Isolation and mapping of ribosomal RNA genes of Caulobacter crescentus

Ribosomal DNA fragments of 1.0, 3.4, 3.7 and 6.1 kb² produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RNA probe. These genomic sequences were further characterized by the isolation of 13 hybrid λ Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Cc1880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to ³²P-labeled rRNA. Thus, the two clones contain different DNA inserts which together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S-5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 × 10⁹ daltons. The relatively simple organization of rDNA sequences in the C. crescentus chromosome compared to Escherichia coli is discussed.     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

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.     

Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.     

Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes³, each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly ³²P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule.When heat-denatured rDNA was rapidly cooled under conditions in which no renaturation would occur between separated complementary strands of DNA, molecules of half the size of the original rDNA molecule were produced. These were double-stranded DNA molecules as evidenced by their resistance to digestion with S1 nuclease. Moreover, they could be digested with EcoRI to produce fragments of sizes which would be predicted from the assumption that each single strand of the original rDNA molecule had folded back on itself to form a “hair-pin” double-stranded DNA structure. Hybridization experiments between ribosomal RNA and purified rDNA showed that each rDNA molecule contains two genes for rDNA. Hybridization of the isolated EcoRI fragments of rDNA with 25 S or 17 S rRNA suggested that the two structural genes for 17 S rRNA are located near the center of the rDNA molecule and the two genes for 25 S rRNA are found in distal positions.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

Mosquito ribosomal RNA genes: Characterization of gene structure and evidence for changes in copy number during development

A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been characterized by restriction mapping, Southern-blotting and S1-nuclease analyses. The 18S rRNA coding region measured 1800 bp and contained a conserved Eco RI site near the 3′-end. The 28S rRNA coding region was divided into α and β sequences, comprising 1750 and 2000 bp, respectively, which were separated by a 350 bp sequence that is removed from the rRNA precursor during processing. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region that contained a series of PvuI repeats upstream of the 18S rRNA coding sequence. During development of the mosquito, Aedes aegypti, the rRNA gene copy number per haploid genome increased from about 400 in larvae to about 1200 in adults.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Molecular systematics of Brassica and allied genera in subtribes Brassicinae, Raphaninae, Moricandiinae, and Cakilinae (Brassicaceae, tribe Brassiceae); the organization and evolution of ribosomal gene families

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of ribosomal DNA (rDNA) of Brassica and allied genera. The total genomic DNA of 95 accessions of 52 species representing 16 genera was restricted with six enzymes, and the restriction fragments were probed with three ribosomal clones (pTA71, Ver 18‐6, and Ver 6‐5). Eleven repeat unit length classes were recognized. The repeat unit size classes of 8.9 kb and 9.5 kb were observed most commonly, being represented in 17 and 14 species, respectively. The restriction enzyme SacI produced three to six (generally three) bands with detectable hybridization to the probe pTA71. This probe–enzyme combination indicated a remarkable uniformity amongst Brassica and allied genera in the coding region of repeat units. By contrast, an extensive size variation in the restriction fragments could be localized in the intergenic spacer (IGS) region. Eleven IGS‐containing length variants were detected. Complex hybridization patterns, resulting from extensive repeat unit heterogeneity and taxon‐specific methylation of one or more cleavage sites, were obtained with the EcoRI + pTA71 combination. The relative homologies between the coding regions were evident from the presence of 1.5 kb in all the taxa, and 0.4‐, 1.3‐, and 1.7‐kb fragments in 33, 27, and 24 species, respectively. The SacI + pTA71 and EcoRI + pTA71 combinations were generally able to distinguish taxa both within and between genera. Three restriction endonuclease digests probed with three ribosomal clones yielded essentially identical fragmentation patterns across all the accessions within the cultivated species Brassica campestris, B. oleracea, and B. juncea. In B. napus, three and seven accessions exhibited restriction profiles similar to one and both diploid progenitor species, respectively. Overall, rDNA repeat unit length polymorphism showed good correlation with the cytodeme‐based classification of Brassica and allied genera. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 545–557.     

Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

Molecular analysis of the histone gene cluster of Psammechinus miliaris: II. The arrangement of the five histone-coding and spacer sequences

Histone DNA of Psammechinus miliaris was obtained in an enriched form by buoyant density gradient centrifugation and was cleaved into 6 kb repeat units (Birnstiel et al., 1975a) by the action of the specific endonucleases EcoRI and HindIII. Since it was suspected that the 6 kb unit harbored all five histone-coding sequences, the histone DNA unit was subdivided into five segments with the aim of providing five fragments carrying just one coding sequence each. This was achieved by the combined use of EcoRI HindII, HindIII, and Hpa I. A physical map was constructed from the overlaps arising in these restriction experiments. Each of the five segments was shown to hybridize uniquely with just one of the five highly purified histone mRNAs (Gross et al., 1976a). By this procedure, the order of the mRNA sequences on the histone DNA was found to be a, c, d, b, e (Gross et al., 1976a), and hence of the protein coding sequences H4, H2B, H3, H2A, and H1. Further evidence is presented that the 6 kb repeat unit, amplified by means of a Murray λ vector phage, contains AT-rich DNA sequences which would be expected not to code for histone proteins.     

Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr₁ to hr₅, occur at or near the following EcoRI fragment junctions: hr₁EcoRI-B—EcoRI-I (0.0 map units); hr₂, EcoRI-A—EcoRI-J (19.8 map units); hr₃, EcoRI-C—EcoRI-G (52.9 map units); hr₄, EcoRI-Q—EcoRI-L (69.8 map units); and hr₅, EcoRI-S—EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr₂ and hr₅, respectively. The sizes of the homologous regions were about 800 base pairs for hr₂, 500 base pairs for hr₅, and less than 500 base pairs for hr₁, hr₃, and hr₄. A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.     

The Organization of rRNA Genes in Vicia faba

Vicia faba DNA was digested with restriction endonucleases andfractionated on 1% agarose gels. The physical map for EcoRI,BamHI and XbaI cleavage sites in V. faba cytosol rDNA was determinedusing the Southern blot hybridization technique. XbaI and BglIIdigestion or partial EcoRI digestion showed that the lengthof a major repeat unit of V. faba rDNA is 6.7 x 10⁶ daltons.EcoRI and BamHI cleaved this unit into two and five DNA fragments,respectively. (Received April 23, 1981; Accepted July 20, 1981)     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA0 is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10?752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100?kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180?bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80?kb?<85% <120?kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.