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.     

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.     

Organization of the chloroplast genome in the red alga Porphyra yezoensis

Summary A comprehensive assessment of the origin and evolution of plastids will require more information on the nature of plastid genomes from non-green algae. I have constructed a physical map of the chloroplast genome from the red alga Porphyra yezoensis. The 185 kb circular genome contains ribosomal RNA encoding inverted repeats (6.6 kb), and is divided into small and large singlecopy regions of approxiamtely 16 kb and 156 kb respectively. The Porphyra genome contains several genes not found in higher plant chloroplasts. Genes encoding the pigmented, light-harvesting phycobiliproteins are organized relatively close to one another on the genome, and represent components of a multi-gene family. the phycocyanin biliprotein genes (ppcBA) map in two single-copy regions, suggesting either duplicated genes or a transsplicing mechanism. In contrast to higher plants, the tufA and rbcS genes are chloroplast-encoded in Porphyra, and rbcS is clustered with the rbcL gene, suggesting an operon type of arrangement. The Porphyra chloroplast genome is distinctive, also, in that part of it has sequence homology to plasmid-like DNA molecules which co-isolate with the chloroplast DNA.     

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.     

Physical mapping of the plastid genome from the chlorophyll c-containing alga,Cryptomonas Φ

Summary A physical map of the circular plastid genome of Cryptomonas has been constructed using the enzymes SacI, BamHI, SmaI, SalI, PstI and XhoI. In addition, fine-structural mapping of the inverted repeat region has been performed using AvaI, BglII, EcoRI and XbaI. The inverted repeat is very small, encompassing no more than 6 kb and containing only genes for the rRNAs. It divides the plastid genome into a small singlecopy region of 12–13 kb which contains genes for phycoerythrin and the 32 kd photosystem II polypeptide, and a large single-copy region of 93–94 kb, giving a total size of 118 kb. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) and the beta subunit of ATP synthase CF₁ are encoded in the large single-copy region. The evolutionary significance of the organization of this plastid genome, the first presented from the chlorophyll c-, phycobiliprotein-containing group of algae, is discussed.     

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.     

A new gene encoding tRNAPro (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts

The chloroplast genome of black pine (Pinus thumbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA^Pro (GGG) and tRNA^Arg (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA^Gly (UCC) gene and full copies of tRNA^His (GUG), tRNA^Thr (GGU) and tRNA^Ser (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolutio. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the two-out-of-three and the U:N wobble mechanisms.     

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     

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.     

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     

Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications

Summary The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.     

Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae

In order to find further genes of the mitochondrial fatty acid synthase, we searched the genome of Saccharomyces cerevisiae for sequences that are homologous to conserved regions of bacterial fatty acid synthase genes. We found the gene products of ORF YKL055c (EMBL Accession No. X75781) and of YOR221C (EMBL Accession No. X92441) to be homologous to bacterial 3-oxoacyl-(acyl carrier protein) reductases and to malonyl-CoA:ACP-transferases, respectively. We disrupted these two genes which in both cases led to a respiratory deficient phenotype, as is the case for the genes encoding a mitochondrial acyl carrier protein and a β-ketoacyl-ACP synthase. We propose to call the above mentioned genes OAR1[3-oxo-acyl-(acyl carrier protein) reductase] and MCT1 (malonyl-CoA:ACP transferase). They are presumed to be part of a type-II mitochondrial fatty acid synthase, a relic of the endosymbiontic origin of mitochondria, delivering substrates for phospholipid re-modelling and/or repair. Received: 25 April / 16 September 1997     

Initial results of a genome survey for novel alzheimer's disease risk genes: Association with a locus on the X chromosome

As the initial step in a systematic genome survey, 16 simple sequence tandem repeat polymorphisms that span the X chromosome at an average spacing of 10 cM were examined for allelic associations with typical-onset Alzheimer's disease (AD). The efficiency of this survey was substantially enhanced by genotyping pools of genomic DNA from 50 autopsy-confirmed AD cases and 50 autopsied controls who were similar in sex ratio, race, and age at death. The frequency of the DXS1047 202-bp allele was twice as common among AD cases (0.45 ± S.E. 0.06) than controls (0.22 ± S.E. 0.05), a finding that was reproduced in an independent and geographically disparate sample. Consistent with Hardy-Weinberg equilibrium, the proportion of women with AD who carried the 202-bp allele, 73% was nearly double that observed for men with AD, 38%. However, the frequency of the 202-bp allele was similar for men and women and the presence of this allele did not affect the age at onset of dementia in either sex. Furthermore, the frequency of the DXS1047 202-bp allele in AD cases and controls was unaffected by the APOE genotype, indicating that these two loci modulate AD risk independently. Finally, the frequency of the 202-bp allele among 50 autopsy-confirmed cases of Parkinson's disease (0.29 ± S.E. 0.06) was indistinguishable from the control value, reflecting relative specificity for this allelic association with AD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:98–107, 1998. © 1998 Wiley-Liss, Inc.     

