Variation in copy number of a 24-base pair tandem repeat in the chloroplast DNA of Oenothera hookeri strain Johansen

Summary A highly variable region of chloroplast DNA has been analyzed from three isolates of Oenothera hookeri strain Johansen. The variability results from the presence of two, four or seven copies of a discrete 24-base pair tandem repeat in a segment of the chloroplast DNA within the inverted repeat. Alignment of this DNA region with the published tobacco cpDNA sequence shows that in Oenothera, the repeats are insertions within a large unidentified reading frame, with each repeat unit specifying an eight amino acid in-frame addition. A model to explain the frequent alterations in the copy number of this 24-bp unit is proposed: imprecise alignment and recombination between the two large inverted repeats followed by copy correction could result in an amplification or deletion of the 24-bp segments.This paper is dedicated in memory of Barry K. Chelm     

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     

Strategy for deletion of complete open reading frames in Saccharomyces cerevisiae

The classical disruption method for yeast genes is by using in vitro deletion of the gene of interest, or of a part of it, with restriction enzymes. We are now routinely using a strategy that takes advantage of polymerase chain reactions (PCRs) which amplify large pieces of DNA. Since this approach results in a complete, precise deletion of the open reading frame, which is replaced by a unique restriction site, the ligated PCR can be used for the insertion of different markers of for two-step gene disruptions without an inserted marker. As we have now used this strategy for the deletion of more than ten genes we have in this report included some hints based on our experience.     

Organization and nucleotide sequence of the broad bean chloroplast genes trnL-UAG,ndhF and two unidentified open reading frames

Summary We have determined the nucleotide sequence of a 6.9 kbp BamHI-Xbal fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-CIal) is known to contain three tRNA genes (trnL-CAA, trnlrUAA and trnF). We have now further identified a gene coding for the third tRNA^Leu isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-Xbal fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.     

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.     

The endpoints of an inversion in wheat chloroplast DNA are associated with short repeated sequences containing homology toatt-lambda

Summary The physical mapping of Aval, BstEII and EcoR1 restriction sites on the chloroplast genome of the green alga Chlamydomonas eugametos is presented. The circular map, with a size of 243 kilobase pairs, is the largest yet reported for a chloroplast genome. It features a large inverted repeat sequence, part of which encodes the 16S and 23S ribosomal RNAs (rRNAs), the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and the 32-kdodalton thylakoid membrane protein (psbA). Such an rRNA-encoding inverted repeat sequence is also found in the chloroplast genomes of Chlamydomonas reinhardtii and most land plants. These genomes, however, differ from that of C. eugametos by the absence of the rbcL gene from the inverted repeat sequence of C. reinhardtii and by the absence of both the rbcL and psbA genes from the inverted repeat sequence of land plants. Possible evolutionary implications of these differences are discussed.Abbrevations cpDNA chloroplast DNA - kbp kilobase pairs - psbA 32 kilodalton thylakoid membrane protein gene - rbcL ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit gene - rRNA ribosomal RNA     

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.     

Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo

Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

Summary Recently, the nucleotide sequences for three mitochondrial plasmids associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian URF1 gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones

Summary Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.     

Electrophoretic karyotype of Dipodascus (Endomyces) magnusii: two main intraspecific chromosomal polymorphisms associated with the difference in total genome size

This study describes the karyotype of strain 270 of the yeast-like fungus Endomyces magnusii. It consists of 13 chromosomal DNA molecules, the size of which range between 1.2 and 5.7 Mb producing a genome size of approximately 38 Mb. By comparing the karyotype of six strains of E. magnusii, we revealed two main chromosome length polymorphisms (CLPs) associated with a pronounced difference in the total genome size (roughly 50%). Karyotype heterogeneity between two main CLPs was demonstrated by Southern analysis with three heterologous probes. The same species affiliation of six E. magnusii strains was confirmed by morphological and cytological studies, protein fingerprint comparisons, as well as restriction analysis of mitochondrial DNA and genomic Southern analysis.