Frequency of telomere repeat units in the Drosophila miranda genome

Cloned DNA fragments of Drosophila miranda which label all chromosome ends show a basic tandem repeat unit of 4.4 kb. The D. miranda telomere specific tandem repeats do not cross-hybridize with genomic D. melanogaster DNA which itself contains telomere repeat units of 3 kb. For a more detailed analysis of the functional criteria of telomere specific sequences we determined the repetition frequency of the tandem repeat units. As a low estimate we found a repetition frequency of 20 for female D. miranda DNA. This is on average equivalent to 2 telomere repeat units per chromosome end in the female D. miranda karyotype. However, a variable number of tandem repeat units per chromosome end would describe more closely the obtained differences in the labeling intensity between the individual chromosomes (X¹L-5). For the D. miranda male DNA we determined a repetition frequency of 90. The frequency difference of 70 copies between male and female DNA must be due to the Y-chromosome.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

Evolutionary changes in the organization of the major LCP gene cluster during sex chromosomal differentiation in the sibling speciesDrosophila persimilis,D. pseudoobscura andD. miranda

The major larval cuticle protein (LCP) genes I–IV ofDrosophila melanogaster are clustered on the right arm of the second chromosome. By cross-hybridization we cloned the corresponding genes from three different members of theobscura group:D. persimilis, D. pseudoobscura andD. miranda. InD. pseudoobscura andD. persimilis the gene cluster maps to autosome3. In contrast, inD. miranda it was found on theX2 andY sex chromosome. Hence, this exceptional karyotypic situation offers a unique opportunity to analyse the molecular processes underlying the phenomenon of chromosome degeneration. Comparison of LCP genes I–IV in theX2 andY chromosomal region inD. miranda revealed extensive DNA rearrangements at the latter. TheY chromosomal LCP cluster is characterized by DNA insertions which are absent in the correspondingX2 chromosomal DNA, suggesting that these DNA sequences must have invaded this area. In addition, part of the analysedY chromosomal region is duplicated.     

Analysis of chromosomal homologies between two species of the subgenus sophophora: D. miranda and D. melanogaster using cloned DNA segments

Chromosomal homology between two species of the subgenus Sophophora, D. miranda and D. melanogaster, belonging to the obscura and the melanogaster group respectively, was probed by DNA in situ cross hybridizations. A set of recombinant plasmids with inserts derived from the D. melanogaster genome were cross hybridized to the D. miranda karyotype. Vice versa, recombinant Lambda phages isolated from a genomic D. miranda library were localized in D. miranda and probed for localization in D. melanogaster. In the main, the results support the homology relations proposed on the basis of cytogenetic data. However, the location of both tandemly repetitive genes tested, 5S RNA genes and the histone genes, is not in accordance with expectation. The 5S RNA genes, when probed with the D. melanogaster plasmid 12D8 (Artavanis-Tsakonas et al., 1977), were found to occur at two sites in both, D. miranda and D. pseudoobscura.     

Chromosomal homologies betweenDrosophila lebanonensis andD. melanogaster determined by in situ hybridization

Twelve biotin-labelled recombinant DNA probes were hybridized to polytene chromosomes ofDrosophila melanogaster andD. lebanonesis. Probes were chosen in order to cover the whole chromosomal complement. Six probes correspond to known genes fromD. melanogaster (RpII215, H3–H4, MHC, hsp28/23, hsp83, hsp70), four probes are clones isolated from aD. subobscura library (Xdh, DsubS3, DsubG3, DsubG4) and the remaining two probes correspond to the Adh gene ofD. lebanonensis and to one sequence (262), not yet characterized, from the same species. The chromosomal homologies obtained from the in situ hybridization results allow us to determine that Muller's C and D chromosomal elements are fused in the karyotype ofD. lebanonensis and constitute the large metacentric chromosome. Single pericentric inversions in theE andB elements have generated the medium and small metacentric chromosomes, respectively. No great changes are detected in Muller'sA element, which remains acrocentric. The changes detected in the karyotypic evolution ofD. lebanonensis are frequently observed inDrosophila evolution, as deduced from chromosomal homologies of severalDrosophila species. The results are also consistent with Muller's proposal that chromosomal elements have been conserved during the evolution ofDrosophila.     

