A group III twintron encoding a maturase-like gene excises through lariat intermediates.

The 1605 bp intron 4 of the Euglena gracilis chloroplast psbC gene was characterized as a group III twintron composed of an internal 1503 nt group III intron with an open reading frame of 1374 nt (ycf13, 458 amino acids), and an external group III intron of 102 nt. Twintron excision proceeds by a sequential splicing pathway. The splicing of the internal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpaired adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phosphodiester bonds. This bond is susceptible to hydrolysis by the debranching activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycf13 mRNA accumulates primarily as a linear RNA, although a lariat precursor can also be detected. The ycf13 gene encodes a maturase-like protein that may be involved in group III intron metabolism.     

The chloroplast genome of Anomochloa marantoidea (Anomochlooideae; Poaceae) comprises a mixture of grass-like and unique features

Features in the complete plastome of Anomochloa marantoidea (Poaceae) were investigated. This species is one of four of Anomochlooideae, the crown node of which diverged before those of any other grass subfamily. The plastome was sequenced from overlapping amplicons using previously designed primers. The plastome of A. marantoidea is 138412 bp long with a typical gene content for Poaceae. Five regions were examined in detail because of prior surveys that identified structural alterations among graminoid Poales. Anomochloa marantoidea was found to have an intron in rpoC1, unlike other Poaceae. The insertion region of rpoC2 is unusually short in A. marantoidea compared with those of other grasses, but with atypically long subrepeats. Both ycf1 and ycf2 are nonfunctional as is typical in grasses, but A. marantoidea has a uniquely long ψycf1. Finally, the rbcL-psaI spacer in A. marantoidea is atypically short with no evidence of the ψrpl23 locus found in all other Poaceae. Some of these features are of noteworthy dissimilarity between A. marantoidea and those crown grasses for which entire plastomes have been sequenced. Complete plastome sequences of other Anomochlooideae and outgroups will further advance our understanding of the evolutionary events in the plastome that accompanied graminoid diversification.     

Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii.

The sequence of the coding region of the rRNA operon of rat-derived Pneumocystis carinii has been completed, including the genes for 5.8S and 26S rRNA. These genes show homology to the rRNA genes of yeast, and an apparent group I self-splicing intron is present in the 26S rRNA gene. Like a similar intron in the 16S rRNA gene, this intron is in a phylogenetically conserved region. Variation in the 26S rRNA sequence was noted between P. carinii organisms isolated from two different sources.     

Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

We cloned and sequenced the psbK gene, coding for a small photosystem II component (PSII-K), from the transformable cyanobacterium, Synechocystis sp. PCC 6803, and determined the N-terminal sequence of mature PSII-K. The psbK gene product is processed by cleaving off eight amino acid residues from the N terminus. A mutant lacking psbK was constructed; this mutant grew photoautotrophically, but its growth rate was reduced. The number of photosystem II reaction centers on a chlorophyll basis was decreased by less than a factor of 2 in the psbK-deletion mutant. In Synechocystis sp. PCC 6803, the psbK gene is transcribed as a single gene and is not part of an operon. Single-site mutations were introduced into psbK leading to early termination or deletion of the presequence. The phenotype of these mutants strongly resembles that of the psbK deletion mutant, indicating that indeed the change in phenotype in the deletion mutant is directly correlated with PSII-K. PSII-K is not essential for photosystem II assembly or activity but is needed for optimal photosystem II function.     

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.     

Elimination of a group II intron from a plastid gene causes a mutant phenotype

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.     

A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.     

Chloroplast genomes of the diatoms <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Thalassiosira pseudonana</Emphasis>: comparison with other plastid genomes of the red lineage

The chloroplast genomes of the pennate diatom Phaeodactylum tricornutum and the centric diatom Thalassiosira pseudonana have been completely sequenced and are compared with those of other secondary plastids of the red lineage: the centric diatom Odontella sinensis, the haptophyte Emiliania huxleyi, and the cryptophyte Guillardia theta. All five chromist genomes are compact, with small intergenic regions and no introns. The three diatom genomes are similar in gene content with 127-130 protein-coding genes, and genes for 27 tRNAs, three ribosomal RNAs and two small RNAs (tmRNA and signal recognition particle RNA). All three genomes have open-reading frames corresponding to ORFs148, 355 and 380 of O. sinensis, which have been assigned the names ycf88, ycf89 and ycf90. Gene order is not strictly conserved, but there are a number of conserved gene clusters showing remnants of red algal origin. The acpP, tsf and psb28 genes appear to be on the way from the plastid to the host nucleus, indicating that endosymbiotic gene transfer is a continuing process.     

Extensive structural conservation exists among several homologs of two Euglena chloroplast group II introns

82 of the 155 chloroplast introns in Euglena gracilis have been categorized as group II introns. Because they are shorter and more divergent than group II introns from other organisms, the assignment of these Euglena introns to the group II class has been questioned. In the current study, two homologs of E. gracilispetB intron 1 and four homologs of psbC intron 2 have been isolated from related species and characterized. Based on a comparative sequence analysis of intron homologs, the intron core and four of the six helical domains present in the canonical group II intron structural model are conserved in E. gracilispetB intron 1 and psbC intron 2 and all of their homologs. Distal portions of domain I, which are involved in most of the tertiary interactions, are less well conserved than the central core.     

Ycf12 is a core subunit in the photosystem II complex

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of approximately 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.     

