Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon.

The Escherichia coli aadA gene product, which confers resistance to spectinomycin and streptomycin, has been widely used as a dominant selectable marker for chloroplast transformation of Chlamydomonas and tobacco. An aadA transformation cassette was adapted for expression in Euglena gracilis chloroplasts by replacing the Chlamydomonas promoter and 3' untranslated region (UTR) with the E. gracilis psbA promoter and 3' UTR. Transgenic DNA was introduced into E. gracilis chloroplasts by biolistic transformation. Streptomycin- and spectinomycin-resistant colonies were obtained, which screened positively for the presence of the transforming vector by PCR amplification. Although integration of the transforming DNA into the chloroplast genome was not detected, transforming DNA was stably maintained in the chloroplast as an episomal element during continuous selection on antibiotics. The aadA cassette was also inserted into a transformation vector which contained the independently expressed psbK operon from either E. gracilis or a closely related species, E. stellata. The psbK operon contained at least two group III introns and a group III twintron, was highly expressed, and was only 1.5 kb in length. In transgenic E. gracilis chloroplasts, a truncated E. stellata psbK operon was transcribed, and the resultant pre-mRNA was accurately spliced. This system should allow the first direct analysis of group II and group III intron-splicing mechanisms. In addition, it could prove useful in the study of many other Euglena transcription and processing events.

Comparison of psbK operon organization and group III intron content in chloroplast genomes of 12 Euglenoid species.

A novel mixed operon has been identified in the photosynthetic protist E. gracilis. The genes for psbK, ycf12, psaM, and trnR are co-transcribed. The resulting tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and intron splicing to form three mature monocistronic mRNAs and a tRNA. A group III twintron and a group III intron are located in psbK. Another group III intron is found in ycf12. The psbK operon has been cloned by PCR amplification from nine related Euglenoid species. In each species, the gene order and content of the psbK operon is conserved. The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements. Intron content varies significantly from species to species. Based on a comparison of the intron content with the results of phylogenetic analysis, group III intron evolution within the Euglenoid lineage is much more complex than previously believed.

A maturase-encoding group III twintron is conserved in deeply rooted euglenoid species: are group III introns the chicken or the egg?

The fourth intron of the Euglena gracilis chloroplast photosystem II gene, psbCi4, is a 1,605-bp twintron composed of two group III introns and a coding locus for a 458-aa polypeptide, mat1, located in the internal intron. psbCi4 homologs have been identified in seven euglenoids, including E. myxocylindracea, E. viridis, E. deses, E. pisciformis, Cryptoglena pigra, Eutreptia sp., and Lepocinclis beutschlii. All of the species examined contain both the group III twintron and the mat1 locus, revealing a more widespread occurrence of group III introns than previously known. The L. beutschlii mat1 locus is interrupted by two novel mini-group II introns of 224 and 258 nt, the smallest group II introns yet identified. Reverse transcriptase polymerase chain reaction analysis confirmed the splicing boundaries of the external and internal E. myxocylindracea, E. viridis, and E. deses introns as well as the novel L. beutschlii mat1 introns. As determined by comparative phylogenetic analysis, group III introns contain a structural homolog of group II intron domain VI. The mat1 loci encode peptide motifs characteristic of group II intron maturases. A group III intron-encoded protein whose predicted sequence is similar to group II intron-encoded maturases and a bona fide domain VI within group III introns are compelling evidence for a common ancestor of group II and group III introns.

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.

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. gracilis petB 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. gracilis petB 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.

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.

The Euglena gracilis intron-encoded mat2 locus is interrupted by three additional group II introns.

The 4,144 nt Euglena gracilis chloroplast psbC intron 2 has been characterized as a single, cis-spliced 593 nt group II intron interrupted by an open reading frame of 758 codons in the loop region of domain IV. The 2,277 nt coding region of orf 758 is interrupted by two additional group II introns of 369 nt and 352 nt. Another 553 nt group II intron is located in the 5' untranslated leader region of orf 758. Because the psbC intron 2 orf encodes a maturase-like protein that has reverse transcriptase domains and a domain X characteristic of group II intron-encoded proteins, the locus has been designated mat2. The psbC intron 2 is the first member of a new category of twintron, characterized by introns within a gene within another intron. A potential role of psbC intron 2 as a "founder" intron involved in the spread of introns to new sites in the plastid genome of the Euglenophycae is discussed.

Alternative splicing of the Euglena gracilis chloroplast roaA transcript.

