Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa

Summary Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3-terminal protion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.     

Genes for components of the chloroplast translational apparatus are conserved in the reduced 73-kb plastid DNA of the nonphotosynthetic euglenoid flagellate Astasia longa

The colourless, nonphotosynthetic protist Astasia longa is phylogenetically related to Euglena gracilis. The 73-kb plastid DNA (ptDNA) of A. longa is about half the size of most chloroplast DNAs (cpDNAs). More than 38 kb of the Astasia ptDNA sequence has been determined. No genes for photosynthetic function have been found except for rbcL. Identified genes include rpoB, tufA, and genes coding for three rRNAs, 17 tRNAs, and 13 ribosomal proteins. Not only is the nucleotide sequence of these genes highly conserved between A. longa and E. gracilis, but a number of these genes are clustered in a similar fashion and have introns in the same positions in both species. The results further support the idea that photosynthetic genes normally encoded in cpDNA have been preferentially lost in Astasia, but that the chloroplast genes coding for components of the plastid translational apparatus have been maintained. This apparatus might be needed for the expression of rbcL and also for that of still unidentified nonphotosynthetic genes of Astasia ptDNA.These sequence data will appear in the EMBL/Gen Bank/DDBJ nucleotide sequence data base under accession numbers X75651, X75652 and X75653     

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)     

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 map and clone bank of tomato plastid DNA

Summary Tomato plastid DNA has been isolated from leaf chloroplasts andPst1 fragments cloned in the plasmid vector pUC8. Recombinant plasmids containing all but the largestPst1 fragment were identified by colony hybridisation. A restriction map of the plastid DNA for four restriction endonucleases was generated by digestion of the clonedPst1 fragments and by hybridisation to Southern blots. The plastid DNA is a circular molecule of 156.6–159.4 kbp with a large inverted repeat, and shows high conservation of restriction sites with plastid DNA from tobacco andPetunia.     

The rice psb-A chloroplast gene has a standard location

Summary The chloroplast DNA-encoded gene psbA codes for the 32 kD protein of photosystem II. It has been mapped in several monocots to a location in the large single copy segment very near to the end of one of the inverted repeats present in the chloroplast genome of most land plants. The psbA gene of rice has been located much further from the inverted repeat, suggesting that rice differs from related species in the location of this gene. Indirect evidence reported here suggests that this gene actually maps to the usual cereal location, at least in the seven varieties tested, which include representatives of three subspecies.     

Euglena gracilis chloroplast DNA: analysis of a 1.6 kb intron of the psb C gene containing an open reading frame of 458 codons

Summary We retrieved a 1.6 kbp intron separating two exons of the psb C gene which codes for the 44 kDa reaction center protein of photosystem II. This intron is 3 to 4 times the size of all previously sequenced Euglena gracilis chloroplast introns. It contains an open reading frame of 458 codons potentially coding for a basic protein of 54 kDa of yet unknown function. The intron boundaries follow consensus sequences established for chloroplast introns related to class II and nuclear pre-mRNA introns. Its 3-terminal segment has structural features similar to class II mitochondrial introns with an invariant base A as possible branch point for lariat formation.     

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.     

The chloroplast genome of Euglena gracilis: the mosaic structure of a DNA segment linking the extra 16S rRNA gene with the rrn operon A

Summary We have completed the analysis of a DNA segment of the chloroplast genome of Euglena gracilis Klebs, Z-strain, which links the 3 end of the extra 16S rRNA gene with the 5 end of the 16S rRNA gene of the rrn operon A. This region is a mosaic of several structural elements and contains an intact rrn interoperon spacer of 1,080 bp, an extra 5S rRNA gene, an open reading frame for 406 codons (ORF 406) which is flanked by short inverted repeats and a short direct repeat originating from the rrn interoperon spacer. It seems that a once complete rrn operon underwent in the past an insertion/deletion event leaving intact the 16S and 5S rRNA but totally excising the 16S-23S intergenic spacer and the 23S rRNA gene. Instead a protein coding gene of yet unknown function was inserted along with other structural elements.     

