Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Drosophila samples of wild type, aly, can, zaa and zab testes

ABSTRACT: Drosophila melanogaster males mutant for any meiotic arrest gene have defective spermatogenesis, with cells arresting as primary spermatocytes, and failing to progress to later stages. This phenotype is remarkably similar to meiosis I maturation arrest azoospemia in humans. Drosophila meiotic arrest genes can be categorised as "aly-class" or "can-class". The aly-class (aly, zaa and zab) regulates both meiotic division and male germ line differentiation by controlling transcriptional activation of a large set of target genes in primary spermatocytes. This set includes genes required for cell cycle progression and those required for spermatid differentiation. The can-class control transcription of the same set of differentiation genes, but not cell cycle genes. aly belongs to a protein family conserved from humans to plants. Aly protein locates to chromatin in wild type individuals, and probably controls transcriptional activity of target promoters through interaction with a DNA binding protein. Objectives: So far aly- and can-class target genes have been identified by the small-scale approach of individually testing cDNA clones (total 18). Expression of 7 genes was unaffected by the mutations (group A), transcription of 8 depended on both classes of genes (group B), finally three genes required the aly- but not the can-class for their expression (group C). The aly-class targets (group C) are particularly intriguing, since they include a gene, boule, whose human homologue deleted in azoospermia (DAZ) is essential for male fertility. This project will address the following questions: How many genes in total are regulated by the meiotic arrest genes? What types of proteins do they encode? Are they all testis specific, or are they expressed at other stages of the life cycle? What is their relevance for fertility in other species?

Plan: We will prepare samples of wild type, aly, can, zaa and zab testes. Those transcripts expressed in wild type testes will be placed into group A, B or C based on analysis of the competitive hybridisation of wild type samples vs each mutant in turn. Microarrays will also enable us to determine whether there is a group D, consisting of genes whose down-regulation in spermatocytes is dependent on the meiotic arrest genes. We have dissected testes from 0-1 day old males of the relevent genotypes. We estimate to get >20 ug total RNA from 100 flies, so have dissected at least 500 males of each mutant genotype, and 2000 of wild type (red e, the background chromosome for aly and can). In total we have about 5000 flies worth of testes in the freezer. We have been careful to only include testes and seminal vesicles, and have excluded other tissues from the genital tract. All the males are collected from bottles that are emptied daily, to ensure they are all the same age. Because of their age relatively few of the males will have mated. For aly and can we have used well characterised null alleles. For zab we have used the only mutant allele, which we also know is a null. We havn't cloned zaa so have no idea whether the only existing mutant allele is null. The testes were dissected in testis buffer over a period of 30 min. Then they were transfered into a very small drop of testis buffer in an eppendorf and frozen in liquid nitrogen. They have been stored at -80C. Before being sent to Cambridge we will defrost the samples, add trizol, and pool samples to supply 3 or 4 tubes per genotype.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Debashis Rana

PROVIDER: E-FLYC-5 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis.

Ayyar Savita S Jiang Jianqiao J Collu Anna A White-Cooper Helen H White Robert A H RA

Development (Cambridge, England) 20030701 13

We have investigated the role of TGIF, a TALE-class homeodomain transcription factor, in Drosophila development. In vertebrates, TGIF has been implicated, by in vitro analysis, in several pathways, most notably as a repressor modulating the response to TGFbeta signalling. Human TGIF has been associated with the developmental disorder holoprosencephaly. Drosophila TGIF is represented by the products of two tandemly repeated highly similar genes, achintya and vismay. We have generated mutations th ...[more]

PMID: 12756169

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Project description:The Drosophila-Wolbachia system is being used to study the molecular nature of the interactions between a host and a symbiont. This system offers a unique opportunity for such a study since the Drosophila genome sequence is available, several Wolbachi genomes will also be available soon and there are at least three known Wolbachia strains infecting Drosophila: a) mod+ strain that induces cytoplasmic incompatibility, b) mod- strain that cannot induce cytoplasmic incompatibility, and c) popcorn strain, a virulent strain which reduces in half the adult lifespan of Drosophila due to its massive proliferation in adult brain. The Drosophila-Wolbachia interaction manifests itself in 3 main ways; first, destruction of the CNS in infected adults, second, induction of some kind of modification or imprinting in the male germ-line resulting in an early failure in embryonic development, (cytoplasmic incompatability (CI)) and third, modification of the female germ-line resulting in resistance to modified sperm. We are interested in identifying Drosophila genes with changes in expression due to Wolbachia infection. We have generated a series of isogenic fly lines (those being used in the IGF P-element project) which we have infected with Wolbachia strains, infection is readily cured by growth on medium containing tetracycline. Thus, we have equivalent genetic background with and without the parasite. We have tested all of the transgenic lines for the level of CI and find strain-specific levels ranging from 0-50%. We also have a strain of D. simulans that shows over 95% CI. Plan: For our initial experiments we wish to make 4 comparisons, in all cases 2 day old males will be collected and for each comparison we will isolate 3 independent biological replicates: Melanogaster no CI [tet] x Melanogaster no CI [+wol] Melanogaster high CI [tet] x Melanogaster high CI [+wol] We will therefore identify genes with changed expression levels in the male upon Wolbachia infection by comparing the melanogaster strains with high or no CI in the presence of tetracycline and Wolbachia. We also hope to identify similar genes in simulans (where we expect the magnitude of the effect to be larger), differences between melanogaster and simulans are controlled for in the mel v sim comparison.

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Dataset Information

Transcription profiling of Drosophila samples of wild type, aly, can, zaa and zab testes

Publications

Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis.

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