Gracilis Chloroplast Genome Research Articles

Deep sequencing analysis of chloroplast transcription and splicing in Euglena gracilis

The transcription and splicing of the Euglena gracilis (E. gracilis) chloroplast have been studied only on the scale of individual genes, and a comprehensive genome-scale analysis is lacking. We addressed this question in this study using transcriptomic RNA sequencing (RNA-seq) technology. Upon mapping tens of millions of RNA-seq reads onto the E. gracilis chloroplast genome, the transcription of protein-coding genes, including monocistronic genes and genes in operons, was visualized, and the expression levels of these genes were quantified. The splicing of intron-containing genes in the E. gracilis chloroplast genome was also visualized and quantified, with introns, including some twintrons, and exons being clearly discerned. The correct coding sequences of psbD, rpl23 and rpl16 were deduced based on read coverage of the corresponding genes and confirmed experimentally. Additionally, a new group III intron was identified from the 5’ UTR of petB. This study updates our knowledge on the transcription and splicing of the E. gracilis chloroplast, and will be instrumental in the mechanistic exploration of gene transcription and intron splicing in the E. gracilis chloroplast.

Complete chloroplast genome of the Malus baccata var. gracilis provides insights into the evolution and phylogeny of Malus species.

Malus baccata (L.) var. gracilis (Rehd.) has high ornamental value and breeding significance, andcomparative chloroplast genome analysis was applied to facilitate genetic breeding for desired traits andresistance and provide insight into the phylogeny of this genus. Using data from whole-genomesequencing, a tetrameric chloroplast genome with a length of 159,992 bp and a total GC content of36.56% was constructed. The M. baccata var. gracilis chloroplast genome consists of a large single-copy sequence (88,100 bp), a short single-copy region (19,186 bp), and two inverted repeat regions, IRa(26,353 bp) and IRb (26,353 bp). This chloroplast genome contains 112 annotated genes, including 79protein-coding genes (nine multicopy), 29 tRNA genes (eight multicopy), and four rRNA genes (allmulticopy). Calculating the relative synonymous codon usage revealed a total of 32 high-frequencycodons, and the codons exhibited a biased usage pattern towards A/U as the ending nucleotide.Interspecific sequence comparison and boundary analysis revealed significant sequence variation in thevast single-copy region, as well as generally similar expansion and contraction of the SSC and IRregions for 10 analyzed Malus species. M. baccata var. gracilis and Malus hupehensis were groupedtogether into one branch based on phylogenetic analysis of chloroplast genome sequences. Thechloroplast genome of Malus species provides an important foundation for species identification, geneticdiversity analysis, and Malus chloroplast genetic engineering. Additionally, the results can facilitate theuse of pendant traits to improve apple tree shape.

Tracing Patterns of Chloroplast Evolution in Euglenoids: Contributions from Colacium vesiculosum and Strombomonas acuminata (Euglenophyta)

The chloroplast genomes of two photosynthetic euglenoids, Colacium vesiculosum Ehrenberg (128,889 bp), and Strombomonas acuminata (Schmarda) Deflandre (144,167 bp) have been sequenced. These chloroplast genomes in combination with those of Euglena gracilis, Eutreptia viridis, and Eutreptiella gymnastica provide a snapshot of euglenoid chloroplast evolution allowing comparisons of gene content, arrangement, and expansion. The gene content of the five chloroplast genomes is very similar varying only in the presence or absence of, rrn5, roaA, psaI, psaM, rpoA, and two tRNAs. Large gene rearrangements have occurred within the C. vesiculosum and S. acuminata chloroplast genomes. Most of these rearrangements represent repositioning of entire operons rather than single genes. When compared with previously sequenced genomes, C. vesiculosum and S. acuminata chloroplast genomes more closely resemble the E. gracilis chloroplast genome in size of the genome, number of introns, and gene order than they do those of the Eutreptiales. Overall, the chloroplast genomes of these five species show an evolutionary trend toward increased intron number, a decrease in gene density, and substantial rearrangement of gene clusters.

Comparative chloroplast genomics between Euglena viridis and Euglena gracilis (Euglenophyta)

Bennett M.S., Wiegert K.E. and Triemer R.E. 2012. Comparative chloroplast genomics between Euglena viridis and Euglena gracilis (Euglenophyta). Phycologia 51: 711–718. DOI: 10.2216/12-017.1The chloroplast genomes of Euglena gracilis, Eutreptia viridis, Eutreptiella gymnastica, Colacium vesiculosum, Strombomonas acuminata and the colourless Euglena (Astasia) longa, which had secondarily lost the ability to photosynthesize, were previously reported. These studies had shown that there was a great diversity in the size of euglenoid chloroplast genomes and in the arrangement of gene clusters. However, while these genomes provided important insights into the evolution of the chloroplast genome across genera, they did not address genomic variability within a genus. In an effort to continue with these investigations, we sequenced the chloroplast genome of Euglena viridis and compared this to the E. gracilis chloroplast genome in order to explore intrageneric chloroplast evolution. The chloroplast genome of E. viridis was also compared to those of the other previously published euglenoid chloroplast genomes. Our results showed that while the chloroplast genome of E. viridis most closely resembled that of E. gracilis, the chloroplast genomes did show significant differences. The chloroplast genome of E. viridis was far more compact, had a gene cluster that was reversed in both gene order and strand orientation, had a region that was comprised almost entirely of open reading frames and had substantially fewer introns. However, despite these differences, it was clear from the chloroplast genome sequence that E. viridis and E. gracilis were very closely related and that the majority of euglenoid chloroplast evolution probably occurred before the divergence of the genus Euglena.

Euglena gracilis chloroplast psbB, psbT, psbH and psbN gene cluster: regulation of psbB-psbT pre-mRNA processing.