Subcloning and functional analysis of aBacillus subtilis β-1,3-1,4-glucanase gene (bglS) inEscherichia coli

This article describes a 7.1kb EcoRI DNA fragment carrying aBacillus subtilis -1,3-1,4-glucanase gene (bglS). By subcloning, a 1.5kb EcoRI-PstI DNA fragment was inserted into the polylinker cloning sites of the plasmid pUC19 and transferred toEscherichia coli JM101. The fragment directed the synthesis inE. coli of a-1,3-1,4-glucanase that specifically degrades barley glucan and lichenan. The largest proportion (>50%) of the total enzyme activity was cellular enzyme; 25% of the-1,3-1,4-glucanse activity was present in the extracellular fractions. Through enzyme analysis, the enzymes purified fromE. coli or fromBacillus subtilis 1.88 were proved to be identical.     

Nucleotide sequence comparison of the Adh gene in three drosophilids

Summary The alcohol dehydrogenase (Adh) gene has been isolated fromDrosophila simulans andD. mauritiana by screening clone libraries of each with a previously cloned Adh gene fromD. melanogaster. The isolated clones were subcloned and partially sequenced to determine the relatedness of these species and to examine details of evolutionary change in the structure of the Adh gene. We report the sequence of the first 704 nucleotides of each gene as well as 127 bases in the 5 untranslated region. When these sequences are compared,D. melanogaster differs fromD. simulans andD. mauritiana by 2.8% and 3.1%, respectively.D. simulans andD. mauritiana differ by only 1.8%, implying that they are more closely related to each other than either is toD. melanogaster. This is consistent with phylogenetic relationships established by a variety of genetic, biochemical, and morphological means and illustrates that DNA sequencing of a single gene may be used to assess the evolutionary relationships of species.     

Analysis ofDrosophila chromosome4 using pulsed field gel electrophoresis

Previous estimates of the size ofDrosophila melanogaster chromosome4 have indicated that it is 1% to 4% of the genome or 6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules ofD. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from thecubitus interruptus andzfh-2 genes, which are located on chromosome4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome4. In DNA obtained from embryos of variousD. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. TheD. melanogaster chromosome4 probes also identified a single, 2.4 Mb band in embryonic DNA fromDrosophila simulans. We conclude thatD. simulans chromosome4 is substantially smaller than that ofD. melanogaster, presumably owing to diffirences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome4 DNA and will facilitate the molecular mapping of this chromosome.     

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1–Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2. Received: 27 December 1995 / Accepted: 30 April 1996     

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopic (EM) analysis was performed on regions of Drosophila melanogaster polytene chromosomes that contain inserted DNA segments of 19 and 8 kb. These segments had been inserted by P-elementmediated transformation. The 19 kb segment includes both the Drosophila hsp70 gene fused to the Escherichia coli -galactosidase gene and the rosy gene (Lis et al. 1983). This insert generates a new moderate-size band at the 9D4-9E1-2 region in polytene chromosomes. Upon heat shock, a puff originates from a portion of the new band. The 8 kb segment includes the Sgs7 and Sgs3 genes (Richards et al. 1983). This insert generates very diffuse thin bands that decondense at the stage of activation of the Sgs genes to produce wide interbands or small puffs. In all of the above cases, the insertion appears to occur at interband regions, and the genetically complex DNA segments that are inserted generate only a single detectable band.     

Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudoobscura and D. subobscura

Enzyme loci, visible marker genes and -cloned DNA-sequences from a D. miranda library were mapped cytologically on the chromosome elements C and E of D. pseudoobscura and D. subobscura. New data are incorporated into the linkage maps of the two species. Homologous segments can now be localized in the polytene chromosomes with these markers. A comparison of the chromosome elements E of D. melanogaster and D. subobscura shows 12 conserved subsections which have been rearranged by paracentric inversions in the evolution of the two lineages.     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Multiple sex chromosomes in Drosophila miranda: A system to study the degeneration of a chromosome

Drosophila miranda possesses an intriguing sex chromosome constitution. While female metaphase plates have 10 chromosomes (diploid set), in males only 9 chromosomes can be identified. The missing homologue has been translocated to the Y, forming a neo-Y chromosome which is polytenized in the salivary gland cells. This report presents a detailed characterization of DNA, isolated from D. miranda flies. In situ hybridizations, using cRNA transcribed from unfractionated D. miranda DNA, reveal hybridization to the neo-Y with label distributed over the entire chromosome. The original partner of the translocated chromosome, X², is essentially unlabelled. These results suggest that repetitive DNA sequences invade the translocated chromosome. This result is discussed with reference to the hypothesis of degeneration of the Y chromosome, formulated by Muller (1918, 1932a).     

