Sequence analysis of the junction of the large single copy region and the large inverted repeat in the petunia chloroplast genome

Summary We have determined the nucleotide sequence at the junction of the large single copy (LSC) region and the right and left members of the large inverted repeat, IRA and IRB, respectively, of the petunia chloroplast (cp) genome. As in Nicotiana debneyi and spinach (Zurawski et al. 1984), coding sequences of rps19 of petunia overlap the junction of IRB and LSC. Immediately into the LSC region upstream of IRA in the petunia cp genome are two small insertions relative to N. debneyi that occur at sites just inside IRA of N. debneyi. We discuss how these additions in one copy of the large inverted repeat of an N. debneyi like ancestor to petunia resulted in shortening of the petunia large inverted repeat by 8 bases and in the resultant slight movement of rps19 farther into LSC. On a larger scale, the large inverted repeat in the tobacco, N. debneyi and petunia lineage relative to a spinach-like ancestor may have sustained several contractions due to deletions between short direct repeats found within IRA and the IRA/LSC junction. We also show how the large inverted repeat of N. debneyi instead may have been expanded relative to a tobacco-like ancestor by insertion into the large inverted repeat of bases between short inverted repeat sequences in LSC and the LSC/IRB junction.     

Organization and sequence of five tRNA genes and of an unidentified reading frame in the wheat chloroplast genome: evidence for gene rearrangements during the evolution of chloroplast genomes

Summary The genes for the initiator tRNA_CAU ^Met. tRNA_UCC ^Gly, tRNA_GGU ^Thr, tRNA_UUC ^Glu and tRNA_GUA ^Tyr and an open reading frame of 62 codons have been identified by sequencing a 2,358 by BamHl and a 1,378 by BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 by intergenic region between the genes for the initiator tRNA^Met and the gene for tRNA_UCC ^Gly is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 by of the intergenic region and the first 28 by of the tRNA^Met gene, the second copy is found downstream of the tRNA^Met gene.     

Organisation of the chloroplast genome of kiwifruit (Actinidia deliciosa)

Summary A restriction map of the chloroplast genome has been determined for kiwifruit, Actinidia deliciosa. Single and multiple enzyme digests of kiwifruit chloroplast DNA were hybridised to a set of Brassica chloroplast probes, and the kiwifruit bands aligned with the known Brassica map. The chloroplast DNA of kiwifruit is typical of the majority of angiosperm chloroplast genomes; it is 160 kb in size, contains a 15–34 kb inverted repeat, and its gene content and gene order are similar to those of the Brassica chloroplast genome.     

Insertion/deletion mutations in the Zea chloroplast genome

Summary The chloroplast (cp) genomes of Zea species are distinguished by at least four restriction fragment length (insertion/deletion) mutations. All four mutations occur in the large unique sequence region of the genome. Restriction fragments containing three of these mutations were cloned. The large and small forms of two of the mutated fragments were sequenced. This revealed 80 and 83 by insertion/deletions. The inserted/deleted segments are not parts of tandem repeats nor were they flanked by direct repeats. Two other insertion/deletion mutations were not sequenced, but their sizes were estimated to be 150 and 250 by by size fractionation on agarose gels. Use of Tripsacum pilosum and Sorghum bicolor as outgroups suggests that three of the fragment length mutations arose via deletions. The fourth could not be polarized. The three species of section Luxuriantes of Zea were identical to one another for each of the four length mutations, and they were consistantly distinguished from the taxa of section Zea by these mutations. These data support the division of Zea into the above named sections.Abbreviations bp base pairs - kb 1,000 bp - cpDNA chloroplast DNA - SDS sodium dodecyl sulfate     

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.     

Characterization of DNA synthesis and chloroplast DNA replication initiation in a Petunia hybrida chloroplast lysate system

Summary An in vitro chloroplast DNA synthesizing lysate system prepared from purified chloroplasts of Petunia hybrida leaves has been developed. Both co-isolated endogeneous chloroplast (cp)DNA and externally added DNA can be used as DNA templates in the system. The system contains a -like DNA polymerase as determined by using DNA polymerase-specific inhibitors and synthetic templates. The molecular weight of this enzyme is about 85 kd. Part of the DNA synthesizing activity is repair synthesis. When a chimaeric plasmid containing a fragment with a potential cpDNA replication origin is used as a template (pPCY62), specific initiation of DNA synthesis is observed on this fragment which strongly suggests that the in vitro chloroplast lysate system is also capable of replication initiation.     

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     

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.     

AZFc区gr/gr及DAZ基因拷贝缺失与原发性男性生精障碍的相关性研究

目的探讨Y染色体AZFc区gr/gr缺失及DAZ基因拷贝缺失与男性生精障碍的相关性。方法运用PCR与PCR-RFLP检测技术,对252例正常生精男性,430例原发性生精障碍男性患者进行Y染色体AZFc区gr/gr缺失及DAZ基因拷贝缺失分析。结果正常生精男性组gr/gr缺失率为5.2%,原发性生精障碍组gr/gr缺失率为10.2%,P=0.021,差异有统计学意义;正常生精男性组DAZ1/DAZ2基因拷贝共缺失率为2.0%,原发性生精障碍组中DAZ1/DAZ2基因拷贝共缺失率为7.0%,P=0.004,差异亦有统计学意义;正常生精男性组DAZ3/DAZ4基因拷贝共缺失率为3.2%,在生精障碍组中DAZ3/DAZ4基因拷贝的共缺失率为3.3%,P=0.954,差异无统计学意义。结论在原发性男性生精障碍患者中存在较高频率的gr/gr缺失及DAZ1/DAZ2基因共缺失,提示gr/gr缺失及DAZ1/DAZ2基因拷贝的共缺失是男性不育的高风险因子。     

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.     

