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.     

Molecular cloning of microdissected lampbrush loop DNA sequences of Drosophila hydei

We microdissected a Y chromosomal lampbrush loop pair from primary spermatocyte nuclei of Drosophila hydei and cloned the DNA directly at the microscale. Four of the 12 recombinant DNA clones recovered display in situ hybridization to mitotic metaphase Y chromosomes, preferentially in the chromosomal region identified as the origin of the lampbrush loop pair. All clones, however, also hybridize to autosomal and X chromosomal loci in polytene chromosomes. Y chromosomal DNA sequences of D. hydei again prove to be members of different families of repeated sequences distributed throughout the genome. These microcloning experiments, which were carried out under very unfavourable experimental conditions (low DNA content of the lampbrush loops in the presence of large amounts of RNA) prove that almost any chromosomal structure detected by light microscopy is directly accessible to molecular cloning experiments by micromethods.     

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.     

Isolation and structural analysis of ribosomal protein genes in Xenopus laevis. Homology between sequences present in the gene and in several different messenger RNAs

The ribosomal protein genes are present in two to four copies per haploid genome of Xenopus laevis. Using cloned complementary DNA probes, we have isolated, from a genomic library of X. laevis, several clones containing genes for two different ribosomal proteins (L1 and L14). These genes contain intervening sequences. In the case of the L1 gene, the exons are 100 to 200 base-pairs long and the introns, on average, 400 base-pairs. Along the genomic fragments, two different classes of repetitive DNA are present: highly and middle repetitive DNA. Both are evolutionarily unstable as shown by hybridization to Xenopus tropicalis DNA. Several introns of the gene coding for protein L1 contain middle repetitive sequences. Hybridization and hybrid-released translation experiments have shown that sequences inside the two genes hybridize to several poly(A) messenger RNAs. Some of the products encoded by these mRNA have electrophoretic properties of ribosomal proteins.     

Evidence for rolling circle replication of tandem genes in Drosophila

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Cytological and genetic analysis of the Y chromosome of Drosophila melanogaster

By applying quinacrine-, Hoechst- and N-banding techniques to neuroblast prometaphase chromosomes the Y chromosome of Drosophila melanogaster can be differentiated into 25 regions defined by the degree of fluorescence, the stainability after N-banding and the presence of constrictions. Thus these banding techniques provide an array of cytological landmarks along the Y chromosome that makes it comparable to a polytene chromosome for cytogenetic analysis. — 206 Y-autosome translocations (half of them carrying Y-linked sterile mutations) and 24 sterile y ⁺ Y chromosomes were carefully characterized by these banding techniques and used in extensive complementation analyses. The results of these experiments showed that: (1) there are four linearly ordered fertility factors in Y ^L and two fertility factors in Y ^S . (2) These fertility factors map to characteristic regions of the Y chromosome, specifically stained with the N-banding procedure. (3) The most extensively analyzed fertility factors are defined by a series of cytologically non-overlapping and genetically noncomplementing breaks and deficiencies distributed over large chromosome regions. For example, the breakpoints which inactivate the kl-5 and ks-1 loci are scattered along regions that contain about 3,000 kilobases (kb) DNA. Since these enormous regions formally define single genetic functions, the fertility genes of the Y chromosome have an as yet unappreciated physical dimension, being larger than euchromatic genes by two orders of magnitude.     

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.     

Selection of oligonucleotide probes and experimental conditions for multiplex hybridization experiments

Different DNA probes hybridize under different conditions. I examine the constraints of the design of oligonucleotide probes that are meant to hybridize to different unique sites in human genomic DNA under a single set of hybridization conditions as a parallel array. In 522 kb of human genomic DNA, 75% of 12-base and 89% of 22-base are unique, as opposed to 90% and 100% as expected of unstructured DNA, and this is not due solely to repetitive elements in the DNA. Hybridization in TMAC to reduce A + T content effects on melting temperature allows only 90% of unique targets to be hybridized under one set of conditions if a 2°C difference between matched and mismatched sequences is required. Standard hybridization conditions allow no more than 60% of unique probes to be used together. This suggests that probe, hybridization conditions, and instrument design for multiple-probe hybridization applications will be harder than previously suggested.     

Molecular Characterization of Ribosomal Genes on the Ybb- Chromosome of DROSOPHILA MELANOGASTER

The question of whether the Ybb- chromosome contains ribosomal genes has been examined by using Southern blot analysis and comparing rDNA hybridization patterns for X/X and X/Ybb- DNA. The results demonstrate that the Ybb- chromosome contains sequences that hybridize to an rDNA probe under stringent conditions. Differential hybridization of some of these sequences with DNAs corresponding to different regions of a complete ribosomal gene repeat provides evidence that some of the genes on the Ybb- chromosome are type 2 repeats. Because data obtained by other workers suggest that type 2 repeats are transcribed only to low levels, these repeats may be classed as "nonfunctional". A further finding is that the ribosomal genes on the Ybb- chromosome do not undergo multiple rounds of DNA replication during polytenization of X/Ybb- cells.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.     

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.