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.     

Die Funktionsstrukturen des Y-Chromosoms in den Spermatocytenkernen von Drosophila hydei,D. neohydei,D. repleta und einigen anderen Drosophila-Arten

Ohne ZusammenfassungHerrn Prof. Dr. W. Beermann danke ich für sein ständiges Interesse am Fortgang der Arbeiten und für häufige, wertvolle Diskussionen, Frl. G. Thies und Frl. D. Fleischmann für sorgfältige Assistenz.     

Involvement of Y chromosomal loci in the synthesis of Drosophila hydei sperm proteins

A comparative study of the protein patterns in testes of wild-type and X/O flies of Drosophila hydei revealed quantitative differences in at least three major protein fractions. One protein component of a Mr 155,000 fraction and a protein of Mr 35,000 are completely absent in X/O testes. The amount of protein in a Mr 55,000 fraction is considerably reduced. The tubulins, which are part of this fraction, are also reduced in amount. All three proteins were found as constituents of sperm tails. Studies of Y chromosomal mutants revealed that the presence of at least two of these proteins depends on the activity of loci O, P, and Q of the Y chromosome. However, preliminary evidence indicates an autosomal location of the genes of these sperm proteins. This suggests a regulatory role of Y chromosomal genes in the production of some major sperm proteins.     

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.     

A group of non-Y encoded Drosophila hydei primary spermatocyte nuclear glycoproteins exhibits epitopes depending on formation of Y chromosomal giant lampbrush loops

In wild-type Drosophila hydei (genotype X/Y) four different primary spermatocyte nuclear glycoproteins, classified as non-Y encoded because of their occurrence in X/O genotypes, were demonstrated to possess a few epitopes that depended on formation of the Y chromosomal giant lampbrush loops threads (th; Mr 55000 proteins) or pseudonucleolus (ps; Mr 38000, 58000 and 98000 proteins). The epitopes reacted with lectins and/or antibodies in vitro (lectin-/immunoreplica of primary spermatocyte total nuclear protein), and were lacking in mutants not possessing the respective loops. Those dependent on ps reacted with human sera. Epitopes restricted to proteins from th-forming spermatocytes reacted with lectin Con A (specific for d-Man and/or d-Glc) and antibodies directed against mouse immunoglobulins (AIA). In situ experiments (immunofluorescence microscopy of primary spermatocyte nuclei) revealed antibody cross-reactions with the respective loops. The reagents stained the distal (fused) sections and proximal (compact) parts of ps (human sera) or the proximal (compact) parts of th (AIA). Reaction with the latter loops was significantly repressed after absorption of AIA with the l-Fuc carbohydrate unit, classifying the AIA as fucosyl specific, and the epitopes along th as l-Fuc carbohydrate units.Abbreviations D-man D-mannose - D-Glc D-glucose - L-Fuc L-fucose - D-GlcNac N-acetyl-D-glucosamine - D-GalNac N-acetyl-D-galactosamine - AIA anti-immunoglobulin antibody - hnRNP heterogeneous nuclear ribonucleoprotein - DNP deoxyribonucleoprotein - FITC fluorescein isothiocyanate     

Analysis of nuclear proteins in primary spermatocytes of Drosophila hydei: the correlation of nuclear proteins with the function of the Y chromosomal loops

The protein content of spermatocyte nuclei from X/Y males and mutants of D. hydei which lack different Y chromosomal loop forming sites, was compared with that of X/0 males in ¹⁴C/³H double labelling experiments. Proteins of 45,000, 52,000, 54,000, 66,000, 80,000, 84,000, and 170,000 Dalton are found to be enriched in nuclei containing two or more active Y chromosomal loop forming sites. These proteins are also present in the nuclei of X0 males. In the complete absence of the Y-chromosomal loops proteins of 35,000, 46,000, 58,000 and 110,000 Dalton become enriched in the spermatocyte nuclei. — Analysis of the nuclear RNP of spermatocytes led to the isolation of an hnRNP-containing fraction with an S-value of >900S (RNP-PP). — In the RNP-PP of XY males labelled protein material associated with hnRNA is enriched by a factor of 3 in respect to the X0 genotype. The nuclear RNP has a heterogenous buoyant density in CsCl of p = 1.33 to 1.43 g/cm³. RNase T₁ treatment of the crude nuclear RNP from XY males prior to sucrose gradient analysis shows that the 66,000 Dalton protein which is also strongly enriched in the nuclei in the presence of active Y chromosomal loop forming sites, is the main protein associated with protected RNA-sequences of 80–120 and 200–300 nucleotides in length. Competitive nitrocellulose filter binding assays reveal that the 66,000 Dalton protein predominantly forms in 2 M NaCl stable RNA/protein complexes with the poly A ⁺hnRNA of the RNP-PP. These RNP complexes have a buoyant density of p = 1.43 g/cm³ in CsCl. The results are discussed in relation to the nuclear structure and the function of the Y chromosomal loops during spermatogenesis in Drosophila hydei.     

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.     

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.     

Salivary gland function and chromosomal puffing patterns in Drosophila hydei

Summary The salivary glands of D. hydei larvae show differences between the cells in the distal (posterior) part and those of the proximal (anterior) part during the third instar. The first sign of these differences is an increase in cellular and nuclear volume in the distal cells of the gland, beginning at 103 hours after oviposition. After 125 hours the cytoplasm of the extreme distal cells acquires a reticulated structure, and at 130 hours these cells contain large granules or droplets of mucoprotein. From this moment up to puparium formation the number of cells containing these granules increases and the boundary of this type of cells shows a shift in the proximal direction. Just before puparium formation the granules disappear from the cells and a glue substance is secreted by the larvae. At this moment only a few cells in the extreme proximal part still lack granules. Electron-microscopical observations indicate that these cells were active in secretion, whereas all cells containing large granules are inactive in this respect during most of the third instar.During the early third instar a change in cell function occurs, i.e. from synthesis of substances presumed to be digestive enzymes which are secreted, to a synthesis of a glue substance which is stored. This change begins in the extreme distal cells of the gland.Investigation of the chromosomal puffing pattern revealed that a total number of 148 puffs were present during some period of the third instar, prepupal, and early pupal stages. The activity of 110 puffs was evaluated during a series of successive time intervals. Changes in the puffing pattern during puparium formation were compared with those observed during pupation.Proximal and distal nuclei differ in the activity level of a number of puffs, but only puff 47 B is restricted in activity to the distal cells. This puff becomes active at 119 hours and disappears 4 hours before puparium formation (156 hours). Determination of nuclear diameter and DNA in nuclei of both parts of the gland revealed a correlation between a particular DNA content and the function of the cell. Distal cells show higher nuclear diameters than proximal cells after the onset of granule production. The first differences in nuclear diameter can be seen at 103 hours. Cells in the transitional part of the gland, located between distal granulecontaining and proximal granule-negative cells, always show the same DNA content. These cells are found at different locations within the gland during the third instar. This zone of cells shows a shift in proximal direction during the third instar, identical to that of the neighbouring granule-containing cells.The possible interrelation between nuclear DNA content, the activity of puff 47 B, and the production of the glue substance were discussed.     

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.