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.     

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.     

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.     

Sequence arrangement of the rRNA genes of the dipteran Sarcophaga bullata

Velocity sedimentation studies of RNA of Sarcophaga bullata show that the major rRNA species have sedimentation values of 26S and 18S. Analysis of the rRNA under denaturing conditions indicates that there is a hidden break centrally located in the 26S rRNA species. Saturation hybridization studies using total genomic DNA and rRNA show that 0.08% of the nuclear DNA is occupied by rRNA coding sequences and that the average repetition frequency of these coding sequences is approximately 144. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA was determined by the examination of the structure of rRNa:DNA hybrids in the electron microscope. Long DNA strands contain several gene sets (18S + 26S) with one repeat unit containing the following sequences in order given: (a) An 18S gene of length 2.12 kb, (b) an internal transcribed spacer of length 2.01 kb, which contains a short sequence that may code for a 5.8S rRNA, (c) A 26S gene of length 4.06 kb which, in 20% of the cases, contains an intron with an average length of 5.62 kb, and (d) an external spacer of average length of 9.23 kb.     

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.     

Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

Ribosomal genes of the loach Misgurnus fossilis L. Isolation and restriction analysis

The ribosomal DNA of the teleost fish--loach has been isolated from sperm DNA by CsCl density gradient centrifugation. The rDNA sediments on density gradients by two heavy satellites beta = 1.715 and greater than 1.720. The DNA of the first satellite (1.715) was separated and treated by restrictases EcoRI and BamHI. It was shown that there are two EcoRI-sites in rDNA of loach, locating in 18S and 28S rRNA coding sequences. From tandem of repeating ribosomal genes EcoRI cuts out the fragment with homogeneous length-3 megadaltons (constant fragment) and heterogeneous population of fragments 11-13 megadaltons (major) and 7-8 megadaltons (minor fraction). The constant fragment contains mostly 28S coding sequence, and the heterogeneous fragment--18S coding sequence. The data indicate that the ribosomal genes of the loach as well as other higher eucaryotes were organized in genome as tandem of repeating units with heterogeneous length (10-16 megadaltons, 14.5-24 kb) due to heterogeneity of the length of nontranscribed spacer.     

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.     

Primary structure and sequence organization of the 16S–23S spacer in the ribosomal operon of soybean (Glycine max L.) chloroplast DNA

Summary The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from soybean chloroplasts has been determined. The spacer region is over 3000 bp long and contains two tRNA genes, coding for rRNA^Ile and tRNA^Ala which contain intervening sequences of 953 and 811 base pairs respectively. There is a strong homology between the two introns suggesting that they have a common origin. These spacer tRNAs are synthesized as part of a kb precursor molecule containing 16S and 23S rRNA sequences.     

Sequence analysis of the transcribed and 5'' non-transcribed regions of the ribosomal RNA gene in Dictyostelium discoideum

The nucleotide sequence of Dictyostelium discoideum rDNA extending over almost the entire transcribed region and a part of the 5' non-transcribed spacer region has been determined. Computer analysis revealed that there were several conserved sequences in the 17S, 5.8S and 26S coding regions when compared with the sequences at analogous positions in some eukaryotic rRNA genes. The data also showed that the D. discoideum rDNA contains several extra sequences, which have not been found in other eukaryotes' rDNAs , near the 3' terminus of the 17S coding region and the 5' terminus of the 26S coding region.     

Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes

We have determined the complete nucleotides sequence (2168 bases) of the immunoglobulin mu gene cloned from newborn mouse DNA. The cloned 13kb fragment contained the entire constant region gene sequence that is interrupted by three intervening sequences at the junction of domains as previously shown in the gamma 1, gamma 2 b and alpha genes. The amino acid sequence predicted by the nucleotide sequence agrees with that of the mu chain secreted by a myeloma MOPC104E except for 8 residues out of 448 residues. The homologous domains of the mu, gamma 1 and gamma 2b genes are more similar to each other than the different domains of the mu genes are. The result implicates that the class of the immunoglobulin heavy chain genes diverged after the heavy chain genes established the multi-domain structure. The short intervening sequences of the mu and gamma genes are more conserved than the coding sequences except for the COOH-terminal domains. The results implicate that the nucleotide sequence of the intervening sequence is under selective pressure, possibly to maintain a secondary structure of the nuclear RNA to be spliced.