Restriction and gene maps of plastid DNA from Capsicum annuum

Summary Chloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to chromoplasts in Capsicum annuum does not involve any large rearrangements of the plastid chromosome. A restriction map of Capsicum annuum plastid DNA was constructed using the same restriction enzymes in single digests and in all possible pair combinations. Overlapping restriction fragments were identified by digesting each product of a single digest with each of the other three enzymes. The resulting restriction map is similar to that of chloroplast DNA from other members of the Solanaceae with respect to most restriction sites. The genome size corresponds to 143 kbp. The locations of 24 genes, coding for ribosomal RNAs and for proteins of Photosystem I (PSI), Photosystem II (PSII), ATP synthase, cytochromes, the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) (RuBPC), and ribosomal proteins were determined by probing Southern blots of Capsicum chloroplast DNA with probes of genes from spinach and tobacco. The gene locations are completely conserved with respect to those of other members of the Solanaceae and the majority of higher land plants.Abbreviations and notations atpA, atpB, atpE, and atpF genes for the , , , and I subunit, respectively, of ATP synthase - cpDNA chloroplast DNA - petA, petB, petD genes for cytochrome f, cytochrome b ₆, and subunit IV of cytochrome b ₆/f complex, respectively - psaA, psaB, psaC genes for the P₇₀₀ apoproteins - psbA gene for Q_B - psbB and psbC genes for the 51-kDa and 44-kDa proteins, respectively, of PSII - psbD gene for the Q_B-like polypeptide of PSII - psbE gene for cytochrome b ₅₅₉ - rbcL gene for the large subunit of RuBPC - rpl2 gene for ribosomal protein L2 - rpoA gene for the subunit of RNA polymerase - rps11, rps12 and rps19 genes for ribosomal proteins S11, S12, and S19, respectively - rps19 open reading frame for a protein with N terminus similar to that of S19 - RuBPC ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) - trnH gene for histidine transfer RNA - URF39 and URF509 unidentified reading frames for polypeptides of 39 and 509 amino acids, respectively Gene names follow the convention of Hallick and Bottomley (1983)     

Restriction site and genetic map of Cucurbita pepo chloroplast DNA

Summary A detailed restriction map of squash chloroplast DNA (cpDNA) was constructed with five restriction endonuclease, SalI, PvuII, BglI, SacII, and PstI. The cleavage sites were mapped by sequential digestion of cpDNA using low-gelling temperature agarose. The restriction map shows that squash cpDNA is an approximately 153 kilobase (kb) circle with a large inverted repeat sequence of 23.3 kb, separated by a large (83.7 kb) and a small (22.7 kb) single copy region. Genes for a number of chloroplast polypeptides were localized on the map by hybridizing the cpDNA restriction fragments to heterologous gene-specific probes from tobacco, pea, tomato, maize, and spinach chloroplasts. The gene locations and organization of squash cpDNA are highly conserved and similar to chloroplast genomes of tomato, pepper, and Ginkgo.Abbreviations cpDNA chloroplast DNA - kb kilobases - IR inverted repeat. Gene names follow the nomenclature recommendation of Hallick and Bottomley (1983)     

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

Summary A 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.     

Molecular analysis of mitochondrial DNA from tomato cell suspension cultures

Summary Mitochondrial DNA (mtDNA) from Lycopersicon esculentum was purified from cell suspension cultures. The DNA, isolated from mitochondria purified by two successive sucrose density gradients, was uncontaminated with nuclear DNA or DNA from proplastids. The total molecular weights of BamHI, BglI, and BglII fragments indicate a mitochondrial genome size of at least 270 kb. Cross hybridization between tomato mtDNA and cloned spinach plastid genes revealed some homology. In hybridization experiments using cloned mitochondrial rRNA genes and BamHI digested total mtDNA the presence of recombination repeats is demonstrated.     

Deleted forms of plastid DNA in albino plants from cereal anther culture

Summary Wheat and barley albino plants derived from anther culture contain plastid genomes which have suffered deletion. DNA molecules of the size of unit genomes exist in these plants. In some cases these may be linear genomes. In all cases a region near the T8 fragment of barley or the corresponding region of the wheat plastid genome has been retained. This region may therefore represent sequences sufficient for replication.     

