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.     

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.     

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.     

Cloning and polypeptide analysis of the leading region in F plasmid DNA transfer

A segment of the F plasmid DNA, located between the origin of transfer and the primary F replication region, is the first to enter the recipient cell during conjugation.PstI,SalI, andSmaI restriction endonuclease sites have been mapped within this leading region in conjugational DNA transfer and chimeric plasmids carrying overlapping fragments of the region have been constructed. Analyses of polypeptides synthesized by maxicells carrying these chimeric plasmids have shown four new polypeptides ofM_r 27,800, 23,100, 14,400, and 11,000 to be encoded by sequences within the leading region.     

Evidence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA

Closely linked genes for 18S and 5S rRNAs have been located on four different cloned SalI restriction fragments of wheat (Triticum aestivum L.) mitochondrial DNA. Restriction analysis has revealed that in each of the cloned fragments, the 18S and 5S rRNA genes are contained within the same basic structural unit, R, which is at least 4 kbp long. This unit is flanked by sequences designated u (0.8 kbp), v (13.7 kbp), w (0.7 kbp), and y (1.4 kbp), in the orientations v-R-w, v-R-y, u-R-w, and u-R-y in the four different SalI fragments. We conclude that 18S + 5S rRNA genes are located at several distinct sites in the wheat mitochondrial genome, and suggest that reciprocal intra- and/or intermolecular recombination between such repeated sequences could promote extensive genomic rearrangement and thus contribute to the physical heterogeneity that is a hallmark of most plant mitochondrial DNAs.     

The chloroplast genome of an exsymbiotic Chlorella-like green alga

Chloroplast DNA was isolated and cloned from Chlorella, strain N1a, exsymbiotic with Paramecium bursaria. BamHI, SalI, SstI, KpnI and XhoI restriction fragments of the DNA were assembled into a circular map. The genome consists of approximately 120 kbp of DNA, has a G/C content of 38%, and contains only a single copy of the rRNA cistron. The rRNA cistron is small, 5000–8000 bp, and the 16S and 23S genes are separated by less than 2000 bp.     

Localization of the genes coding for the 51 kDa PSII chlorophyll apoprotein,apocytochrome b6, the 65–70 kDa PSI chlorophyll apoproteins and the 44 kDa PSII chlorophyll apoprotein in pea chloroplast DNA

Summary DNA probes isolated from previously mapped spinach genes were used to locate 5 genes on pea ctDNA by heterologous hybridization. The genes mapped include psbC, psaA, psaB, psbB, and petB. PsbB and petB mapped to a 6.7 kbp XbaI DNA fragment adjacent to the petD gene. Northern probes from within the DNA which codes for psbB and petD hybridized to 6 RNAs ranging from 1.2 to 5.6 kbp. The psaA and psaB genes, which code for 65–70 kDa proteins of Photosystem I, were mapped to a 7.5 kbp. XbaI DNA fragment. A 5.8 kbp RNA is transcribed from the region which contains the psaA and psaB genes suggesting that these genes are co-transcribed. Finally the psbC gene which codes for a 44 kDa chlorophyll-protein of Photosystem II was mapped to a 12.3 kbp PstI DNA fragment. The pea psbC open reading frame overlaps the psbD coding sequence and this gene pair is within 3 kbp of the psaA-psaB genes. Overall, the organization of the 3 gene clusters analyzed in peas is similar to that reported for spinach.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

Chloroplast genome differences between Paspalum dilatatum Poir and the related species P. notatum Flugge

 Chloroplast DNA (cpDNA) of Paspalum dilatatum and P. notatum was digested singly or in combination with the restriction endonucleases PstI, PvuII, SalI, KpnI and XhoI. Data obtained from filter hybridization experiments with barley and wheat cpDNA probes were used to construct restriction site maps of the chloroplast genomes of the Paspalum species. The cpDNA fragments were ordered into a circular configuration of approximately 139.3 kbp that contained two inverted repeat regions of approximately 23 kbp and a small and large single-copy region of approximately 11 kbp and 83 kbp, respectively. The cpDNA maps showed that P. dilatatum and P. notatum shared a large number of restriction sites with the proportion of shared restriction sites S=0.90. No restriction site differences were detected in the KpnI maps. Eight species-specific restriction site differences that could be used to identify the cytoplasm of each Paspalum species were identified in the PstI, PvuII, SalI, and XhoI cleavage maps. The overall structural organization of the Paspalum cpDNAs is rather similar to those of most cpDNAs from other plants. The results presented in this study will be of value for exploring further phylogenetic relationships within the genus Paspalum. Received: 27 February 1997 / Accepted: 7 March 1997     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

The physical map of the chloroplast DNA from Asparagus officinalis L.

The genus Asparagus consists of 100–300 species of both dioecious and hermaphrodite plants. Since there are diploid, tetraploid, and hexaploid plants in this genus, RFLP (restriction fragment length polymorphism) analysis of chloroplast DNA (ctDNA) is suitable for examining the phylogenetic relationships. We have constructed a physical map of the ctDNA of garden asparagus (A. officinalis L. cv Mary Washington 500 W) using five restriction endonucleases, namely, BamHI, PstI, SalI, HindIII, and XhoI. Asparagus ctDNA was digested with restriction enzymes and cloned into plasmid and phage vectors, and a clone bank was constructed that covered 70% of the genome. A physical map was constructed by Southern hybridization of total DNA from asparagus with homologous and heterologous probes. The asparagus ctDNA was about 155 kb long and it contained two inverted repeats (23kb each) separated by a large single-copy region (90kb) and a small single-copy region (19kb). Fifteen genes, encoding photosynthesis-related proteins, rDNAs, and tRNAs, were localized on the physical map of asparagus ctDNA. Comparing the length and the gene order of asparagus ctDNA with that of other plants, we found that asparagus ctDNA was similar to tobacco ctDNA but different from rice ctDNA. The restriction patterns of the ctDNAs from several varieties of A. officinalis and three species of Asparagus were analyzed. The restriction patterns of the varieties of A. officinalis were very similar, but polymorphisms were detected among the three species of Asparagus.     

Effect of DNA methylation on protein-DNA interaction of HL-60 cells

HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.     

Effect of DNA methylation on protein-DNA interaction of HL-60 cells

HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as inducer in the presence of optimum concentration of 10 pmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonucleaseHae III,Sma I,Sal I,XhoI andHind III which are sensitive to 5-methyicytoside were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.     

Cloning and characterization of the xyl genes from Escherichia coli

Summary Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase (xylA), xylulokinase (xylB), and regulatory (xylR) or transport (xylT) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10–15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp BglII fragment and a 2.6 kbp HindIII-SalI fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

A physical map of the sorghum chloroplast genome

Summary The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the bcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.USDA-ARS     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.