Initial results of a genome survey for novel alzheimer's disease risk genes: association with a locus on the X chromosome

As the initial step in a systematic genome survey, 16 simple sequence tandem repeat polymorphisms that span the X chromosome at an average spacing of 10 cM were examined for allelic associations with typical-onset Alzheimer's disease (AD). The efficiency of this survey was substantially enhanced by genotyping pools of genomic DNA from 50 autopsy-confirmed AD cases and 50 autopsied controls who were similar in sex ratio, race, and age at death. The frequency of the DXS1047 202-bp allele was twice as common among AD cases (0.45 ± S.E. 0.06) than controls (0.22 ± S.E. 0.05), a finding that was reproduced in an independent and geographically disparate sample. Consistent with Hardy-Weinberg equilibrium, the proportion of women with AD who carried the 202-bp allele, 73% was nearly double that observed for men with AD, 38%. However, the frequency of the 202-bp allele was similar for men and women and the presence of this allele did not affect the age at onset of dementia in either sex. Furthermore, the frequency of the DXS1047 202-bp allele in AD cases and controls was unaffected by the APOE genotype, indicating that these two loci modulate AD risk independently. Finally, the frequency of the 202-bp allele among 50 autopsy-confirmed cases of Parkinson's disease (0.29 ± S.E. 0.06) was indistinguishable from the control value, reflecting relative specificity for this allelic association with AD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:98–107, 1998. © 1998 Wiley-Liss, Inc.     

The trpA gene on the plastid genome of Cyanidium caldarium strain RK-1

The trpA gene (for the subunit of tryptophan synthase) was found on the plastid genome of the primitive unicellular red alga Cyanidium caldarium strain RK-1. This is the first example of an actively-transcribed gene for tryptophan synthase encoded on a plastid genome. In contrast to trpA, trpB (the gene for the subunit of tryptophan synthase) was encoded in the cell nucleus. Considering the primitive characteristics of C. caldarium, trpB must have been lost from the plastid genome before trpA.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the following accession number D17791     

Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome

Campoletis sonorensis virus (CsV) (Polydnaviridae) is a large eucaryotic DNA virus with a structurally complex genome consisting of 28 or more superhelical (SH) DNA segments. Little is known of the relationship between different SH DNAs, but some SH DNAs do cross hybridize, indicating a relatedness between certain SH DNAs. In a previous study of viral expression in parasitized Heliothis virescens larvae, several partially homologous viral mRNAs were also identified (G. W. Blissard, S. B. Vinson, and M. D. Summers, 1986, J. Virol. 57, 318-327). To study the organization of the viral genome and the relationship between two partially homologous viral mRNAs, we analyzed cDNA clones and a cloned SH DNA segment of the CsV genome. Of the two CsV mRNAs examined (1.6 and 1.0 kb), both mRNAs were abundant at 48 hr after parasitization and the 1.6-kb mRNA was detected as early as 2 hr after parasitization. Nucleotide sequence analyses of cDNA clones representing the two partially homologous CsV mRNAs (1.6 and 1.0 kb) show that the two CsV mRNAs share five regions of imperfect homology (68 to 88%) which include a large part of each mRNA. These data indicate that the two mRNAs are transcribed from two separate but closely related CsV genes. Comparison of predicted amino acid sequences shows that the two related viral genes encode proteins with divergent amino acid sequences. Northern and Southern hybridization analyses using cloned cDNAs as probes showed that one CsV mRNA (1.6 kb) is homologous to CsV SH DNAs W, R, and M, while the other mRNA (1.0 kb) shows strong homology only to SH DNA W. By cloning and Southern hybridization mapping of the 15.8-kbp SH DNA W, we demonstrate that the genes for the related 1.6- and 1.0-kb mRNAs are located on different regions of this single 15.8-kbp SH DNA. Alignment of nucleotide sequences from a cloned viral genomic DNA and a cDNA demonstrates that the CsV gene encoding the 1.6-kb mRNA is a spliced gene containing at least two introns. Conservation of splice junctions between the two mRNAs suggests that the 1.0-kb mRNA is also spliced. These data represent the detailed analysis of two closely related CsV mRNAs abundantly expressed in parasitized H. virescens larvae, the first observation of related viral genes in a eucaryotic DNA virus, the demonstration of splicing in the Polydnaviridae, and the cloning and mapping of one of the largest SH DNA segments of the CsV genome.     

The genes of both subunits of ribulose-1,5-bisphosphate carboxylase constitute an operon on the plastome of a red alga

Summary Plastid (pt) DNA from the red alga Porphyridium aerugineum was purified by CsCI gradient centrifugation. An EcoRI library of the ptDNA was screened with a gene probe specific for the gene encoding the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco EC 4.1.1.39) from spinach. A 5.8 kb EcoRI clone containing the LSU gene (rbcL) was isolated and the DNA sequence of the Porphyridium rbcL gene and its flanking regions was determined. An open reading frame was found 130 by downstream from the rbcL gene that shows homology to genes coding for the small subunit of Rubisco (rbcS) from higher plants and cyanobacteria. Both genes (rbcL + rbcS) are cotranscribed. Comparison of rbcL and rbcS sequences from Porphyridium, higher plants and cyanobacteria seems to reveal a remarkable evolutionary distance between the plastids of the red algae (rhodoplasts), chloroplasts and cyanobacteria.     

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