The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

The complete sequence of the chloroplast genome of cassava (Manihot esculenta, Euphorbiaceae) has been determined. The genome is 161,453 bp in length and includes a pair of inverted repeats (IR) of 26,954 bp. The genome includes 128 genes; 96 are single copy and 16 are duplicated in the IR. There are four rRNA genes and 30 distinct tRNAs, seven of which are duplicated in the IR. The infA gene is absent; expansion of IRb has duplicated 62 amino acids at the 3′ end of rps19 and a number of coding regions have large insertions or deletions, including insertions within the 23S rRNA gene. There are 17 intron-containing genes in cassava, 15 of which have a single intron while two (clpP, ycf3) have two introns. The usually conserved atpF group II intron is absent and this is the first report of its loss from land plant chloroplast genomes. The phylogenetic distribution of the atpF intron loss was determined by a PCR survey of 251 taxa representing 34 families of Malpighiales and 16 taxa from closely related rosids. The atpF intron is not only missing in cassava but also from closely related Euphorbiaceae and other Malpighiales, suggesting that there have been at least seven independent losses. In cassava and all other sequenced Malphigiales, atpF gene sequences showed a strong association between C-to-T substitutions at nucleotide position 92 and the loss of the intron, suggesting that recombination between an edited mRNA and the atpF gene may be a possible mechanism for the intron loss.     

The complete structure of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.     

Extensive structural conservation exists among several homologs of two Euglena chloroplast group II introns

82 of the 155 chloroplast introns in Euglena gracilis have been categorized as group II introns. Because they are shorter and more divergent than group II introns from other organisms, the assignment of these Euglena introns to the group II class has been questioned. In the current study, two homologs of E. gracilispetB intron 1 and four homologs of psbC intron 2 have been isolated from related species and characterized. Based on a comparative sequence analysis of intron homologs, the intron core and four of the six helical domains present in the canonical group II intron structural model are conserved in E. gracilispetB intron 1 and psbC intron 2 and all of their homologs. Distal portions of domain I, which are involved in most of the tertiary interactions, are less well conserved than the central core. Received: 27 June 1997 / Accepted: 6 August 1997     

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

Chickpea (Cicerarietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5'exon, similar to other legumes. Two genes have lost their introns, one in the 3'exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate "IR-lacking clade" (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering.     

Six newly sequenced chloroplast genomes from prasinophyte green algae provide insights into the relationships among prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species

Background

Because they represent the earliest divergences of the Chlorophyta, the morphologically diverse unicellular green algae making up the prasinophytes hold the key to understanding the nature of the first viridiplants and the evolutionary patterns that accompanied the radiation of chlorophytes. Nuclear-encoded 18S rDNA phylogenies unveiled nine prasinophyte clades (clades I through IX) but their branching order is still uncertain. We present here the newly sequenced chloroplast genomes of Nephroselmis astigmatica (clade III) and of five picoplanktonic species from clade VI (Prasinococcus sp. CCMP 1194, Prasinophyceae sp. MBIC 106222 and Prasinoderma coloniale) and clade VII (Picocystis salinarum and Prasinophyceae sp. CCMP 1205). These chloroplast DNAs (cpDNAs) were compared with those of the six previously sampled prasinophytes (clades I, II, III and V) in order to gain information both on the relationships among prasinophyte lineages and on chloroplast genome evolution.

Results

Varying from 64.3 to 85.6 kb in size and encoding 100 to 115 conserved genes, the cpDNAs of the newly investigated picoplanktonic species are substantially smaller than those observed for larger-size prasinophytes, are economically packed and contain a reduced gene content. Although the Nephroselmis and Picocystis cpDNAs feature a large inverted repeat encoding the rRNA operon, gene partitioning among the single copy regions is remarkably different. Unexpectedly, we found that all three species from clade VI (Prasinococcales) harbor chloroplast genes not previously documented for chlorophytes (ndhJ, rbcR, rpl21, rps15, rps16 and ycf66) and that Picocystis contains a trans-spliced group II intron. The phylogenies inferred from cpDNA-encoded proteins are essentially congruent with 18S rDNA trees, resolving with robust support all six examined prasinophyte lineages, with the exception of the Pycnococcaceae.

Conclusions

Our results underscore the high variability in genome architecture among prasinophyte lineages, highlighting the strong pressure to maintain a small and compact chloroplast genome in picoplanktonic species. The unique set of six chloroplast genes found in the Prasinococcales supports the ancestral status of this lineage within the prasinophytes. The widely diverging traits uncovered for the clade-VII members (Picocystis and Prasinophyceae sp. CCMP 1205) are consistent with their resolution as separate lineages in the chloroplast phylogeny.     

蕨类植物叶绿体基因ycf94的分子进化研究

ycf94基因是近年来在叶绿体基因组中新发现的一个基因,在蕨类植物中表现高度保守。该研究共选取94种蕨类植物,在系统发育背景下,对ycf94基因的结构特征、密码子偏好性、进化速率和适应性进化进行分析。结果表明, ycf94基因的密码子偏好性较弱,偏好使用以A/U结尾的密码子,且不同物种间的偏好性存在一定差异。密码子偏好性的形成主要受到突变压的影响,同时也存在其他因素的作用;基于凤尾蕨科和其他蕨类中ycf94基因的结构特征存在区别,对两者的分子替换速率进行了比较,表明颠换率、非同义替换率和ω值间存在显著差异;仅检测出1个正选择位点74A,强烈的负选择作用表明ycf94基因的结构和功能基本趋于稳定。这为蕨类系统发育分析提供了新依据,并提供了解析ycf94基因功能的线索。