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.     

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.     

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.     

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.     

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.     

Amplification of rDNA and type I sequences in Drosophila males deficient in rDNA

rDNA magnification is a heritable change in rDNA content that occurs in D. melanogaster males when chromosomes deficient in rDNA are placed together for several generations. We have examined the restriction endonuclease cleavage pattern of the rDNA from an X chromosome undergoing magnification, and find no evidence for the selective amplification of either uninterrupted rDNA units or those containing insertion sequences. In addition, we observe an amplification of rDNA in the first generation of extremely bobbed male progeny to a level exceeding that of wild-type flies, but that reduces to the wild-type level in subsequent generations. The type I rDNA insertion elements also occur as tandem arrays, independently of rDNA. Southern hybridizations indicate that the majority of these sequences are located in the heterochromatin surrounding the nucleolus organizer on the X chromosome, and we find that they, too, amplify transiently in the first generation of magnifying males.     

Widely differing degrees of sequence conservation of the two types of rDNA insertion within the melanogaster species sub-group of Drosophila

We have examined the distribution of sequences homologous to the type I and type II rDNA insertions of Drosophila melanogaster in its sibling species. Each of the six species we have examined has sequences homologous to the type I insertion, which have undergone extensive divergence by the criterion of their EcoRI, BstI and HindIII restriction patterns. We have isolated cosmid clones containing type I sequences from D. simulans and D. mauritiana, the two species most closely related to D. melanogaster. Southern hybridisation analysis of these clones indicates that, as in D. melanogaster, the type I sequences can exist independently of rDNA and can also dissociate to give sub-components homologous to the right hand segment of the D. melanogaster type I insertion. The type II sequences, on the other hand are present in five out of the six species, but their restriction endonuclease cleavage profile is highly conserved. The differences in the degree of conservation of the two types of insertion sequence are discussed.     

Intervening sequences in the ribosomal RNA genes of Ascaris lumbricoides: DNA sequences at junctions and genomic organization

An rDNA size class in the genome of the nematode Ascaris lumbricoides is described which is interrupted by a 4.5-kb long intervening sequence located in the 26S coding region. This molecular form occurs in approximately 15 copies per haploid genome and amounts to approximately 5% of the total nuclear rDNA. Intervening sequences are present only in the 8.8-kb rDNA, but not in the 8.4-kb rDNA repeating units of A. lumbricoides. Cloning of the interrupted rDNA units revealed, in addition to the main 4.5-kb insertion, shorter intervening sequences of 4-kb and 119-bp length. Both shorter rDNA forms are present in the single copy range of the haploid genome. Sequence analyses of the intervening sequence/rDNA junctions show an identical right-hand junction for all of the three different rDNA forms. The two shorter intervening sequences are a coterminal subset of the right-hand end of the main 4.5-kb insertion, whereas all three insertions have a different left-hand junction with the coding region of rDNA. Each intervening sequence is flanked by a short direct repeat of variable length, being only once present in the uninterrupted rDNA. The intervening sequences of A. lumbricoides show striking similarity to the organization of type I insertion family in dipteran flies, even though they are inserted at different positions in the 26S coding region. Additional rDNA intervening sequences may be present outside of the rDNA cluster, but in not more than 15-20 homologous copies per haploid genome.     

The Integration Machinery of ZAM, a Retroelement from Drosophila melanogaster, Acts as a Sequence-Specific Endonuclease

Retroviruses and retrotransposons insert into the host genome with no obvious sequence specificity. We examined the target sites of the retroelement ZAM by sequencing each host-ZAM junction in the genome of Drosophila melanogaster. Our overall data provide compelling evidence that ZAM integration machinery recognizes and leads to ZAM insertion into the sequence 5'-GCGCGCg-3'. This unique property of ZAM will facilitate the development of new tools to study the integration process of retroelements.     

Identification of a new hobo element in the cabbage moth, Mamestra brassicae (Lepidoptera)

A complete hobo-like element, called Mbhobo, was identified in the cabbage moth, Mamestra brassicae. This element has a high sequence similarity to the HFL1 hobo element of Drosophila melanogaster. Amplification of Mbhobo termini indicated that transposition occurred into a 5'-GTGGGTAC-3' target sequence that was duplicated upon insertion. This target site conforms to the consensus sequence established for the insertion sites of insect hAT elements. Mbhobo has a single 1935 bp long ORF with significant homology to the D. melanogaster HFL1 hobo transposase. FISH experiments evidenced Mbhobo clusters located in heterochromatic regions of Z and W sex chromosomes and in heterochromatic areas of chromosome pair 10.     

The sequence of the 3'' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.     