A 2.4 kb region of the Euglena gracilis chloroplast genome containing the genes psbT, psbH and psbN was characterized. The mRNAs transcribed from psbB, psbT, psbH and psbN were analyzed by northern hybridization, S1 nuclease protection analysis and primer extension RNA sequencing. The gene pairs psbB-psbT and psbH-psbN are cotranscribed from opposite strands. The 5' end of the psbN-psbH transcript and the intercistronic cleavage sites between psbB-psbT and psbN-psbH were determined. The extent of psbB-psbT intercistronic cleavage is greater during photoautotrophic than heterotrophic growth and thus may be developmentally regulated. Processing is absent in the non-photosynthetic E. gracilis mutant Y9Z1NaL.

Gene structure and expression of a novel Euglena gracilis chloroplast operon encoding cytochrome b6 and the ? and ? subunits of the H+-ATP synthase complex

The genes encoding cytochrome b6 of the chloroplast cytochrome b6/f complex (petB) and the ATP synthase CF1-beta subunit (atpB) and epsilon-subunit (atpE) were identified on the EcoD fragment of the Euglena gracilis chloroplast genome. The complete nucleotide sequence of these three genes was determined. The petB-atpB-atpE genes are cotranscribed as a tricistronic operon. This gene organization differs from that of land plants in which atpB-atpE form a discistronic operon, and petB is within the psbB-ycf8-psbH-petB-petD operon. Euglena cytochrome b6 and the beta-subunit of the chloroplast ATP synthase are very similar in derived amino acid sequence to the corresponding gene products from other organisms. The epsilon-subunit of the chloroplast ATP synthase complex is more divergent. In Euglena, the petB-atpB-atpE genes contain introns, including two twintrons, at eight different positions. All of the intron positions were confirmed by analysis of cDNAs. Two independent intercistronic RNA processing events and 11 splicing reactions lead to the accumulation of the mature petB, atpB and atpE monocistronic mRNAs.

Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns

Two new and important features of introns have emerged from analysis of the Euglena gracilis chloroplast genome. One is a new class of introns, designated group III, that may be the closest contemporaries to nuclear pre-mRNA introns. The second is introns that are interrupted by other introns termed twintrons.

Intercistronic group III introns in polycistronic ribosomal protein operons of chloroplasts

A novel ribosomal protein operon in the Euglena gracilis chloroplast genome was characterized. It encodes the genes for ribosomal proteins S4 and S11 (rps4 and rps11). The coding region of the rps11 gene is interrupted by two introns of 107 and 100 bp. The introns belong to a distinct class known as group III introns. The major transcript from this operon was characterized as a fully spliced dicistronic rps4-rps11 mRNA by RNA blot analysis, primer extension sequencing, and cDNA cloning and sequencing. An additional 95 nucleotide (nt) group III intron was identified in the 123 nt rps4-rps11 intercistronic region. The identification of the intercistronic intron between the rps4 and rps11 genes was unexpected. Other RNA transcripts from regions of the genome that could potentially contain intercistronic introns were re-examined and two other intercistronic, group III introns were found. These are located in a large ribosomal protein operon between the genes for the ribosomal proteins L23 and L2, and between L14 and L5. There are at least 50 group III introns in the E. gracilis chloroplast genome. All but 6 are found in genes encoding protein components of the transcriptional and translational apparatus. The distribution of group III introns and the unusual location of intercistronic group III introns may reflect some aspect of gene expression, or provide some insight into the mechanism of their splicing.

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa

Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3'-terminal portion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.

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

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 greater than A greater than G greater than 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.

The Euglena gracilis chloroplast genome: structural features of a DNA region possibly carrying the single origin of DNA replication

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.

Structure of the Euglena gracilis chloroplast gene ( psbA) coding for the 32-kDa protein of Photosystem II

An EcoRI fragment from Euglena gracilis chloroplast DNA, called Eco.I (4.9 kbp), contains the gene for the ‘32-kDa’ thylakoid membrane protein of Photosystem II ( psb A gene), the tRNA 2 Leu gene and additional sequences which possibly code for two proteins of unknown function. The transcription polarity is the same for all these coding sequences. The psb A gene is split: 4 introns (size range, 433–616 bp) separate 5 exons (size range, 39–579 bp) which code for a protein of M r 38380 (345 amino acids). The Euglena protein is about 87% homologous with the higher plant counterparts but it contains 5 lysine residues. Euglena gracilis Chloroplast genome psbA trnL

Location of the single gene for elongation factor Tu on the Euglena gracilis chloroplast chromosome.

The structural gene for elongation factor Tu (EF-Tu) has been mapped by heterologous hybridization to a 2900 base pair sequence of Euglena gracilis Klebs Strain Z Pringsheim chloroplast DNA within the EcoRI fragment, Eco N. The hybridization probes were obtained from a HhaI restriction fragment containing internal sequences to Escherichia coli EF-Tu, located in the tufA gene locus, and from an EcoRI restriction fragment of chloroplast DNA from the eukaryotic algae Chlamydomonas reinhardii, containing the 3' end of the chloroplast EF-Tu gene. This is the second identified protein gene locus to be mapped on the E. gracilis chloroplast genome, and the first using prokaryotic DNA as a probe.

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.

Location of a phenylalanine tRNA gene on the physical map of the Euglena gracilis chloroplast genome

A tRNA phe from Euglena gracilis bacillaris chloroplasts was purified and hybridized with restriction fragments of chloroplast DNA. From the hybridization pattern, the location of the corresponding gene on the physical map of the chloroplast genome was determined.

Restriction endonuclease map of Euglena gracilis chloroplast DNA

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.