A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.     

Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.     

DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

Chromosomal location of the 28S ribosomal RNA gene of channel catfish by in situ polymerase chain reaction

This study describes the use of the polymerase chain reaction for physical mapping of fish genes. A 287–base pair (bp) fragment of the 28S ribosomal RNA gene (28S rDNA) of channel catfish Ictalurus punctatus was isolated and sequenced with human-derived primers. The nucleotide (nt) sequence of this fragment was 20 bp shorter than that of the corresponding region of the human 28S rDNA. The gene was mapped to chromosomes of channel catfish by fluorescence in situ hybridization (FISH) and in situ polymerase chain reaction (ISPCR). A major locus and a minor locus of 28S rDNA were found on chromosomes of channel catfish. The major locus was associated with the active nucleolus organizer region (NOR) sites. The minor locus was highly resolved and not detectable by silver staining, suggesting that this locus was not involved in synthesis of ribosomal RNA and possessed fewer copies of 28S rDNA. Both loci contained GC-rich DNA elements that could be components of 28S rDNA repeated units. In this study, a potential method of comparative mapping of the channel catfish genome has been presented by using human-derived oligonucleotide sequences. These data demonstrate that ISPCR is highly specific and will be useful in physical mapping of fish genomes.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster.

The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers and analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy.     

Molecular phylogenetic analysis of ant subfamily relationship inferred from rDNA sequences

The relationships among ant subfamilies were studied by phylogenetic analysis of rDNA sequences of 15 species from seven subfamilies. PCR primers were designed on the basis of the rDNA sequence of the Australian bulldog ant, Myrmecia croslandi, previously determined. Phylogenetic trees were constructed using sequences of a fragment of 18S rDNA (1.8 kb), a fragment of 28S rDNA (0.7 kb excluding variable regions) and a combination of the 18S and 28S rDNAs, by neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). rDNA sequences corresponding to the same fragments from three non-ant hymenopteran species (a sawfly, a bee and a wasp) were employed as outgroups. These trees indicated that the ant subfamilies were clustered singly, and, among the seven subfamilies examined, Ponerinae and six other subfamilies are in a sister-groups relationship. The relationship among the six subfamilies, however, was not clarified. The phylogenetic trees constructed in the present study are not in contradiction to the tree from cladistic analysis of morphological data by Baroni Urbani et al. (1992) and the tree from morphological and molecular data (Ward and Brady, 2003), but are inconsistent with the traditional phylogeny. The present results thus raise a question as to the status of some traditionally employed "key" morphological characters. The present results also call for a reexamination of Amblyopone traditionally treated as a member of Ponerinae as belonging to a new subfamily.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Identification of rDNA-specific non-LTR retrotransposons in Cnidaria

Ribosomal RNA genes are abundant repetitive sequences in most eukaryotes. Ribosomal DNA (rDNA) contains many insertions derived from mobile elements including non-long terminal repeat (non-LTR) retrotransposons. R2 is the well-characterized 28S rDNA-specific non-LTR retrotransposon family that is distributed over at least 4 bilaterian phyla. R2 is a large family sharing the same insertion specificity and classified into 4 clades (R2-A, -B, -C, and -D) based on the N-terminal domain structure and the phylogeny. There is no observation of horizontal transfer of R2; therefore, the origin of R2 dates back to before the split between protostomes and deuterostomes. Here, we in silico identified 1 R2 element from the sea anemone Nematostella vectensis and 2 R2-like retrotransposons from the hydrozoan Hydra magnipapillata. R2 from N. vectensis was inserted into the 28S rDNA like other R2, but the R2-like elements from H. magnipapillata were inserted into the specific sequence in the highly conserved region of the 18S rDNA. We designated the Hydra R2-like elements R8. R8 is inserted at 37 bp upstream from R7, another 18S rDNA-specific retrotransposon family. There is no obvious sequence similarity between targets of R2 and R8, probably because they recognize long DNA sequences. Domain structure and phylogeny indicate that R2 from N. vectensis is the member of the R2-D clade, and R8 from H. magnipapillata belongs to the R2-A clade despite its different sequence specificity. These results suggest that R2 had been generated before the split between cnidarians and bilaterians and that R8 is a retrotransposon family that changed its target from the 28S rDNA to the 18S rDNA.     

Nonautonomous inverted repeat Alien transposable elements are associated with genes of both monocotyledonous and dicotyledonous plants

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5′-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.     

A cytomegalovirus DNA sequence containing tracts of tandemly repeated CA dinucleotides hybridizes to highly repetitive dispersed elements in mammalian cell genomes.

A single 880-base-pair region within the genome of simian cytomegalovirus strain Colburn contains sequences that hybridize intensely with both human and mouse total genome DNA probes. This sequence was also found in a second simian cytomegalovirus isolate and was retained in both plaque-purified virus subclones and in plasmid DNA clones containing the SalI P fragment. Cleaved genomic DNAs from several mammalian species all exhibited strong dispersed hybridization with the SalI-P probes, and over 70% of the lambda clones in a mouse genomic library plus several selected clones containing globin, 45S rDNA, or 5S rDNA genes all formed hybrids with SalI-P. The appropriate region of cytomegalovirus SalI-P contains relatively A + T-rich unique sequences interrupted by three stretches of the simple alternating dinucleotides, (CA)15, (CA)22, and (CA)21, which we show to be responsible for most of the cell-virus homology. We conclude that discrete, tandemly repeated (CA) dinucleotide tracts capable of forming left-handed Z-DNA helices punctuate mammalian genomes at greater than 10(5) copies per cell and that three adjacent copies of what appear to be a family of interspersed repetitive elements containing these (CA)n stretches are carried in the genomes of simian cytomegaloviruses.     

Interdigitated arrangement of two oligo(A)-terminated DNA sequences in Drosophila.

A cluster of repeated sequences composed of three distinguishable units has been isolated from Drosophila melanogaster, and characterized. The region, cloned as pDmI 158, contains a segment that is homologous to the type 1 ribosomal insertions, a member of the F family of transposable sequences, and a newly described repeated sequence that we have named G. F elements are transposable sequences that lack terminal repeats, generate target site duplications at the point of insertion, and contain an oligo(A) stretch at one end. G sequences are structurally similar though non-homologous to F in that they also carry an oligo(A) stretch. The structure of the 158 region of the genome is best explained by assuming three consecutive events. An F element did insert into a ribosomal insertion-like sequence, followed by the introduction of a G sequence into F. Subsequently, a DNA segment comprising a portion of G and F was tandemly triplicated to yield the arrangement observed. The nested interspersion of repeated sequence elements may be a common feature of eukaryotic genomes.     

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.     

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.