Chloroplast DNA from Adiantum capillus-veneris L., a fern species (Adiantaceae); clone bank,physical map and unusual gene localization in comparison with angiosperm chloroplast DNA

Summary We cloned all of Adiantum capillus-veneris chloroplast DNA PstI fragments longer than 1.0 kb, which cover 98% of the genome. These cloned fragments were used to construct a physical map for five restriction enzymes. The genome of A. capillus-veneris is approximately 153 kb long and contains a 24 kb inverted repeat. Mapping of 12 chloroplast DNA genes and heterologous hybridization, involving A. capillus-veneris chloroplast DNA and angiosperm chloroplast DNA probes, demonstrated that chloroplast DNA of A. capillus-veneris has a different gene order from typical angiosperm cpDNA (e.g., tobacco) in the inverted repeat region and the flanking segment of the large single copy region.     

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.     

The Euglena gracilis chloroplast genome: structural features of a DNA region possibly carrying the single origin of DNA replication

Summary We sequenced a Bg1II-HindIII DNA fragment of the Euglena gracilis chloroplast genome which most likely carries the single origin of DNA replication according to recent electronmicroscopic mapping studies (Koller and Delius 1982a; Ravel-Chapuis et al. 1982). This DNA fragment contains a polymorphic region (Schlunegger et al. 1983) which is composed, as will be shown, of multiple tandem repeats (54 bp, 87% A+T). Furthermore we located on this DNA fragment a short inverted repeat element (96 positions) observed in the electronmicroscopic studies (Koller and Delius 1982b). Between the borders of the polymorphic region and the nearby inverted repeat (distance of 179 positions) we retrieved an exact copy of parts of the rDNA leader (105 positions) including 49 positions of the chloroplast trnW gene. A computer search for bacterial type Ori-regions did not reveal any significant sequence homology. However, the polymorphic region and its immediate vicinity have the capacity to form multiple stem and loop structures which may be involved in DNA replication initiation.     

Modifications of chloroplast DNA during streptomycin induced mutagenesis in Euglena gracilis

Summary The evolution of chloroplast DNA was analysed during streptomycin induced mutagenesis in Euglena gracilis strain bacillaris and strain Z. In addition to a massive reduction of the cellular level of chloroplast DNA, several structural modifications have been observed in early stages of mutagenesis but they are generally eliminated during the later stages. The ribosomal cistrons are regularly rearranged: two of the three tandemly arranged cistrons occuring in wild type chloroplast DNA decrease while the third one is relatively more conserved and amplified during mutagenesis and in bleached mutants.     

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.     

Mitochondrial DNAs of the fungus Armillaria ostoyae: restriction map and length variation

A restriction-enzyme-site map is presented for the 147-kb mtDNA of North American Armillaria ostoyae. The locations of five structural genes, atp6, atp8, coxI, coxIII, and cob, along with the location and orientation of the large and small ribosomal RNA genes, were determined through Southern hybridizations with cloned genes from other fungal mtDNAs. Based on this map, the variation in mtDNA suggested geographic structure at two different levels. On a large geographic scale, 17 mtDNA types from North America were distinct, with respect to both size and restriction maps, from three mtDNA types from Europe. At the local scale, identical mtDNA types were evident among several different genetic individuals located no more than 1 km apart at a site in Michigan. No mtDNA type occurred more than once among genetic individuals from different regions of North America, although the occurrence of similar mtDNAs in isolates from distant regions suggested that this may occur at a low frequency with large sample sizes. Among the North American mtDNA types, analysis of discrete length variants was inconsistent with the hypothesis that the mtDNA of A. ostoyae evolves as a clonal lineage in which each length mutation represents a unique event. The two remaining hypotheses, that similar mutational events have occurred independently and that genetic exchange and recombination occurs among mtDNAs in natural populations of this species, remain to be tested.     

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning,physical mapping and gene location

Summary The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.     

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