Mireille Schäfer

Restriction sites containing CpG show a higher frequency of polymorphism in human DNA

Unique loci in the human genome were examined with restriction enzymes in order to detect restriction fragment length polymorphisms (RFLPs). Of 31 arbitrary loci, nine were detectably polymorphic, reflecting ten polymorphic restriction sites. Nine of the ten polymorphic sites were revealed with two restriction enzymes, Msp I and Taq I, whose recognition sequences have in common the dimer sequence CpG. The cytosines in the CpG sequence are known to be frequently methylated in mammals, and the occurrence of significant variation in Msp I and Taq I sites supports the view that methylated cytosine residues are hotspots for mutation in mammalian DNA.

Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons.

BACKGROUND Male-derived Sex-peptide (SP) elicits egg laying and rejection of courting males in mated Drosophila females. Little is known about the genes that specify the underlying neuronal circuits and mediate this switch in female sexual behavior. RESULTS Here we show that the egghead gene involved in glycosphingolipid biosynthesis provides an essential component to the SP response. We have isolated viable alleles of the vital egghead gene that abolish egghead expression from a distal promoter resulting in the absence of the largest transcript of this complex transcription unit. Temporally and spatially restricted expression of egghead revealed a requirement for egghead early in the development of apterous-expressing ventral nerve cord neurons to rescue the SP response. In viable egghead alleles, these ascending interneurons, three per abdominal and seven per thoracic hemisegment, fail to innervate the central brain. egghead expression in apterous neurons rescues neuronal targeting and the response to SP. Furthermore, neurotransmission in apterous neurons is required to elicit the SP response. CONCLUSION Together with the former finding of SP binding to afferent nerves , these results suggest that SP-mediated modification of sensory input switches female sexual behavior from the virgin to the mated state.

The fatty acid elongase noa is necessary for viability and has a somatic role in Drosophila sperm development

The essential gene noa (CG 3971; also known as Baldspot) encodes a very long chain fatty acid elongase which is most similar to the mammalian elongase ELOVL6. noa is expressed in the nervous system from embryogenesis on, in imaginal discs, the fat body, malpighian tubules and in the gonads of both sexes. Its function is dose dependent, since reduced levels of noa RNA lead to impaired motility and severely reduced viability. In testes, noa RNA is detected in the cyst cells during the postmeiotic phase of germ cell development. An RNAi construct selectively driven in cyst cells leads to male sterility, demonstrating the necessity of noa function for male germline development and the interaction of the somatic cyst cells with the developing sperm.

The essential Drosophila melanogaster gene wds (will die slowly) codes for a WD-repeat protein with seven repeats

Abstract. We have isolated and characterized the will die slowly (wds) gene of Drosophila melanogaster, formerly known as l(1)zw8 or l(1)3Ad. The gene codes for a 2.0-kb RNA that is transcribed at all stages of development. The RNA has been localized by in situ hybridization to imaginal discs, larval brain, to nurse cells in the ovary, and to spermatogonia and spermatocytes in the testis. The putative translation product contains seven WD-repeats and is, therefore, a new member of the family of WD-proteins. Clear homologues of the Drosophila WDS protein exist in three other fully sequenced higher eukaryotes – human, Caenorhabditis elegans and Arabidopsis. A genomic fragment containing the wds transcription unit is able to rescue two different lethal wds alleles, thus proving that we have indeed isolated the wds gene.

The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster.

Oogenesis in Drosophila is a useful model for studying cell differentiation. We have analyzed the role of the egh gene in these processes with the aid of a newly isolated viable but female sterile allele. This mutation results in diverse variable defects in oogenesis. The most frequent defect being follicles that have either more or less than the normal number of 16 germ cells. This is caused by erroneous splitting and/or fusion of correct clusters of 16 cystocytes. The entire follicle has a rather flexible structure in this allele, most obvious by a highly variable position of the oocyte within the follicle. Moreover, a second oocyte can also develop in egh clusters. This is exclusively observed in aberrant follicles that are generated by the aforementioned splitting/fusion process. Surprisingly, even a germ cell which is distinct from the two pro-oocytes can differentiate into an oocyte under these circumstances. Hence, determination of the oocyte is definitely not fixed when germ cell clusters are enveloped by prefollicular cells, and interactions between follicle cells and germ cells must play an important role in oocyte specification. Molecular analysis proves that the oocyte-specific transcript of the egh gene is drastically reduced in this viable allele.

