Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

Sequence analysis of the junctions between a large inverted repeat and single-copy regions in tobacco chloroplast DNA

Summary Tobacco chloroplast DNA contains a large inverted repeat sequence of 26 kilobase pairs (kbp). The inverted repeat is separated by 20 kbp small single-copy and 90 kbp large single-copy regions. We have cloned four DNA fragments containing each junction between the inverted repeat and the single-copy regions. The sequence analysis revealed the exact edges of the inverted repeat. A putative coding region for a ribosomal protein CS19 was found 4 base pairs (bp) away from the inverted repeat on the left margin of the large single-copy region. A sequence AGGAG, which is complementary to the 3 terminal sequence of tobacco chloroplast 16S rRNA, was found within the inverted repeat. A tRNA^His gene was found 5 bp away from the inverted repeat on the right-hand margin of the large single-copy region.     

Construction of a SalI/PstI restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis

The restriction endonucleases SalI and PstI cleave circular chloroplast DNA of spinach (Spinacia oleracea) into 12 and 10 fragments, respectively. The sum of the fragment sizes in each of the series is equivalent to the contour length of the molecule (about 95 Md). A physical map was constructed by sequential digestions using low-gelling-temperature agarose to avoid the necessity of extracting the fragments from the gel. The circular DNA molecule of spinach chloroplasts consists of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single-copy regions of different sizes (about 52 and 13 Md).     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.     

The chloroplast genome of a chlorophylla+c-containing alga,Odontella sinensis

The chloroplast genome of a marine centric diatom,Odontella sinensis, was cloned and sequenced. The circular genome is 119,704 bp in length (AC=Z67753;). It contains an inverted repeat sequence of 7,725 bp separating two single-copy regions of 38,908 and 65,346 bp, respectively, and 174 genes and open reading frames, of which nine are duplicated within the inverted repeat segments.     

Distribution of inverted repeat sequences in nuclear DNA from Physarum polycephalum.

Inverted repeat sequences, capable of forming stable intra-chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7,000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as 'bubbled' hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non-foldback sequences of variable sizes up to 15,000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single-chain segments of DNA, are not random. Instead, they appear to form a regular, arithmetic series of lengths. These observations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and unform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases extend over distances up to 50,000 nucleotides in length.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

The replication origin of proplastid DNA in cultured cells of tobacco

Summary When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.     

Eucaryotic transposable genetic elements with inverted terminal repeats

DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.     

Organisation of inverted repeat sequences in hamster cell nuclear DNA.

Hamster cell nuclear DNA is shown to contain inverted repeat (foldback) sequences, in some respects similar to the foldback fraction in DNA from other animal cell types. Using electron microscopy the majority of foldback duplexes are shown to be located in simple hairpin-like DNA structures, formed from individual pairs of complementary inverted repeated sequences 50--1000 nucleotides in length, in some cases arranged in tandem, and in other cases separated by intervening sequences, up to 16000 nucleotide residues long. In addition, a novel class of foldback structure, referred to as 'bubbled hairpins' is reported, which appear to be formed from clusters of inverted repeat sequences that are separated from adjacent clusters of complementary inverted repeats by large intervening sequences which vary in length from 5000 to over 20000 nucleotide residues. Due to the special pattern of distribution of these latter inverted repeat sequences, 'bubbled hairpins' are observed only in long foldback DNA. Evidence is presented that the distribution of foldback sequences in hamster cell DNA is highly ordered. The lengths of the intervening single chains in foldback structures appear to vary non-randomly. This gives rise to a localised periodic pattern of organisation that is believed to be a consequence of regular alternating arrangements of foldback and non-foldback sequences in the segments of DNA from which foldback structures are derived.     

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley-Liss, Inc.     

A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase

Summary The restriction endonucleases SalGI and PstI have been used to construct a physical map of wheat ctDNA. The molecule was found to contain approximately 135 kbq, and in common with many other higher plant ctDNAs about 15% of the sequences are repeated in an inverted orientation. It was established by electron microscopy that, in wheat, each segment of the inverted repeat contains 21.0 kbp, and that the single copy regions separating the two repeated segments contain 12.8 kbp and 80.2 kbp. Blot hybridisation showed that one set of ribosomal genes is located in each segment of the inverted repeat region and the sizes of these genes were accurately determined by measuring the dimensions of hybrids between the chloroplast rRNAs and the identified Sal and Eco fragments on electron micrographs: the genes for the 16S and 23S rRNAs contain 1530 bp and 2850 bp respectively and are separated by a spacer region of 2350 bp. The Bgl fragment of maize ctDNA known to contain the structural gene for the large-subunit (LS) of ribulose 1,5-bisphosphate carboxylase was used as a probe to locate the LS gene in wheat ctDNA. A small (2.8 kbp) Eco fragment was found to contain most of the wheat LS gene and is derived from the larger single-copy region, 23.5 kbp away from one segment of the inverted repeat and 54.8 kbp from the other.     

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

Summary The 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNA^Ala and tRNA^Ile, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.     

Deoxyribonucleic acid sequence organization of a yeast plasmid.

Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-mum DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-mum DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed.     

The chloroplast DNAs of flax genotrophs

The chloroplast DNA of L. usitatissimum var. Stormont Cirrus has been mapped with respect to the recognition sites for the enzymes SalP1, Sst1 and SalG1. It is a circular molecule of about 160 kilobasepairs, with an inverted repeat containing the rDNA. Comparisons between chloroplast DNA of uninduced and induced flax genotrophs show there to be no major structural differences.     

Intramolecular integration within Moloney murine leukemia virus DNA

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features.