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.     

Rearrangement of chloroplast ribosomal cistrons by unequal crossing-over in Euglena gracilis

Summary The chloroplast DNA of a wild type photosynthetic variant of Euglena gracifs (ATCC n° 10616) with five ribosomal cistrons has been analyzed by restriction mapping. The results complete the electron microscope study of Koller and Delius (MGG 188, 305, 1982); they support a model of formation of the variant DNA by rearrangement of the wild type ribosomal cistrons through unequal crossing-over. The recombination sites have been determined. The recombination model proposed also explains the formation of the Z-S variant with a single ribosomal cistron (Wurtz and Buetow 1981).     

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.     

Intraspecific variation in the structural organization and redundancy of chloroplast ribosomal DNA cistrons in Euglena gracilis

Summary Chloroplast DNAs from six different laboratory collections of Euglena gracilis strain Z and var. bacillaris were analyzed with restriction endonucleases EcoRI and Bam HI. The most variable portion of the organelle genome is the region containing the ribosomal cistrons. Intraspecific differences occur in both ribosomal DNA cistron number (one or three) and structural organization among those strains designated as strain Z and bacillaris. One culture previously designated as Z is most likely bacillaris.     

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.     

Fractionation and identification of Euglena gracilis cytoplasmic and chloroplastic tRNAs and mapping of tRNA genes on chloroplast DNA

Summary The cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by two-dimensional gel electrophoresis and identified by aminoacylation. Purified chloroplast tRNAs, labeled in vitro with |³²P|, were hybridized to endonuclease restriction fragments of chloroplast DNA, allowing the corresponding tRNA genes to be localized on the physical map of Euglena chloroplast DNA.     

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.     

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

Summary The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5 and 3 ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3. Abreviations: Gene names follow the convention of Hallick and Bottomley (1983): rp123, rpl2, rpl22 are, respectively, genes for the L23, L2, L22 polypeptides of the 50S ribosomal subunit; rps19 and rps3 are genes for the S19 and S3 polypeptides of the 30S ribosomal subunit     

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

Summary We have determined the complete nucleotide sequence of the two genes encoding the P₇₀₀ chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5 GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3 and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains. Abbreviations and notations: Gene names follow the convention of Hallick and Bottomley (1983: psaA, psaB, genes for the P₇₀₀ apoprotein; psbE an psbF, genes foe the subunits of cytochrome b ₅₅₉; orfN, open reading frame of N condons     

Evidence for the presence of hairpin chloroplast DNA molecules in barley cultivars

Summary Total DNA was extracted from young green and etiolated barley leaf shoots and run on 2D neutral/alkaline agarose gels. This technique showed the presence of molecules which behaved as though their single-stranded length was twice their double-stranded length. This behaviour was sensitive to S1 nuclease. Our conclusion is that these DNAs are hairpin molecules. They are homologous to sequences found throughout the barley chloroplast genome. This type of molecule is present in uncut DNA with a continuous range of sizes varying between 0.5 and 15 kbp in our experimental conditions. The origin and possible biological function of these molecules is discussed.     

Common features of three inversions in wheat chloroplast DNA

Summary We have determined the DNA sequences of regions involved in two of the three inversions known to have occurred during the evolution of wheat chloroplast DNA. This establishes the extent of the second largest of the three inversions. Examination of these sequences suggests that although short repeated sequences are present, the endpoints of the second and third inversions are not associated with repeated sequences as long as those associated with the first inversion. However the endpoints of all three inversions are all adjacent to at least one tRNA gene, and there is evidence that three of the tRNA genes have been subjected to partial duplication, possibly at the time of inversion. This suggests that tRNA genes might be involved with rearrangements of chloroplast DNA, as has also been postulated for mitochondrial DNA.     

