Characterisation of complete type II insertions in cloned segments of ribosomal DNA from Drosophila melanogaster

We have isolated cloned segments of ribosomal DNA that have EcoRI restrictable (type II) insertions in their 28 S genes. The type II insertions in these plasmids are homologous sequences and have three characteristic cleavage sites for EcoRI. One of these clones is unusual in that it has undergone a deletion of part of the 28 S gene at or near the site of the type II insertion. A second is unusual in that, in addition to the type II insertion in the rDNA, the transcribed spacer sequences are interrupted by an unidentified sequence. This sequence differs in its arrangement of restriction sites from the sequence that interrupts the transcribed spacer of cDm207 (Glover, 1977). The type II sequences in all these clones share homology with the unusually long ‘insertion’ that interrupts the 28 S gene of cDm207. We have re-examined the nature of the additional sequences linked to the type II sequences of cDm207 and find them to be related to type I rDNA insertion sequences.     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.     

Localization of ribosomal DNA insertion elements in polytene chromosomes of Drosophila simulans,Drosophila mauritiana and their interspecific hybrids

The locations of the ribosomal DNA (rDNA) insertion elements type I and type II along the polytene chromosomes of three Drosophila species of the melanogaster subgroup-D. simulans, D. mauritiana and D. melanogaster-have been compared. In situ hybridization has shown that the intragenomic distribution of type I as well as of type II insertions is different for these related species. In particular, we have revealed rDNA-free autosomal sites, containing type II element sequences within the D. simulans and D. mauritiana chromosomes. This finding confirms the ability of this type of insertion to transpose, as was demonstrated earlier for Bombyx mori. The appearance of the rDNA not associated with the nucleolar organizers, evident by additional nucleoli, occurred with species-specific frequency. At the same time, for all three species the pattern of such changes (an attachment of the nucleoti to varying sites of the chromosomes and the presence of ectopic contacts between them, a composition of the rDNA repeats in the nucleolar material not integrated at the nucleolar organizer) was similar. The number of additional nucleoti in the hybrid polytene nuclei corresponded to the value of the parental species exhibiting nucleolar replicative dominance.     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Comparative studies on the rDNA of the silkworm,Bombyx mori and its presumed ancestor

• 1.1. The transcribed region of Bombyx mandarina rDNA was identical to that of B. mori rDNA when the restriction maps and partial nucleotide sequences were compared. The result supports the assumption that Bombyx mandarina is an ancestor of Bombyx mori and that the two were subdivided very recently.
• 2.2. Non-transcribed spacer (NTS) of four clones derived from the two insects was slightly different from one another, which seemed to be due to the difference in the number of repeated sequences distributed in the spacer.
• 3.3. The five clones from B. mandarina had the type I insertion sequence (IS) homologous to that in Bombyx mori 28S rDNA. There was micro-heterogeneity in the structure of IS.

     

The ribosomal DNA of Drosophila melanogaster is organized differently from that of Drosophila hydei

On the X chromosome of Drosophila melanogaster there is a single tandem array of 240 ribosomal RNA genes. The majority of these contain an insertion, known as type I, in the 28 S coding region. Previous genetic and electron microscopic studies indicated that genes bearing the type I insertion (ins⁺) are interspersed at random with those lacking it (ins^?). In contrast, Renkawitz-Pohl et al. (1981) have analyzed the restriction pattern of X chromosomal ribosomal DNA in Drosophila hydei and demonstrated that in this case ins⁺ genes are segregated from ins^?. This suggests either that the rDNA is organized differently in these two species or that the restriction enzyme technique reveals significant clustering not detected by previous methods. By using an appropriate restriction enzyme, we demonstrate that ins⁺ and ins^? genes are intermingled at random in D. melanogaster. These experiments also indicate that genes containing the short form of the insertion are flanked by a larger spacer upstream than downstream.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

A DNA segment from D. melanogaster which contains five tandemly repeating units homologous to the major rDNA insertion

