Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday     

He-T family DNA sequences in the Y chromosome of Drosophila melanogaster share homology with the X-linked Stellate genes

The genome of Drosophila melanogaster contains a class of repetitive DNA sequences called the He-T family, which is unusual in being confined to telomeric and heterochromatic regions. The specific He-T fragment designated Dm665 was cloned in yeast by selection for an autonomously replicating sequence (ARS). Dm665 contains a restriction fragment length polymorphism (RFLP) that is specific to males and thus derives from the Y chromosome. Deletion mapping using X-Y translocations indicates that sequences homologous to Dm665 occur in at least one major cluster in each arm of the Y chromosome. Among 20 yeast artificial chromosome (YAC) clones containing Drosophila sequences homologous with Dm665, four clones derive from defined regions of the long arm of the Y and two from the short arm. The sequence of Dm665 is 2443 bp long, consists of 59% A+T, and contains no significant open reading frames or direct or inverted repeats. However, Dm665 contains a region of 650 bp that shares homology with portions of the X-linked locus Stellate.by W. Hennig     

Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus

Summary The technique of chromosome walking was used to isolate approximately 60 kb of DNA from the region containing the complementation group uncoordinated of Drosophila melanogaster, located in that part of the X chromosome which spans the euchromatin-heterochromatin junction. The cloned DNA can be divided into two distinct regions. The first contains sequences that are low copy number or unique and are largely conserved between strains. The second region is characterized by units repeated in tandem arrays and is polymorphic within, and between, strains. Each repetitive unit is separated by a member of an abundant sequence family, part of which is homologous to the ribosomal type 1 insertion sequence of D. melanogaster. The molecular organization of the cloned DNA was compared with that of sequences isolated from regions of intercalary heterochromatin and also with genes which have been characterized from more conventional euchromatic regions.     

Change in distribution of mobile genetic elements in genome of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: Cause or consequence of selection for quantitative traits?

The distribution pattern of the hobo transposon and Dm412 retrotransposon hybridization sites on the salivary gland polytene chromosomes from larvae of the Drosophila melanogaster isogenic strain 51 used to analyze the effect of the transposition of transposable elements (TEs) on selection for quantitative traits was studied. It was shown that no more than half of the Dm412 hybridization sites were retained 15 years after isogenization; the frequency of the Dm412 transposition varied from 2.0 × 10⁻⁴ to 8.8 × 10⁻⁵ sites per genome for generation depending on whether the appearance of the same hybridization sites in a part of individuals was considered as independent events or as the manifestation of the appearing sample heterogeneity. The distribution patterns of hobo hybridization sites in two isofemale strains derived from the isogenic strain 51 differed much more markedly; the number of the hobo sites in one of the derivative strains was threefold smaller than in the other one and only some of the sites were common. Within each derivative strain, the TE distribution was uniform, which suggests that inbreeding had no effect on Dm412 activity in this strain. The rates of change in the distribution patterns of various TE in the strain 51 corresponded to their spontaneous transposition rates. Since the isogenic strain accumulates polymorphism in the TE distribution without selection, the TEs are more likely to be the markers of selection events rather than their inducers. Thus, when studying the effects of various environmental factors on TE transposition even in isogenic strains, it is necessary to perform additional close inbreeding to reduce the potential polymorphism.     

Analysis of the chromocenter DNA composition in polytene chromosomes of <Emphasis Type="Italic">Drosophila orena</Emphasis> ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.     

The pogo transposable element family of Drosophila melanogaster.

Summary A 190 by insertion is associated with the white-eosin mutation in Drosophila melanogaster. This insertion is a member of a family of transposable elements, pogo elements, which is of the same class as the P and hobo elements of D. melanogaster. Strains typically have many copies of a 190 by element, 10–15 elements 1.1–1.5 kb in size and several copies of a 2.1 kb element. The smaller elements all appear to be derived from the largest by single internal deletions so that all elements share terminal sequences. They either always insert at the dinucleotide TA and have perfect 21 bp terminal inverse repeats, or have 22 by inverse repeats and produce no duplication upon insertion. Analysis by DNA blotting of their distribution and occupancy of insertion sites in different strains suggests that they may be less mobile than P or hobo. The DNA sequence of the largest element has two long open reading frames on one strand which are joined by splicing as indicated by cDNA analysis. RNAs of this strand are made, whose sizes are similar to the major size classes of elements. A protein predicted by the DNA sequence has significant homology with a human centrosomal-associated protein, CENP-B. Homologous sequences were not detected in other Drosophila species, suggesting that this transposable element family may be restricted to D. melanogaster.     

Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Tirant: A New Retrotransposon-Like Element in Drosophila melanogaster

In this paper we report a new retrotransposon-like element of Drosophila melanogaster called Tirant. This sequence is moderately repeated in the genome of this species and it has been found to be widely dispersed throughout its distribution area. From Southern blot and in situ analyses, this sequence appears to be mobile in D. melanogaster, since its chromosome location and the hybridization patterns vary among the different strains analyzed. In this way, partial sequencing of Tirant ends suggests that it is a retrotransposon, since it is flanked by two LTRs. The presence of sequences homologous to Tirant has been also investigated in 28 species of the genus Drosophila by means of Southern analyses. These sequences were only detected in species from melanogaster and obscura groups. These data suggest that ancestral sequences of Tirant appeared after the Sophophora radiation and before the divergence of those groups. Received: 1 January 1995 / Accepted: 20 August 1995     

Identification of cytoplasmically transferred mitochondrial DNA in female germlines of Drosophila and its propagation in the progeny

Summary The composition of mitochondrial DNA (mtDNA) was analyzed in single female flies that developed from fertilized Drosophila melanogaster eggs, into which germ plasm of D. simulans had been introduced. HpaII cleavage patterns showed that all 12 individual female flies examined had developed from eggs in which 37%–71% of the total mtDNA was D. simulans mtDNA (Ds mtDNA) and the rest was D. melanogaster mtDNA (Dm mtDNA). The stability of this heteroplasmic state in these isofemale lines was monitored for seven generations at both individual and population levels. Results showed that the heteroplasmy of Dm and Ds mtDNAs was stably transmitted for at least three generations at the population level, but showed stochastic segregation at the individual level. After 4–6 generations, all individuals lost Ds mtDNA. The mechanisms of preferential loss of Ds mtDNA and of transmission of heteroplasmic mtDNA to descendants are discussed.     

Mariner-like sequences are present in the genome of the fruitfly,Drosophila melanogaster

No mariner-like elements (MLEs) have been described until now in the genome of Drosophila melanogaster despite many experiments using molecular methods. However, analyses of sequence data from the Berkeley Drosophila Genome Project show that there are DNA sequences corresponding to pieces of MLE in the genome of D. melanogaster. The sequences of these elements have diverged considerably (about 40%) from any other sequences observed elsewhere. Moreover, the putative amino acid sequences encoded by the best conserved regions reveal that these sequences are clearly homologous to MLEs transposase.     

Histone H1 Genes and Histone Gene Clusters in the Genus Drosophila

Whereas the genomes of many organisms contain several nonallelic types of linker histone genes, one single histone H1 type is known in Drosophila melanogaster that occurs in about 100 copies per genome. Amplification of H1 gene sequences from genomic DNA of wild type strains of D. melanogaster from Oregon, Australia, and central Africa yielded numerous clones that all exhibited restriction patterns identical to each other and to those of the known H1 gene sequence. Nucleotide sequences encoding the evolutionarily variable domains of H1 were determined in two gene copies of strain Niamey from central Africa and were found to be identical to the known H1 sequence. Most likely therefore, the translated sequences of D. melanogaster H1 genes do not exhibit intragenomic or intergenomic variations. In contrast, three different histone H1 genes were isolated from D. virilis and found to encode proteins that differ remarkably from each other and from the H1 of D. melanogaster and D. hydei. About 40 copies of H1 genes are organized in the D. virilis genome with copies of core histone genes in gene quintets that were found to be located in band 25F of chromosome 2. Another type of histone gene cluster is present in about 15 copies per genome and contains a variable intergenic sequence instead of an H1 gene. The H1 heterogeneity in D. virilis may have arisen from higher recombination rates than occur near the H1 locus in D. melanogaster and might provide a basis for formation of different chromatin subtypes. Received: 2 March 2000 / Accepted: 1 June 2000     

Transposable elements map in a conserved pattern of distribution extending from beta-heterochromatin to centromeres in Drosophila melanogaster

In situ hybridisation to mitotic chromosomes shows that sequences homologous to different Drosophila melanogaster transposable elements are widely distributed not only in beta but also in alpha-heterochromatin. Clusters of these sequences are detected in most proximal positions. They colocalise with known satellite sequences in several regions, but are also located in places where no known sequence has been mapped so far. The pattern of hybridisation is dinstinctive and specific for each element, and presents constant features in six different D. melanogaster strains studied. The entirely heterochromatic Y chromosome contains large amounts of these sequences. Additionally, some of these sequences appear to be present in substantial quantities in the smallest minichromosome of Drosophila, Dp(1;f)1187.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).     

Restriction Endonuclease Analysis of Mitochondrial DNA as an Aid in the Taxonomy of Naegleria and Vahlkampfia1

ABSTRACT Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

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.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.     

Movement of Drosophila melanogaster transposable elements other than P elements in a P-M hybrid dysgenic cross

Summary The Drosophila melanogaster mobile DNA sequences P factors and P elements transpose at elevated rates when P strain males are mated to M strain females in a hybrid dysgenic cross (Engels 1983). Isofemale lines derived from such a cross were analysed by in situ hybridisation using cloned copies of the transposable elements copia, 412 and F. It was found that lines derived from dysgenic crosses showed a statistically significant number of new sites for these elements when compared to a non-dysgenic control cross. This result suggests a functional coupling of copia, 412 and F transposition and some component present in the P-M dysgenic system.