Molecular structure of the lampbrush loops nooses of the Y chromosome of Drosophila hydei

The molecular structure of the lampbrush loopforming fertility gene nooses from the short arm of the Y chromosome of Drosophila hydei is described on the basis of cloned DNA sequences which are characteristic for the sequence organization in the lampbrush loop. Y chromosomal lampbrush loops are organized into tandem repeat clusters of loop-specific repetitive DNA sequences and in interspersed repetitive DNA sequences with homologies elsewhere in the genome. In this paper, the basic properties of a repeat unit of the tandemly repeated sequence family ay1 are described. Moreover, it is shown that a loop contains several different domains carrying repeat clusters of the same repeated DNA family but with divergent sequence character. One of these clusters is characterized by an internal duplication of the basic repeat unit. We propose that the tandem repeat DNA family ay1 forms a frame of the lampbrush loop which is required for structural and functional reasons.     

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.     

Selection of DNA sequences from interval 6 of the human Y chromosome with homology to a Y chromosomal fertility gene sequence of Drosophila hydei

Summary An experimental approach towards the molecular analysis of the male fertility function, located in interval 6 of the human Y chromosome, is presented. This approach is not based on the knowledge of any gene product but on the assumption that the functional DNA structure of male fertility genes, evolutionary conserved with their position on the Y chromosome, may contain an evolutionary conserved frame structure or at least conserved sequence elements. We tested this hypothesis by using dhMiF1, a fertility gene sequence of the Y chromosome of Drosophila hydei, as a screening probe on a pool of cloned human Y-DNA sequences. We were able to select 10 human Y-DNA sequences of which 7 could be mapped to Y interval 6 (the pY6H sequence family). Since the only fertility gene of the human Y chromosome is mapped to the same Y interval, our working hypothesis seems to be strongly supported. Most interesting in this respect is the isolation of the Y-specific repetitive pY6H65 sequence. The pY6H65 locus extends to a length of at least 300 kb in Y interval 6 and has a locus-specific repetitive sequence organization, reminiscent of the functional DNA structure of Y chromosomal fertility genes of Drosophila. We identified the simple sequence family (CA)_n as one sequence element conserved between the Drosophila dhMiFi fertility gene sequence and the homologous human Y-DNA sequences.     

Simple Quantitative PCR Approach to Reveal Naturally Occurring and Mutation-Induced Repetitive Sequence Variation on the Drosophila Y Chromosome

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.     

Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster

The gypsy retrovirus of Drosophila is quite unique among retroviruses in that it shows a strong preference for integration into specific sites in the genome. In particular, gypsy integrates with a frequency of >10% into the regulatory region of the ovo gene. We have used in vivo transgenic assays to dissect the role of Ovo proteins and the gypsy insulator during the process of gypsy site-specific integration. Here we show that DNA containing binding sites for the Ovo protein is required to promote site-specific gypsy integration into the regulatory region of the ovo gene. Using a synthetic sequence, we find that Ovo binding sites alone are also sufficient to promote gypsy site-specific integration into transgenes. These results indicate that Ovo proteins can determine the specificity of gypsy insertion. In addition, we find that interactions between a gypsy provirus and the gypsy preintegration complex may also participate in the process leading to the selection of gypsy integration sites. Finally, the results suggest that the relative orientation of two integrated gypsy sequences has an important role in the enhancer-blocking activity of the gypsy insulator.     

Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo

The U5 snRNA loop 1 is characterized by the conserved sequence G₁C₂C₃U₄U₅U₆Y₇A₈Y₉ and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae. Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro. To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted. Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation. These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo. A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1. Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns. The cell cycle arrest is likely attributed to inefficient splicing of α-tubulin pre-mRNA in one mutant and actin pre-mRNA in another. These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo.     

Mobilization of the gypsy and copia retrotransposons in Drosophila melanogaster induces reversion of the ovo dominant female-sterile mutations: molecular analysis of revertant alleles

The ovo locus is required for the maintenance of the female germ line in Drosophila melanogaster. In the absence of an ovo⁺ gene, males are completely normal but females have no germ-line stem cells. Three dominant mutations at the ovo locus, called ovo^D, were observed to revert towards recessive alleles at high frequency when ovo^D males were crossed to females of the strain y v f mal. We have found that this strain contains an inordinately high number of gypsy transposable elements, and crossing it with the ovo^D strains results in the mobilization of both gypsy and copia, with high-frequency insertions into the ovo locus: of 16 revertants examined 12 have gypsy and four have copia inserted at 4E, the ovo cytological site. Using gypsy DNA as a tag we have cloned 32 kb of wild-type DNA sequences surrounding a gypsy insertion and characterized molecular rearrangements in several independent revertants: in 10 of them gypsy appears to be inserted into the same site. The orientation of gypsy is strictly correlated with whether the neighbouring lozenge-like mutation appears in the revertants. A distal limit of the ovo locus was molecularly determined from the breakpoint of a deletion affecting closely flanking regions.     

Identification of a sporulation-specific promoter regulating divergent transcription of two novel sporulation genes in Saccharomyces cerevisiae

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.