Localization of chloroplast ribosomal protein genes on Spirodela oligorhiza chloroplast DNA

Summary Three chloroplast ribosomal protein genes have been located on Spirodela chloroplast DNA. (1) rps14 was physically mapped at the central region of the large single copy region by heterologous hybridization with the corresponding gene of Marchantia chloroplast DNA. (2) Chloroplast ribosomal protein C-S23 was detected by immunoprecipitation with a polyspecific antiserum against 30S chloroplast ribosomal proteins among the in vitro translation products of mRNAs selected by Spirodela Pstl-D chloroplast DNA fragment. (3) We have previously reported that Spirodela BamHI-G chloroplast DNA fragment encodes a 14–15 kD 50S chloroplast ribosomal protein (Posno et al. 1985 Curr Genet 9 : 211–219). Here we show that the homologous Spinacia chloroplast DNA fragment Sa1I-9 additionally directs the synthesis of another 50S chloroplast ribosomal protein in a DNA dependent E. coli cellfree system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. mRNA selection experiments revealed that this additional chloroplast ribosomal protein gene is present on Spirodela chloroplast DNA as well, but is not expressed in the E. coli cellfree system. The identity of this 50S chloroplast ribosomal protein could not be established unambiguously, since two-dimensional gel analysis revealed that this protein comigrated, depending on the experiment, with C-L11 (26 kD) or with C-L24 (17 kD).Abbreviations (k) bp (kilo)basepairs - cp chloroplast - IR inverted repeat - kD kilodalton - LSCR large single copy region - Mw molecular weight - r-protein ribosomal protein - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - SSCR small single copy region     

Characterization of chloroplast DNA in Chlamydomonas eugametos and C. moewusii and its inheritance in hybrid progeny

Summary The density, molecular weight, and cellular repetition of DNA molecules associated with the -DNA satellite of the interfertile algae Chlamydomonas eugametos and C. moewusii are reported. The similarities between these values and those for the chloroplast DNA (cpDNA) in the related alga Chlamydomonas reinhardtii indicate that these satellites represent cpDNA. The buoyant densities of C. eugametos and C. moewusii cpDNAs are indistinguishable from one another, as are those of their respective nuclear DNAs. These densities differ slightly from the densities of the homologous components of C. reinhardtii whole cell DNA. All three species differ with respect to additional minor satellite DNAs and low molecular weight DNAs of unknown cellular location.Differences in the Aval and Smal restriction endonuclease fragmentation patterns of C. eugametos and C. moewusii cpDNAs were employed to study the inheritance of cpDNA in an F1 hybrid which had inherited a non-Mendelian streptomycin resistance marker (sr-2) from the C. eugametos mating-type plus (mt ⁺) parent and in two homoplasmic mitotic segregants from a B 1 hybrid (F₁ × C. moewusii) which had been initially heteroplasmic for the resistance marker. Although the cpDNA patterns in the F1 hybrid were similar to those of the C. eugametos ml ¹ parent, important differences were noted which suggest that recombination between C. eugametos and C. moewusii cpDNA had occurred. Homoplasmic streptomycin resistant and sensitive mitotic segregants recovered from the B1 hybrid product reveal Aval restriction patterns similar to those of the respective resistant and sensitive parents. These data are consistent with the hypothesis that the sr-2 marker is located in cpDNA and that C. eugametos and C. moewusii cpDNA sequences can coexist in the same chloroplast and, at least sometimes, segregate without extensive recombination. The transmission of low molecular weight DNAs characteristic of C. moewusii but of unknown cellular origin shows no direct correlation with the transmission of the sr-2 marker.     

Two new group-II twintrons in the Euglena gracilis chloroplast are absent in basally branching Euglena species

Studies of the phylogeny and chloroplast intron content of selected Euglena species have led to insights in our understanding of the timing of intron acquisition. In the current study, two new twintrons, found in E. gracilis, have been characterized by the analysis of partially spliced pre-mRNAs. Intron 1 of atpE is a 463-nt group-II intron interrupted by a second group-II intron 320 nt long. Intron 1 of psbD is also a group-II twintron with external and internal introns of 635 nt and 463 nt, respectively. The two introns composing the psbD twintron, as well as six additional group-II introns found in the E. gracilispsbD gene, are not present in several basally branching Euglena species, including E. myxocylindracea, E. stellata and E. viridis. The distribution of psbD introns in Euglena is consistent with a late evolutionary acquisition of group-II introns in this lineage. Received: 4 March / 11 September 1996