Cultivar variability for the presence of plastid DNA in pollen of Pisum sativum L.: implications for plastid transmission

Summary The mode of plastid transmission in the garden pea (Pisum sativum L.) was analyzed cytologically using the DNA-fluorochrome 4,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. The reproductive cells of mature pollen obtained from 12 inbred lines and cv Early Alaska were examined for the presence or absence of DAPI-stained plastid DNA aggregates. Plastid DNA was detected in all 13 pea lines examined, although there was variability with regard to the percentage of pollen graines showing plastid DNA aggregates of generative cells (ranging from 3% in accession 82-12r to 65% in accession 82-14n). These cytological results may indicate genetic variability for plastic DNA inheritance in the garden pea. This paper is dedicated to the memory of Gerald A. Marx     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

A detailed restriction endonuclease cleavage map of rat liver mitochondrial DNA

Summary A restriction endonuclease cleavage map of rat liver mitochondrial DNA is presented for the following enzymes: Xba I, Bgl II, Hae II, Bam HI, Hpa I, Hha I, Bcl I, Hind II, Hind III, Eco RI, Hpa II, Hae III, and Sau 3A. It was derived from complete and partial digestions with these enzymes, double digestions, and redigestions of defined fragments obtained with one enzyme with other restriction enzymes. By the use of these and further enzymes (Taq YI, Alu I) the mitochondrial DNA (ca. 15.6 Kb) can be dissected into a large number of defined fragments.Abbreviations mtDNA mitochondrial DNA - bp or Kbp base pairs or kilo base pairs     

Physical maps of the two circular plastid DNA molecules of the brown algaPylaiella littoralis (L.) Kjellm

Summary Two circular molecules of different sizes, both belonging to the chloroplast DNA of the brown algaPylaiella littoralis, have been observed by electron microscopy (Dalmon et al. 1983). Clone banks representing 86% of the small chloroplast circular DNA molecule (58 kbp) and 69% of the large circular DNA molecule (133 kbp) have been established and used as tools in the construction of physical maps. Two rDNA operons have been mapped in a very small inverted repeat on the large circular molecule. One 16S rRNA pseudogene and one split 23S rRNA gene have been mapped on the small DNA molecule, far apart from each other. Using heterologous probes, genes for ten different proteins have also been located on these maps. Their arrangement on the large molecule is different from that found in higher plants and algae. Probes fromrbcL, psbA andrps19 genes hybridize to several separated fragments. Two of them (psbA andrps19) hybridize to both types of molecules.     

Evidence for tissue-specific cytosine-methylation of plastid DNA from Zea mays

Summary Total DNA isolated from leaves, etiolated seedlings, roots, endosperm or embryos of Zea mays was digested separately with each of the restriction enzymes HpaII, MspI and HhaI, and the resulting fragment patterns, which were specific for the plastid rRNA operon, were analyzed by Southern hybridization. While most of the fragment patterns were consistent with previously established physical maps, the partial resistance shown by one HpaII site and one HhaI site, both of which reside in the 16S/23S rDNA spacer region, was observed in DNA isolated from embryo, root tissue and endosperm. The partially resistant HpaII site was susceptible to cleavage with restriction enzyme MspI. From this and from the known inhibition of restriction enzyme Hhal at methylated HhaI sites, we conclude that the partial resistance of the two sites is caused by C-specific methylation of plastid DNA in the respective tissues. The tissue specificity of this DNA methylation is likely to reflect a differential expression of plastome encoded genes.     

Physical map of the plastid genome of the unicellular red alga Cyanidium caldarium strain RK-1

The physical map of the plastid genome of the unicellular red alga Cyanidium caldarium strain RK-1 was constructed. The 150-kbp genome was circular and had an inverted repeat region (IR) which contained the genes for 16 s and 23 s ribosomal RNAs, as is usually seen in most plastid genomes. Since C. caldarium is a very primitive alga, the results suggest that the ancestral cyanobacteria lost most of its genome as an endosymbiont comparatively early in the process of plastid formation. After that, several genes seem to have been lost from plastid genomes, step by step, during the course of evolution.     

Restriction enzyme analysis of mitochondrial DNA in aging human cells

Human diploid fibroblasts show a limited lifespan in vitro. To investigate the integrity of mitochondrial DNA (mtDNA) in aging fibroblasts, whole cell DNA samples from the human cell line IMR-90 have been prepared at 36, 22, and 3 population doublings (PD) from the end of the lifespan (63 PD). These DNA samples were then digested separately with 19 different restriction endonucleases, and the resulting fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Fragment sizes were revealed by hybridization to 32P-labelled mouse mtDNA and autoradiography, and were compared with computer maps of fragments generated from the known sequence of human mtDNA. These 19 enzymes recognize a total of 297 recognition sites comprising 1315 nucleotide base pairs (bp), approximately 8% of the human mtDNA (16 569 bp). Control experiments reveal that a minor component representing as little as 5% of the total mtDNA can be detected. No changes were seen in the restriction fragment pattern with fibroblast cell age. It is concluded that there are no large deletions, insertions, or rearrangements in human mtDNA, and no single base changes in the detectable regions. This suggests efficient maintenance of mtDNA molecules and/or elimination of damaged mtDNA during fibroblast cell lifespan.     

