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 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.     

DNA clones and RNA transcripts of four lampbrush loops from the Y chromosome of Drosophila hydei

Drosophila hydei clones representing transcribed middle-repetitive sequences from four of six major lampbrush loops of the Y chromosome were isolated. Sequences homologous to each clone are clustered in a particular locus on the Y chromosome, but additional euchromatic sites were found for one of the transcribed clones. In situ hybridization to lampbrush-loops RNA permitted the identification of clones homologous with the two "nooses" loops on YS and with the "clubs" and "tubular ribbons" on the YL arm. Loop-specific nuclear RNA molecules range in size from 10S to 60S. Loop RNA is accumulated in the nucleus and remains attached to the loops during the course of primary spermatocyte growth. It disappears, however, along with the loop structures, during the first meiotic prophase. The structure and function of the Y chromosome and its lampbrush loops are briefly considered in the light of these findings.     

Differential staining of Y chromosomal lampbrush loops of Drosophila hydei

The lampbrush loops of the Y chromosome in primary spermatocytes of Drosophila hydei can be stained in a site specific manner employing a modified Giemsa technique. In this communication it is shown that pronase pretreatment changes the staining properties of the various Y loops as well as of the X and autosomes. In addition it is shown that the various chromosomal structures display differences in sensitivity against the action of the enzyme supporting the idea of a site specificity of RNP formation.Dedicated with gratitude to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

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.     

The function of the lampbrush loops formed by the Y chromosome of Drosophila hydei in spermatocyte nuclei

Summary The function of pairs of translocated fragments of the Y chromosome of Drosophila hydei was tested. As the pairs of fragments together had a complete set of Y chromosomal sites, complementation of their function could be predicted according to results of earlier experiments. In contrast to the earlier experiments the development of lampbrush loops during the spermatocyte stage was blocked in one partner of each combined pair. As a consequence, no complementary effect on spermiogenesis is detectable. The results indicate that the formation of lampbrush loops by seven sites in the Y chromosome is a necessary prerequisite for the normal progress of spermiogenesis. This can be considered as further support of the view that the lampbrush loops in spermatocyte nuclei of Drosophila are phenotypic manifestations of the activity of male fertility factors.Supported by the Deutsche Forschungsgemeinschaft.     

Genetic function correlated with unfolding of lampbrush loops by the Y chromosome in spermatocytes of Drosophila hydei

Summary Deficiencies of the Y chromosome of Drosophila hydei including sites which develop lampbrush loops invariably cause sterility of males. Suppression of loop unfolding in one or more sites equally results in similar morphogenetic defects of spermiogenesis. A variegated type repression of lampbrush loop unfolding observed during the spermatocyte stage results in varying morphogenetic effects on spermiogenesis. This demonstrates the existence of causal relationships between the active phase of Y chromosomal factors in spermatocytes and the differentiation processes in spermatids.In some translocated Y fragments the mode of unfolding of a particular pair of lampbrush loops may be permanently changed. As a result, lampbrush loops of a mutant phenotype are developed. Some alterations of this type are correlated with functional alterations resulting in defective spermiogenesis.Three different fragments of the Y chromosome in which lampbrush loop formation was repressed have been tested for possible reversions of loop suppression by means of X irradiations. In none of the three cases reversion has been detected among two thousand tested chromosomes.To the memory of Karl-Heinz Bier.     

Evolution of Y chromosomal lampbrush loop DNA sequences of Drosophila

The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.     

Two separated nucleolus organizers on the Drosophila hydei Y chromosome.

By genetical, cytological, and filter saturation hybridization methods it is shown that the Y chromosome of Drosophila hydei contains two separate nucleolus organizers, one on the short arm, the second near the tip of the long arm.     

Y chromosomal DNA of Drosophila hydei

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.