We describe a cloned segment of D. melanogaster DNA (cDm219) that contains five tandemly arranged sequence units homologous to the type I insertion sequence found in the majority of 28S rRNA genes on the X chromosome. Heteroduplex studies show that two of the units have a deletion corresponding to a 1.1 kb piece of DNA close to the right-hand end of the type I insertion. Another unit has a 7.5 kb sequence (zeta) substituted for a 0.95 kb piece of DNA close to the left-hand part of the type I rDNA insertion. The two remaining units are interrupted by the Col E1 plasmid vector. There are also differences in the restriction endonuclease cleavage maps both between the units of cDm219 themselves and compared to the restriction endonuclease cleavage maps of cloned rDNA segments that contain type I insertions. Quantitation of the gel transfer hybridization of zeta element probes to restriction endonuclease digests of D. melanogaster DNA indicates there are 30--40 copies of zeta sequences distributed in seven major arrangements within the haploid genome. The hybridization of zeta and insertion sequence probes to a library of D. melanogaster DNA segments cloned in bacteriophage lambda indicates at least 4--6 copies of the zeta element could be linked to insertion sequences. The common site of in situ hybridization of zeta sequences is to the chromocentral heterochromatin of polytene chromosomes.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Molecular cloning and characterization of the chicken gene homologous to the transforming gene of rous sarcoma virus

Four molecular clones containing DNA homologous to the Rous sarcoma virus transforming gene (src) have been isolated from a random library of normal chicken DNA. The four clones are distinct overlapping isolates, which together span approximately 33 kb of cellular DNA. The cloned locus appears to represent the major region of chicken DNA homologous to src, since src-containing restriction fragments of this locus account for the fragments detected by hybridization of src-specific probe to restriction digests of total chicken DNA. Analysis of the cloned chicken src locus by restriction and heteroduplex mapping indicates that the locus contains 1.6-1.9 kb of DNA homologous to the viral src gene. The chicken DNA sequences homologous to viral src are interrupted by five or six nonhomologous regions, totaling approximately 6 kb, which presumably represent introns in the cellular src gene.     

Coding region deletions associated with the major form of rDNA interruption in Drosophila

The nucleotide sequences at and around the termini of 5 kb type 1 interruptions in three separate clones of D. melanogaster rDNA repeats have been determined, and have been compared with the sequence of the corresponding region of an insertion-free rDNA repeat. All three interrupted rDNA repeats contain a small deletion of 28S rRNA coding material at the left coding/insertion sequence junction. A second deletion was found in one of the three clones, ad other aberrations were suggested by the results of restriction enzyme digestions of unfractionated rDNA. The termini of 5 kb type 1 rDNA insertions in D. melanogaster were also compared with the corresponding regions of 28S rDNA interruptions in D. virilis: the insertion site is identical in the two species, but the termini of the two species' interruptions show no homology. I sequenced a 1.1 kb region of the 5 kb type 1 D. melanogaster rDNA interruption that covers the sequences of the 1 kb and 0.5 kb insertions. There is 98% homology between the rightmost 1 kb of the 5 kb interruption and the sequences of the shorter insertions. Data suggest that Drosophila rDNA interruptions arose as a transposable element, and that divergence had included length alterations generated by unequal crossing over.     

Characterization of Variability in Some Pathogenic Species of Alternaria Based on the Nucleotide Sequence of Ribosomal DNA

The internal transcribed spacer (ITS) sequences within the ribosomal DNA (rDNA) region were targeted to delineate genetic variability among eight Alternaria species that cause economically important diseases in crops. The rDNA regions of Alternaria species comprising of rRNA genes and the ITS regions were cloned and sequenced. Phylogenetic relationship based on the rDNA sequences and PCR-RFLP of amplified rDNA sequences clustered eight species of Alternaria into three major groups. A. macrospora and A. helianthi accumulated wide genetic variations and are distantly related to rest of the six species which formed two major groups. Group I comprised of three species viz., A. dianthicola, A. brassicae and A. citri, while group II had A. longipes, A. porri and A. alternata. Incorporation of unique stretches of nucleotides and single nucleotide substitutions within relatively conserved ITS1 and ITS2 regions led to clustering of the members of Alternaria species in each group. The divergent sequences within the ITS regions can be employed to design species-specific PCR primer for use in molecular diagnostics.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Evolutionary conservation of the human 7 S RNA sequences

