Chloroplast DNA from the fern Osmunda cinnamomea: physical organization,gene localization and comparison to angiosperm

Summary Chloroplast DNA from the fern Osmunda einnamomea was isolated by a sucrose gradient procedure utilizing PEG to stabilize chloroplasts. Analysis with the restriction endonucleases PvuII, Sacl and BstEII indicates a chloroplast genome size of 144 kb. A physical map of the fragments produced by these three enzymes was constructed by filter hybridizations using purified PvuII fragments as hybridization probes. The Osmunda chloroplast genome is circular and contains an inverted repeat 8–13 kb in size.Gene probes from tobacco, corn and spinach were used to map the positions of six genes on the Osmunda chloroplast chromosome. The 16S and 23S ribosomal RNAs are encoded by duplicate genes which lie within the inverted repeat. Genes for the large subunit of ribulose-1,5-bisphosphate carboxylase, a photosystem II polypeptide, and the alpha and beta subunits of chloroplast coupling factor are located in three different segments of the large single copy region.The Osmunda chloroplast genome is remarkably similar in size, conformation, physical organization, and map positions of known genes, to chloroplast DNA from a number of angiosperms. The major difference between chloroplast DNA from this fern and angiosperms is that the inverted repeat is smaller in Osmunda (8–13 kb) than in angiosperms (22–25 kb).Abbreviations PEG polyethylene glycol 4000 - kb kilobase pairs - bp base pairs - rRNA ribosomal RNA - LS large subunit of ribulose-1,5-bisphosphate carboxylase - PII 32,000 dalton photosystem II polypeptide (Mattoo et al. 1981) - CF alpha subunit of chloroplast coupling factor - CF beta subunit of chloroplast coupling factor     

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)     

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure,gene localization,and characterization of a large inversion

Summary We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.     

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

Summary A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.     

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.     

A chloroplast DNA mutational hotspot and gene conversion in a noncoding region near rbcL in the grass family (Poaceae)

The noncoding DNA region of the chloroplast genome, flanked by the genes rbcL and psaI (ORF36), has been sequenced for seven species of the grass family (Poaceae). This region had previously been observed as a hotspot area for length mutations. Sequence comparison reveals that short duplications, probably resulting from slipped-strand mispairing, account for many small length differences between sequences but that major mutational hotspots are localized in three small areas, two of which show potential secondary structure. Mutation in one of these hotspots appears to be a result of more complex recombination events. All seven species contain a pseudogene for rpl23 and evidence is presented that this pseudogene is being maintained by gene conversion with the functional gene. Different transition/transversion biases and AT contents between the pseudogene and the surrounding noncoding sequences are noted. In the subfamily Panicoideae there is a deletion in which almost 1 kb of ancestral sequence, including the 3 end of the rpl23 pseudogene, has been replaced by a non-homologous 60-base sequence of unknown origin. Two other deletions of almost the same region have occurred in the grass family. The deleted noncoding region has mutational and compositional properties similar to the rbcL coding sequence and the rpl23 pseudogene. The three independent deletions, as well as the pattern of mutation in the localized hotspots, indicate that such noncoding DNA may be misleading for studies of phylogenetic inference.     

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68–127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Physical map and protein gene map of cyanelle DNA from the second known isolate of Cyanophora paradoxa (Kies-strain)

Summary A restriction map of the cyanelle DNA from a different isolate of Cyanophora paradoxa (Kies-strain) was established. The positions of 18 protein genes and the rRNA genes have been located and compared to the positions of these genes from the first isolate of C. paradoxa (Pringsheim-strain). The gene arrangement is absolutely conserved in both cyanelle DNAs. The differences in size (ca. 9 kb) and the unrelatedness in the restriction patterns could be explained by numerous small insertions into intergenic regions of the cyanelle chromosomes.     

Chloroplast DNA differences in the genus Oenothera subsection Munzia: a short direct repeat resembling the lambda chromosomal attachment site occurs as a deletion/insertion within an intron of an NADH-dehydrogenase gene

Summary A small restriction fragment length mutation has been mapped in the large inverted repeats of the chloroplast (cp) DNA of Munzia-Oenothera species (vom Stein and Hachtel 1986). This mutation could be localized within the intron of a reading frame presumably coding for subunit B of an NADH-dehydrogenase (ndhB). Sequence analysis revealed a 24 by duplication/deletion. The predicted secondary structure of the ndhB-intron is altered by this duplication/deletion. Part of the directly repeated segment shows remarkable similarity to the phage lambda attachment site. Evidence is presented for similar sequences in other plastome regions where deletions/insertions have been found. Furthermore, the locations of the genes for other components of the NADH-dehydrogenase (ndhA, ndhC, ndhD, ndhE, ndhF) were established by heterologous hybridization using gene probes from tobacco cpDNA.Abbreviations bp base pairs - kB 1,000 bp - cpDNA chloroplast DNA - Oe Oenothera - att-lambda attachment site of bacterio-lambda     