Genetic map of mitochondrial DNA in Podospora anserina

Summary In order to develop an eukaryotic vector with the Podospora plasmid, further characterization is required of the mitochondrial DNA into which this plasmid is integrated, a physical map (restriction sites) of the Podospora chondriome (size 95 kb) has been completed. As prerequisite for the establishment of a genetic (functional) map, 70% of the chondriome was cloned in E. coli vectors. Using mitochondrial genes from Saccharomyces cerevisiae, six structural genes were located on the Podospora chondriome by cross hybridization experiments. There is strong evidence that the plasmid is inserted into the cytochrome b gene. A comparison of the genetic map of the Podospora chondriome with those of Neurospora crassa and Aspergillus nidulans exhibits a rather good accordance with respect to the sequence of genes.     

Paternal plastid inheritance in alfalfa: plastid nucleoid number within generative cells correlates poorly with plastid number and male plastid transmission trength

Summary A previous study on alfalfa determined that the number of plastids/generative cell does not necessarily correlate with male plastid transmission strength in a given genotype. The objectives of the present study were to learn (1) whether plastid nucleoid number/generative cell is comparable to the number of plastids/generative cell, and (2) whether plastid nucleoid number/generative cell correlates with known male plastid transmission behavior in three alfalfa genotypes. Our results, which were based upon 150 generative cells examined by DAPI/epifluorescence microscopy, indicate that the mean plastid nucleoid number/generative cell is much less than the mean number of plastids/generative cell in genotype 7W (60 nucleoids/264 plastids) and genotype 301 (54 nucleoids/165 plastids). In genotype MS-5, mean plastid nucleoid number/generative cell (45) is similar to the mean number of plastids/generative cell (65). The significantly fewer plastid nucleoids/generative cell in MS-5, compared to that of 7W and 301, correlates positively with the relatively poor male plastid transmission strength of this genotype. However, the difference between the mean number of plastid nucleoids/generative cell in 7W and 301 is not significant, yet 301 is a much stronger transmitter of male plastids than is 7W.     

Plastid DNA from Pyrenomonas salina (Cryptophyceae): physical map,genes, and evolutionary implications

Summary Cryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the - and -subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.     

DNA markers define plastid haplotypes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To identify genetic markers in the Arabidopsis thaliana plastid genome (ptDNA), we amplified and sequenced the rpl2-psbA and rbcL-accD regions in 26 ecotypes. The two regions contained eight polymorphic sites including five insertions and/or deletions (indels) involving changes in the length of A or T mononucleotide repeats and three base substitutions. The 27 alleles defined 15 plastid haplotypes, providing a practical set of ptDNA markers for the Columbia, Landsberg erecta and Wassilewskija ecotypes that are commonly used in genetic studies and also for the C24 and RLD ecotypes that are the most amenable for cell culture manipulations.     

Physical and gene mapping of cauliflower (Brassica oleracea) mitochondrial DNA

Summary A physical map of the mitochondrial DNA isolated from B. oleracea (cauliflower) inflorescences was constructed with the restriction endonucleases Sall, Kpnl and Bgll. Physical mapping was made using the multi enzyme method with either unlabeled or labeled DNA fragments isolated by preparative electrophoresis and a clone bank prepared by inserting incomplete Sall restriction digests of mitochondrial DNA into a cosmid vector.The different mapping studies led to a circular map, about 217 kb in size, containing the entire sequence complexity of the genome. The 26S and 18S – 5S ribosomal RNA genes appeared to be separated by about 75 kb in this map. However, the particular cross-hybridization between several restriction fragments and the sequential diversity of some cosmids indicated that intra molecular recombination may occur naturally in higher plant mitochondria. Namely, one recombinational event resulted in the ribosomal RNA genes mapping closer together.Abbreviations mtDNA mitochondrial DNA - kb kilobasepairs - rRNA ribosomal RNA - LGT agarose low gelling temperature agarose     

Points of rearrangements between plastid chromosomes: location of protein coding regions on broad bean chloroplast DNA

Summary By using genes from spinach and tobacco chloroplast DNA as probes we have localized 22 protein genes on a refined map of Vicia faba (broad bean) chloroplast DNA. The genes are rbcL, rpoA, psaA,B, psbA–E, atpA,B,E,FH,I, pet-A,B,D, and the ribosomal protein genes rp12, rps7, rps12, and rps19. In addition, we mapped sequences homologous to a conserved open reading frame previously found in an intron of a trnK gene. The results were used to map the relative positions of rearrangements that distinguish the broad bean and spinach plastid DNAs. By hybridization with small probes several regions on both DNAs with sequence homology to more than one chromosomal position were found. Intermediate segments are rearranged in broad bean relative to spinach. A spinach chloroplast DNA fragment that functions as an autonomously replicating sequence in yeast is shown to be missing in broad bean chloroplast DNA. This ars is located at a position in spinach DNA which has been a target for rearrangement in the broad bean chromosome. These results can be interpreted by assuming that the DNAs contain short repeat sequences at some conserved positions. We suggest a model for chloroplast DNA divergence assuming that short repeats exist which are the substrate for a recombination system. The few rearrangements between chloroplast chromosomes from a variety of plants may be explained as the result of selection against short repeats.Abbreviations Kbp 10³ base-pairs - IR large inverted repeat unit