A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.     

Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.     

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Adjacent repeating units of xenopus laevis 5S DNA can be heterogeneous in length

The distribution of length heterogeneity in adjacent repeating units of X. laevis 5S DNA has been examined by “cloning” 5S DNA in bacteria. Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E. coli. Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length. This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs. The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

Conservation of sequences in related genomes of Apodemus: constraints on the maintenance of satellite DNA sequences.

Satellites from two related species of the Apodemus genus, A. sylvaticus and A. flavicollis, have been analysed with restriction enzymes Taq I, Alu I and Hind III. The restriction maps are closely conserved between species and show a novel feature of two differing internal periodicities within a 375 base pair repeating unit detected by two different restriction enzymes. This places constraints on the introduction of the observed restriction sites according to current models such as unequal crossing-over. The implications of such a conserved sequence and its presence in other species are discussed.     

Characterization of the rDNA repeat units in the MitchellPetunia genome

The principal rDNA repeating unit in the MitchellPetunia genome has a length of 8.5 kb. In addition there is a major variant of length 9.7 kb, and two minor variants of 9.3 kb and 10.4 kb. The size heterogeneity of the rDNA repeating units results from length differences in the non-transcribed spacer regions. These differences may reflect simple insertions into the non-transcribed spacer region of the major ‘short’ repeat; however, additional sequence changes have occurred since the ‘short’ repeat is characterized by restriction endonuclease cleavage sites which are absent in the longer variant units.     

Restriction endonuclease cleavage map of mitochondrial DNA from Aspergillus nidulans.

Mitochondrial DNA of the ascomycete fungus Aspergillus nidulans, a circular molecule of 31 500 base pairs, is cleaved by restriction endonucleases Eco R I, Hind II, Hind III and Bgl II into 3, 7, 9 and 5 fragments, respectively. The relative positions of the cleavage sites could be mapped by analysis of fragments obtained by double enzyme digestions of whole DNA and by complete and partial redigestion of isolated restriction fragments.     

Ribosomal RNA genes in biotypes ofScilla peruviana (Liliaceae)

Scilla peruviana biotypes have different chromosome numbers due to changes in the nucleolar chromosomes and polyploidy. We have examined two diploid (2n = 15 and 2n = 16) and two tetraploid biotypes (2n = 28 and 2n = 32). From the results of rRNA/DNA filter hybridizations it appears that rDNA percentages of the diploid biotypes are, approximately, 2.2-fold higher than those of the tetraploid biotypes. To examine the rRNA gene structure we have utilizedSouthern blot hybridization after DNA digestions with three restriction enzymes: Eco RI, Hind III and Bam HI. From the band analysis of both single and double digestions it has been possible to reveal the presence, in the diploid biotypes, of three gene types, heterogeneous both for length and for nucleotide sequences in the external spacer. The three rRNA genes are 12 600, 12 700, and 12 800 base pairs long and they have a different position of the Hind III sites in the external spacer. On the other hand, a single gene type of 12 600 base pairs, identical to the first type of the diploid biotypes, surprisingly exists in the tetraploid biotypes. Considerations on the rRNA gene regulation and evolution are made.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes.

Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: Exhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde cross-linked into any SDS-resistant nucleoprotein structure nor topologically "locked" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. These findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the "late" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al., 1978).     

Length variation in the non-transcribed spacer of Calliphora erythrocephala ribosomal DNA is due to a 350 base-pair repeat

The non-transcribed spacer regions in the ribosomal DNA cistrons of Calliphora erythrocephala vary in length. This length variation is shown to be due to variable numbers of small repeated units found in certain areas of the NTS² regions, as has been found in several other systems. In contrast to the other organisms, however, the length variation in C. erythrocephala leads to the formation of only two major size classes with a length difference of about 1 kb. We have investigated the nature of this length difference by means of electron microscope heteroduplex and restriction enzyme digestion analyses. We demonstrate that the length variation in C. erythrocephala is due to a variation in the number of 350 bp repeating units defined by the presence of sites for three restriction enzymes, HhaI, Sau3A and AluI. Furthermore, the existence of an XbaI site within one 350 bp unit and the lack of a HhaI restriction site within another 350 bp unit reveals that at least some sequence differences exist among the repeating units. These restriction site differences can be viewed as genetic markers and lead to the hypothesis that the longer NTS class originated from the shorter NTS class by an unequal crossover event that served to duplicate the region containing repetitive units. An interesting feature of our observation is that not all NTS length classes are equally represented, suggesting special rules for the unequal recombination events often hypothesized as the basis for such variation.