Chromosome Localization of the λ20p1.4 clone of the Drosophila melanogaster Nuclear Lamina DNA in the melanogaster Species Subgroup of the Genus Drosophila (Sophophora)

Chromosome localization of sequences homologous to 20p1.4 of the Drosophila melanogaster nuclear lamina DNA (nlDNA) was established by in situ hybridization in species of the melanogastersubgroup. DNA of the 20p1.4 clone was shown to be located in the chromocenter in all the species examined. Laboratory strains of D. simulans, D. mauritiana, andD. sechellia exhibited interspecific differences in localization of 20p1.4 nlDNA on chromosome arms. In eight natural populations, intraspecific polymorphism of 20p1.4 nlDNA chromosome localization was shown to be present in D. simulans but absent in D. melanogaster. The possible participation of transposable elements in 20p1.4 nlDNA relocation is discussed.     

Chromosome homologies within the Drosophila obscura group probed by in situ hybridization

Probes specific to chromosome elements were used to investigate chromosome homologies between seven species of the Drosophila obscura group by in situ hybridization. Our results were in perfect agreement with the already established chromosome element homologies between D. subobscura, D. pseudoobscura, D. persimilis, and D. miranda. Furthermore, we were able to identify the chromosomal elements of D. obscura, D. ambigua, and D. subsilvestris. Of special interest was the localization of the two D. melanogaster-derived representatives of the tandemly repetitive genes, cDm500 and 12D8. In contrast to the findings with the element-specific probes, the localizations of the repetitive genes varied in the various species. Whereas D. melanogaster, D. subobscura, D. pseudoobscura, D. persimilis, and D. miranda showed only one strong block of label in the cross in situ hybridizations with cDm500, three labeling blocks were found on two elements for both D. ambigua and D. obscura. The two labeling blocks on one element occur in very close proximity, but are clearly separated in both species by cytologically detectable chromosomal material. We used the distribution of the cDm500 labeling sites to postulate a series of chromosomal rearrangements involved in the karyotype evolution of the analyzed species. Our results support the conclusion that the chromosomal elements retain their essential identity and that the observed gross structural rearrangements are due to fusions and paracentric or pericentric inversions. Cytologically obvious translocations were not recorded and are considered by us to be rare. The frequently occurring translocations of the tandemly repeated gene clusters observed in this study are probably due to a different mechanism, which may be an intrinsic property of this category of genes.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday with our best wishes     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.     

DNA sequence comparison of micropia transposable elements fromDrosophila hydei andDrosophila melanogaster

Members of the retrotransposon family micropia were discovered as constituents of wild-typeY chromosomal fertility genes fromDrosophila hydei. Several members of the micropia family have subsequently been recovered fromDrosophila melanogaster and four micropia elements, micropia-DhMiF2, -DhMiF8, -Dm11 and-Dm2, two each fromD. hydei andD. melanogaster, have been totally sequenced (17 kb of micropia sequences and 6.8 kb from insertions)¹. Comparative analysis of micropia sequences revealed a complex pattern of divergence within a singleDrosophila genome. The divergence includes deletions, possibly by a slipped mispairing mechanism, insertions of a retroposon, and of another retrotransposon (copia) and positional nucleotide shuffling within the tandem repeats of the 3 non-protein-coding region of micropia elements. A 10 bp long sequence of each repeat unit of the 3 tandem repeats of micropia elements is highly conserved and is therefore a candidate of functional importance either in transposition events or in regulatory activity on flanking DNA sequences.Abbreviations LTR long terminal repeat - PBS primer binding site - PolII RNA polymerase II - bp base pairs - kb kilobases (pairs) - LINE long interspersed sequence - MHC major histocompatibility complex This paper is dedicated to the 90^th birthday of Prof. Dr. Bernhard Rensch.