The controlling element Ds at the Shrunken locus in Zea mays: structure of the unstable sh-m5933 allele and several revertants

We have analyzed the structure of the Shrunken (Sh) locus in a strain containing an unstable recessive mutation, sh-m5933, caused by the transposable controlling element Dissociation (Ds). We have also analyzed nine spontaneous Sh revertant alleles. The sh-m5933 allele contains a 30 kb insertion at the Sh locus, as well as a duplication that includes part of the insertion and the Sh locus sequence on the 5' side of the insertion site. The revertants continue to show Ds-mediated chromosome breakage at the Sh locus, have an intact Sh locus from which the insertion has been excised, and retain the duplication. One of the nine revertant alleles has a 2 kb deletion at the junction between the Sh locus and the insertion sequence in the duplicated segment of the locus. The revertant also shows a temporal change in the pattern of somatic chromosome breakage, implicating the junction sequence as the site of Ds-mediated chromosome breakage.     

Nucleotide sequences at the boundaries between gene and insertion regions in the rDNA of Drosophila melanogaster

Ribosomal RNA genes interrupted by type 1 insertions of 1 kb and 0.5 kb have been sequenced through the insertion region and compared with an uninterrupted gene. The 0.5 kb insertion is flanked by a duplication of a 14 bp segment that is present once in the uninterrupted gene; the 1 kb insertion is flanked by a duplication of 11 of these 14 bp. Short insertions are identical in their entire length to downstream regions of long insertions. No internal repeats occur in the insertion. The presence of target site duplications suggests that type 1 insertions arose by the introduction of transposable elements into rDNA. Short sequence homologies between the upstream ends of the insertions and the 28S' boundaries of the rRNA coding region suggest that short type 1 insertions may have arisen by recombination from longer insertions.We have sequenced both boundaries of two molecules containing type 2 insertions and the upstream boundary of a third; the points of interruption at the upstream boundary (28S' site) differ from each other in steps of 2 bp. Between the boundary in the 0.5 kb type 1 insertion and the type 2 boundaries there are distances of 74, 76, and 78 bp. At the downstream boundary (28S' site) the two sequenced type 2 insertions are identical. The rRNA coding region of one molecule extends across the insertion without deletion or duplication, but a 2 bp deletion in the RNA coding region is present in the second molecule. Stretches of 13 or 22 adenine residues occur at the downstream (28S') end of the two type 2 insertions.     

Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt.

It is well known that the local conformation of a segment of DNA is dependent upon both the sequence of the segment and the conditions under which the DNA is prepared. In extreme cases, the DNA may contain regions of both right and left-handed conformations, mandating the existence of a conformational junction between the two. These B-Z junctions have been observed in plasmids but, to date, no model systems have been characterized to determine the molecular nature of these junctions. Preliminary CD, UV, and NMR studies on such a model are presented here. A 16 base pair oligonucleotide, containing a potential B-Z junction, has been synthesized and characterized by the above techniques. The results suggest that this molecule contains both right and left-handed conformations under condition of high salt, and thus a B-Z junction.     

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.     

Introns and their flanking sequences of Bombyx mori rDNA.

We obtained two different clones (16 kb and 13 kb) of B. mori rDNA with intron sequence within the 28S-rRNA coding region. The sequence surrounding the intron was found to be highly conserved as indicated in several eukaryotes (Tetrahymena, Drosophila and Xenopus). The 28S rRNA-coding sequence of 16 kb and 13 kb clone was interrupted at precisely the same sites as those where the D. melanogaster rDNA interrupted by the type I and type II intron, respectively. The intron sequences of B. mori were different from those of D. melanogaster. In 16 kb clone, the intron was flanked by 14 bp duplication of the junction sequence, which was also present once within the 28S rRNA-coding region of rDNA without intron. This 14 bp sequence was identical with those surrounding the introns of Dipteran rDNAs.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.     

Genome sequencing of a 239-kb region of rice chromosome 10L reveals a high frequency of gene duplication and a large chloroplast DNA insertion

In this study we describe a 239-kb region on the long arm of rice chromosome 10 that contains a high density (71%) of locally duplicated genes, including 24 copies of a glutathione S-transferase gene. Intriguingly, embedded within this cluster is a large insertion (approximately 33 kb) of rice (Oryza sativa) chloroplast DNA that is derived from two separate regions of the chloroplast genome. We used DNA fiber-based fluorescence in situ hybridization (fiber-FISH) analyses of O. sativa spp. japonica nuclei to confirm that the insertion of organellar DNA was not a cloning artifact. The sequence of the chloroplast insertion is nearly identical (99.7% identity) to the corresponding regions in the published rice chloroplast genome sequence, suggesting that the transfer event occurred recently. PCR amplification and sequence analysis in two subspecies of rice, O. sativa spp. japonica and spp. indica, indicates that the transfer event predated the divergence of these two subspecies. The chloroplast insertion is flanked by a 2.1-kb perfect direct repeat that is unique to this location in the rice genome.