Changes in chloroplast genome composition and recombination during the maturation of zygospores of Chlamydomonas reinhardtii

Summary In crosses of the unicellular green alga Chlamydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems.     

Maternal inheritance of chloroplast genome and paternal inheritance of mitochondrial genome in bananas (Musa acuminata)

Restriction fragment length polymorphisms (RFLPs) were used as markers to determine the transmission of cytoplasmic DNA in diploid banana crosses. Progenies from two controlled crosses were studied with heterologous cytoplastmic probes. This analysis provided evidence for a strong bias towards maternal transmission of chloroplast DNA and paternal transmission of mitochondrial DNA in Musa acuminata. These results suggest the existence of two separate mechanisms of organelle transmission and selection, but no model to explain this can be proposed at the present time. Knowledge of the organelle mode of inheritance constitutes an important point for phylogeny analyses in bananas and may offer a powerful tool to confirm hybrid origins.     

Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation

Summary SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.     

Molecular evolution of noncoding regions of the chloroplast genome in the Crassulaceae and related species

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA (cpDNA) in order to study sequence-length variation in the Crassulaceae and in related species. Several length mutations were observed that are of diagnostic value for evolutionary relationships in the Crassulaceae and the Saxifragaceae. Length variation and sequence divergence in the intergenic spacer between the trnL (UAA) 3 exon and the trnF (GAA) gene among 15 species were studied in detail by nucleotide-sequence analysis. A total of 50 insertion/deletion mutations were observed, accounting for a spacer-length variation in the range of 228–360 bp. Eighteen short direct repeat motifs (4–11 bp) and two inverted repeat motifs (7–11 bp) were found to be associated with length variation. Phylogenetic analysis of the sequence data indicated a pattern of relationships that was largely consistent with a previous analysis of cpDNA restriction-site variation. Evaluation of the level of homoplasy in insertion/deletion mutations within a phylogenetic framework revealed that only 1 out of 34 length mutations longer than 2 bp must have had multiple origins. The feasibility of the noncoding chloroplast DNA regions for molecular evolutionary studies is discussed.     

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.     

Organization of the psbE,psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome

Summary The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE — 8 by spacer —psbF — 110 by spacer — orf38 — 87 by spacer — orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T > A > G > C in the mRNA-like strand and group II-like boundary sequences at their 3 and 5 ends having the consensus 5-GTGTG .. INTRON .. TTAATTTNAT-3. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE psbF orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.Abbreviations and notations DEP diethyl pyrocarbonate; gene names follow the convention of Hallick and Bottomley (1983) - psaA, psaB genes for the P₇₀₀ apoproteins - psbE and psbF genes for the subunits of cytochrome b ₅₅₉ - orfN open reading frame of N codons     

True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas

The ribulosebisphosphate carboxylase/oxygenase-defective Chlamydomonas mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA. In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene.     

The intergenic region between the Vicia faba chloroplast tRNACAA Leu and tRNAUAA Leu genes contains a partial copy of the split tRNAUAA Leu gene

Summary A cluster of three tRNA genes located on fragment Bam6a from Vicia faba chloroplast DNA has been sequenced: it contains the genes for tRNA_CAA ^Leu, tRNA_UAA ^Leu and tRNA^Phe. The two tRNALeu genes are separated by 443 by and are transcribed divergently from different DNA strands. The intergenic region contains a series of short repeats and a partial copy of the split tRNA_UAA ^Leu gene which includes 100 by of the 5 flanking region, 35 by of the 5 exon and the first 42 by of the intron. It is possible that some of these duplications occurred upon the rearrangement of the two tRNA^Leu genes in broad bean (and in pea) or upon the deletion of one copy of the inverted repeat, since in all other higher plant chloroplast genomes studied so far these two tRNA^Leu genes are located far apart on the genome, one being in the inverted repeat region, the other one in the large single copy region. The tRNA^Phe and tRNA_UAA ^Leu are encoded by the same DNA strand, and separated by 110 bp.     

The ribosomal RNA repeats are non-identical and directly oriented in the chloroplast genome of the red alga Porphyra purpurea

A detailed restriction map of the chloroplast genome of the red alga Porphyra purpurea has been constructed. Southern hybridization experiments with cloned or gel-purified restriction fragments and PCR products indicate that the P. purpurea chloroplast genome is approximately 188 kb in size. This circular molecule contains two rRNA-encoding repeats (approximately 4.9 kb) that separate the genome into single-copy regions of 34 kb and 144 kb. Interestingly, these repeats are arranged in a direct orientation. In addition, DNA sequencing of the ends of both repeats revealed that the two rRNA repeats are not identical. No intramolecular recombination between the repeats can be detected. We discuss the possibility that the chloroplast genome of P. purpurea is organized like that of the ancestral chloroplast.