Mutations in a nuclear gene of Chlamydomonas cause the loss of two chloroplast ribosomal proteins,one synthesized in the chloroplast and the other in the cytoplasm

Summary The allelic nuclear mutations of Chlamydomonas reinhardtii, cr-6 and cr-7, result in the loss of two proteins from the large subunit of the chloroplast ribosome. One of these proteins, L-13, is synthesized in the chloroplast and the other, L29, is made in the cytoplasm. The loss of these two proteins is correlated with the inability of the large subunits of the chloroplast ribosomes to form monomers which incorporate labeled phenylalanine at normal rates in response to a polyuridylic acid template. Using antisera raised against L13 and L29, we found that protein L-13 was synthesized in appreciable amounts in pulse labeled cells of cr-6 and cr-7, but protein L-29 was not. We conclude that the inability to synthesize protein L29 is a primary defect in both cr-6 and cr-7 and that this protein is required for the stable assembly of protein L-13 into chloroplast ribosomes. The absence of one or both of these proteins from the large subunit of chloroplast ribosomes of the mutants interferes with the ability of the small and large subunits to associate properly into normal 70S monomers.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

Mitochondrial DNA of Chenopodium album (L): a comparison of leaves and suspension cultures

Summary The mitochondrial genome (mt genome) of Chenopodium album was analysed. It was found to have a size of about 270 kb and a GC content of 46%. The genomes of plant cells and suspension cultures were compared. Restriction fragment pattern analyses and hybridization experiments revealed quantitative and qualitative alterations. However, a comparison of the restriction fragment patterns of two independently established in vitro cultures did not reveal differences as far as the mt chromosomal DNA is concerned. Therefore, alterations in mtDNA induced by in vitro culture seem not to be caused by an entirely undirected process in Chenopodium. A plasmid-like DNA molecule was amplified in only one of the cultures investigated and not in the other or in the plant cells. This molecule has a length of 1,083 by and is referred to as Ca1 mp1.     

Effect of extract from a transplant for eyelid plasty (series ALLOPLANTTM) on DNA synthesis in cultures cells

Extract isolated from a collagen-containing transplant (series ALLOPLANT^TM), which is used in the treatment of benign and malignant neoplasms of the eyelid, inhibits DNA synthesis in the cellin vitro. This effect is nonspecific, reversible, and dose-dependent. The extract is thermostable and resistant to proteolytic enzymes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 1, pp. 75–79, January, 1994     

Polymorphism for ribosomal RNA gene arrangement in the mitochondrial genome of fall rye (Secale cereale L.)

A restriction-fragment-length polymorphism (RFLP) in mitochondrial DNA (mtDNA) was detected between varieties of fall rye (Secale cereale L.) by Southern hybridization with rrn18, the gene encoding the mitochondrial 18S ribosomal RNA. Restriction mapping showed that the RFLP is based on differing numbers of genomic contexts (one vs three) for a recombining-repeat element (the 18S/5S repeat). From examination of other Secale species, we conclude that the one-context state arose relatively recently, putatively by deletion of two of an ancestral set of three distinct genomic loci containing the mitochondrial 18S/5S repeat. This is consistent with our earlier conclusion that the 18S/5S repeat has probably existed in at least two genomic copies throughout much of the history of the grass family (at least 40 million years). Interestingly, the intervarietal difference in the number of distinct rrn18 loci is not accompanied by a major difference in the number of rrn18 copies per unit mass of mtDNA. This suggests the existence of a mechanism that can compensate rather precisely for differences in mitochondrial gene dosage, perhaps by over-replication or stabilization of specific subgenomic molecules.     

DNA sequence analysis of the 24.5 Kilobase pair cytochrome oxidase subunit I mitochondrial gene from Podospora anserina: a gene with sixteen introns

Summary The DNA sequence of a 26.7 Kilobase pair (10³ base pairs = 1 Kb) region of the mitochondrial genomes of races s and A from Podospora anserina was determined. Within this region, the 24.5 Kb cytochrome oxidase subunit I gene was located and its exon sequences determined by computer analysis comparisons with other fungal genes. The Podospora COI gene was interrupted by two group II introns (one in race s) and fourteen group I introns ranging in size from about 2.2 Kb to 404 bp. Earlier studies on secondary structure analysis, as well as comparison of their open reading frames (ORFs), showed that the two group II introns were closely related. The fourteen group I introns were representatives of three subgroupings (IB, C and a new category, subgroup ID). Two of these group I introns were separated by just a single exon codon. The analysis of all these introns is discussed in comparison with other fungal introns as well as with the known Podospora anserina introns.