The cloning and characterization of the cytoplasmic 7 S RNAs of HeLa cells has provided pure probes to study the organization of the corresponding genomic DNA sequences. Such analysis has shown that the 7 S L and K RNAs are derived from families of middle repetitive DNA (Ullu & Melli, 1982; Ullu et al., 1982). In this work we analyze the evolutionary conservation of these sequences in the RNA and DNA of distantly related species. Hybridization of the 7 S recombinants to the RNA of rodents, birds, amphibians and echinoderms suggests high conservation of these sequences throughout evolution. Southern blot analysis of genomic DNAs from the same species shows the presence of families of repeated sequences homologous to the 7 S recombinants and Alu DNAs in the genomes of the same species. We were unable to hybridize the 7 S probes to the RNAs of Drosophila melanogaster or Dictyostelium discoideum, although sequence(s) homologous to the 7 S L probe were found in the genome of D. discoideum and to both 7 S L and K probes in the genome of D. melanogaster.     

Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster.

We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera.     

Duplicated rDNA sequences of variable lengths flanking the short type I insertions in the rDNA of Drosophila melanogaster.

We describe cloned segments of rDNA that contain short type I insertions of differing lengths. These insertions represent a coterminal subset of sequences from the right hand side of the major 5kb type I insertion. Three of these shorter insertions are flanked on both sides by a short sequence present as a single copy in uninterrupted rDNA units. The duplicated segment is 7, 14 and 15 nucleotides in the different clones. In this respect, the insertions differ from the 5kb type I insertion, where the corresponding sequence is found only at the right hand junction and where at the left hand side there is a deletion of 9 nucleotides of rDNA (Roiha et al.,1981). One clone is unusual in that it contains two type I insertions, one of which is flanked by a 14 nucleotide repeat. The left hand junction of the second insertion occurs 380 nucleotides downstream in the rDNA unit from the first. It has an identical right hand junction to the other elements and the 380 nucleotide rDNA sequence is repeated on both sides of the insertion. We discuss the variety of sequence rearrangements of the rDNA which flank type I insertions.     

The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement

The restriction endonuclease pattern of a number of hsp70-homologous clones isolated from a library of heat shock cDNA from Drosophila auraria, a species belonging to the montium subgroup of the melanogaster species group, reveals two types of clones, A and B, differing in a single restriction site. Both types, as well as hsp70-specific probes derived from both hsp70 loci of Drosophila melanogaster, hybridize in situ with a single band at region 32 A of the 2L polytene arm, indicating a clustered organization of the hsp70 gene copies in D. auraria. The longest type B clone was sequenced and it was found that one strand contains an open reading frame (ORF) exhibiting great identity with a previously described hsp70 gene of D. auraria (now denoted as type A) and with its counterparts of D. melanogaster, while its second strand, unlike the type A clone, does not contain a long antiparallel coupled ORF (LAC ORF) because of a base substitution resulting in a premature stop codon. After additional data had been derived from isolation and characterization of hsp70-homologous genomic clones, together with Southern analysis of genomic DNA, we found that two hsp70 gene copies are present at the above locus of D. auraria with an inverted tandem repeat organization, while the presence of a third hsp70 gene is not clearly evident. The above results are compared with those observed at the homologous loci of some melanogaster subgroup species (D. melanogaster and its sibling species), in which, however, the hsp70 locus is duplicated, and with the more distantly related Dipteran Anopheles albimanus. Received: 22 May 1998; in revised form: 18 September 1998 / Accepted: 21 September 1998