate the expression of ABD-BI in neuromeres 10-13, inclusive, and that mutants in iab-9 eliminate expression of ABD-BII in neuromere 14. ABD-B expression is also analyzed in homozygotes for (i) loss-of-function mutants involving the iab-5, iab-6 and iab-7 regions, (ii) gain-of-function mutants Miscadastral pigmentation (Mcp) and Superabdominal (Sab), and (iii) a trans-regulator, Polycomb (Pc). ABD-B expression along the antero-posterior axis is colinear with the chromosomal order of the cis-regulatory regions. The behavior of rearrangement-associated iab-6 and iab-7 mutants suggests that the enhancer-like region, iab-5, and possibly also iab-6, may be shared between the abd-A and Abd-B domains. Such sharing is proposed as a factor that tends to keep gene complexes intact during evolution.

Abdomen↗
PubMed1995

Functional analysis of the chromo domain of HP1.

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.

Amino Acid Sequence↗