Alterations in titer and distribution of high mobility group proteins during embryonic development of Drosophila melanogaster

High mobility group proteins are thought to have an architectural function in chromatin. Here we describe changes in titers, extent of phosphorylation, and cellular distribution of the three abundant HMG proteins during embryonic development of Drosophila. The titers of the HMG proteins HMGD, HMGZ, and D1 are highest in ovaries and at the beginning of embryonic development. They decrease continuously until cellularization of the embryo. Relative to the histone H1 titer, the levels of HMGD and D1 remain almost constant during gastrulation and organogenesis, whereas the titer of HMGZ increases during late organogenesis. Up to gastrulation, the development is accompanied by dephosphorylation of D1. In contrast, HMGD and HMGZ appear to be constitutively phosphorylated. As the high extent of phosphorylation of D1 is also characteristic in ovaries, it is likely that the posttranslational modifications of this protein observed in early embryonic stages are of maternal origin. Using site specific antibodies against helices I and III of HMGD and HMGZ and against the AT-hook motif of D1, protein-specific staining patterns have been observed during embryonic development. Despite high levels of HMG proteins at the beginning of embryonic development, we were unable to detect any of these proteins in nuclei of stage 2 embryos. The accumulation of the HMG proteins correlates with the onset of transcription in stage 3. Our results argue against a proposal of a shared role of HMGD and histone H1 in Drosophila chromatin.

Distribution of spacer length classes and the intervening sequence among different nucleolus organizers in Drosophila hydei

Drosophila hydei rRNA genes from different chromosomes and from different stocks have been studied by restriction enzyme analysis. In DNA from wild-type females, about half of the X chromosomal rRNA genes are interrupted by an intervening sequence within the 28S coding region. In contrast to D. melanogaster, the intervening sequences belong to a single size class of 6.0 kb. Although there are two nucleolus organizers on the Y chromosome, genes containing the intervening sequence seem to be restricted to the X chromosome. — As shown in four cloned rDNA fragments, the nontranscribed spacers differ in length by having varying numbers of a 242 base pair sequence located in tandem in the right section of the spacer. In genomic rDNA, the spacers also differ in length by a regular 0.25 kb interval. Spacers with between 5 and 15 subrepeats occur frequently within the X and Y chromosomal nucleolus organizers in different D. hydei stocks; shorter and longer spacers are also present but are relatively rare. — Although each genotype is characterized by different frequencies of some spacer classes, the prominent spacer length heterogeneity pattern is similar among the different nucleolus organizers and, therefore, seems to be conserved during evolution.

Length variation in the non-transcribed spacer of Calliphora erythrocephala ribosomal DNA is due to a 350 base-pair repeat☆

Abstract The non-transcribed spacer regions in the ribosomal DNA cistrons of Calliphora erythrocephala vary in length. This length variation is shown to be due to variable numbers of small repeated units found in certain areas of the NTS † regions, as has been found in several other systems. In contrast to the other organisms, however, the length variation in C. erythrocephala leads to the formation of only two major size classes with a length difference of about 1 kb. We have investigated the nature of this length difference by means of electron microscope heteroduplex and restriction enzyme digestion analyses. We demonstrate that the length variation in C. erythrocephala is due to a variation in the number of 350 bp repeating units defined by the presence of sites for three restriction enzymes, Hha I, Sau 3A and Alu I. Furthermore, the existence of an Xba I site within one 350 bp unit and the lack of a Hha I restriction site within another 350 bp unit reveals that at least some sequence differences exist among the repeating units. These restriction site differences can be viewed as genetic markers and lead to the hypothesis that the longer NTS class originated from the shorter NTS class by an unequal crossover event that served to duplicate the region containing repetitive units. An interesting feature of our observation is that not all NTS length classes are equally represented, suggesting special rules for the unequal recombination events often hypothesized as the basis for such variation.

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.

A new set of lacZ fusion vectors, pUCPlac, for studying gene expression in Drosophila by P-mediated transformation

A new vector, pUCPlac, was generated by introducing a truncated lacZ structural gene into pUChsneo [Steller and Pirrotta, EMBO J. 4 (1985) 167-171]. In front of this gene a new polylinker was added which will allow the in-frame fusion of sequences containing cis-acting regulatory elements plus translation start site of any given gene. After P-mediated germ-line transformation in Drosophila the action of these regulatory elements can be conveniently monitored by histochemical staining for beta-galactosidase activity. Correct cloning can be easily ascertained by supercoil plasmid sequencing of the constructs using commercially available primers.