A novel gene, roaA (ribosomal operon-associated gene), encoding a potential RNA-binding protein has been identified in the rpl23 ribosomal protein operon of the Euglena gracilis chloroplast genome. The roaA gene is interrupted by one group III and three group II introns. Introns 1 and 2 of roaA can be interpreted as a twintron, formed from the insertion of a group II intron into the 5'splice site of a group III intron. Alternative splicing of the group III intron results in two distinct transcripts encoding proteins of 516 and 514 amino acids. Group III introns may play a role in the generation of alternatively spliced transcripts.

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.

A group III intron is formed from domains of two individual group II introns.

A 1352-nucleotide intron within the Euglena gracilis chloroplast ycf8 gene has been characterized as a complex twintron with overlapping internal introns and alternative splicing pathways. Partially spliced pre-mRNAs were characterized by a combination of cDNA cloning and sequencing, Northern hybridization, and S1 nuclease protection analyses. In the predominant pathway, two internal group II introns (601 and 392 nucleotides) are spliced from subdomain ID of an external group II intron (359 nucleotides). In an alternative pathway, following excision of the 601-nucleotide intron, splicing of a group III intron occurs. This group III intron is recruited from sequences of the external intron and the 392-nucleotide intron. This is the first evidence that a group III intron can be derived from portions of existing group II introns. The mechanism of group III intron formation may also be relevant to the evolution of nuclear introns from putative group II intron ancestors.

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.

Gene structure and expression of a novel Euglena gracilis chloroplast operon encoding cytochrome b6 and the beta and epsilon 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.

The psaA operon pre-mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth.

The chloroplast genome of Euglena gracilis contains a psaA operon which encodes a lysine tRNA gene, trnK; psaA and psaB photosystem I genes, and psbE, psbF, psbL and psbJ photosystem II genes. The pre-mRNA of the psaA operon undergoes a complex processing pathway of 5' and 3' tRNA processing, splicing of 11 group II introns and one group II twintron, plus three intercistronic RNA cleavage events. The accumulated transcripts of the psaA operon have been characterized by Northern hybridization, S1 nuclease analysis and primer extension RNA sequencing. The mature 5' end of the psaA-psaB-psbE-psbF-psbL-psbJ hexacistronic transcript lies 8 nt downstream of the trnK gene, and is the result of intercistronic trnK-psaA cleavage. Other intercistronic processing events occur between the psaA and psaB genes and the psaB and psbE genes. Processing at the latter site produces a dicistronic mRNA of PSI genes and a tetracistronic mRNA of PSII genes. The PSI dicistronic transcript is further processed to monocistronic psaA and psaB mRNAs. Secondary structural motifs within the intercistronic regions may be recognition sites for processing. The steady-state levels of psaA operon mRNAs from Euglena grown under several different conditions have been determined. Accumulated transcripts from all growth conditions are spliced, and a proportion are also processed at the intercistronic sites. The products of intercistronic processing increase from heterotrophic dark- to heterotrophic light-grown Euglena, and from heterotrophic light- to photoautotrophic light-grown Euglena. The differential accumulation of psaA operon mRNAs may be a means of chloroplast gene regulation or, alternatively, a consequence of gene expression during chloroplast development.

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.

Complete sequence of Euglena gracilis chloroplast DNA.

We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.

A complex twintron is excised as four individual introns.

Twintrons are introns-within-introns excised by sequential splicing reactions. A new type of complex twintron comprised of four individual group III introns has been characterized. The external intron is interrupted by an internal intron containing two additional introns. This 434 nt complex twintron within a Euglena gracilis chloroplast ribosomal protein gene is excised by four sequential splicing reactions. Two of the splicing reactions utilize multiple 5'- and/or 3'-splice sites. These findings are evidence that introns with multiple active splice sites can be formed by the repeated insertion of introns into existing introns.

A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns.

The structure of a Euglena gracilis chloroplast operon encoding four subunits of the chloroplast ATP synthase complex and two ribosomal proteins has been determined. These six genes contain 17 introns. This operon is transcribed as a hexacistronic primary transcript which is subsequently processed to monocistronic mRNAs. The linear order of these genes, 5'-rps2-atpI-atpH-atpF-atpA-rps18-3' , encoding ribosomal protein S2, chloroplast ATP synthase subunits CF0IV, CF0III, CF0I, CF1 alpha and ribosomal protein S18, respectively, is similar to the equivalent operons of prokaryotes, cyanelles and land-plant chloroplasts. This operon differs from those of these other organisms in the co-transcription of rps18 and in intron content.

Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.

Chloroplasts.

New features of chloroplast gene expression are continually being discovered, particularly in the area of post-transcriptional RNA processing. RNA editing of chloroplast pre-mRNAs occurs in both monocotyledons and dicotyledons, and involves both initiator and internal codons. The view of introns as mobile genetic elements expands both with the identification of additional twintrons in Euglena chloroplast genes and with studies on the homing group I introns of Chlamydomonas.