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.     

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.     

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.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

Mutation in the U2 snRNA influences exon interactions of U5 snRNA loop 1 during pre-mRNA splicing

The U2 and U6 snRNAs contribute to the catalysis of intron removal while U5 snRNA loop 1 holds the exons for ligation during pre-mRNA splicing. It is unclear how different exons are positioned precisely with U5 loop 1. Here, we investigate the role of U2 and U6 in positioning the exons with U5 loop 1. Reconstitution in vitro of spliceosomes with mutations in U2 allows U5-pre-mRNA interactions before the first step of splicing. However, insertion in U2 helix Ia disrupts U5-exon interactions with the intron lariat-3' exon splicing intermediate. Conversely, U6 helix Ia insertions prevent U5-pre-mRNA interactions before the first step of splicing. In vivo, synthetic lethal interactions have been identified between U2 insertion and U5 loop 1 insertion mutants. Additionally, analysis of U2 insertion mutants in vivo reveals that they influence the efficiency, but not the accuracy of splicing. Our data suggest that U2 aligns the exons with U5 loop 1 for ligation during the second step of pre-mRNA splicing.     

Ultrastructural similarity in landmark loops of amphibian lampbrush chromosomes

Simultaneous transmission and scanning electron microscopy studies were performed on lampbrush chromosomes of Notophthalmus viridescens and Xenopus laevis. The organization of their normal and landmark loop ribonucleoprotein (RNP) matrices was compared to that of Pleurodeles waltl lampbrush loops, previously described. Ultrastructural observations clearly showed that in the three species, the RNP matrix of normal and landmark loops displayed a common basic structure: an RNP fibril packed into tightly juxtaposed RNP particles of remarkably uniform size, ie 30 nm. Furthermore, analysis of the spatial arrangement of these constitutive RNP fibrils allowed us to establish ultrastructural similarities between the different types of loop matrices of the three species studied. Thus, granular loops with the same organization were found to be present in the three species, whereas Pleurodeles was the only one to exhibit, in its lampbrush chromosomes, the typical globular matrices previously described. “Sequential labelling loops” of Notophthelmus were shown to be similar of both “convoluted dense loops” of Xenopus and “dense loops” of Pleurodeles.     

Divergence of U2 snRNA sequences in the genome of D. melanogaster.

Four different U2-snRNA genes/related sequences of D. melanogaster were cloned and characterized. The sequences of all four genes suggest that they were generated by a DNA-mediated mechanism. These genes/related sequences were found to be located in two loci, each locus containing two U2 snRNA sequences. Using coding sequences as well as flanking sequences as hybridization probes against polytene chromosomes of D. melanogaster Oregon R we were able to map these loci separately at positions 34BC and 84C. By Northern analysis we observed that the quantities of U2- and U1-snRNA are coordinated and change during the embryonic development of the fly.     

Structure of the yeast U2/U6 snRNA complex

The U2/U6 snRNA complex is a conserved and essential component of the active spliceosome that interacts with the pre-mRNA substrate and essential protein splicing factors to promote splicing catalysis. Here we have elucidated the solution structure of a 111-nucleotide U2/U6 complex using an approach that integrates SAXS, NMR, and molecular modeling. The U2/U6 structure contains a three-helix junction that forms an extended "Y" shape. The U6 internal stem-loop (ISL) forms a continuous stack with U2/U6 Helices Ib, Ia, and III. The coaxial stacking of Helix Ib on the U6 ISL is a configuration that is similar to the Domain V structure in group II introns. Interestingly, essential features of the complex--including the U80 metal binding site, AGC triad, and pre-mRNA recognition sites--localize to one face of the molecule. This observation suggests that the U2/U6 structure is well-suited for orienting substrate and cofactors during splicing catalysis.     

Solid-phase processing of U2 snRNA precursors

HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.