E-FLYC-5 - Transcription profiling of Drosophila samples of wild type, aly, can, zaa and zab testes
Released on 7 May 2004, last updated on 27 March 2012
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.
transcription profiling by array, transcript identification
Helen White-Cooper, unknown unknown, Debashis Rana <firstname.lastname@example.org>