Clostridium difficile IStron CdISt1: discovery of a variant encoding two complete transposase-like proteins

Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.     

Novel group I intron in the tRNA(Leu)(UAA) gene of a gamma-proteobacterium isolated from a deep subsurface environment

A group I intron has been found to interrupt the anticodon loop of the tRNA(Leu)(UAA) gene in a bacterium belonging to the gamma-subdivision of Proteobacteria and isolated from a deep subsurface environment. The subsurface isolate SMCC D0715 was identified as belonging to the genus Pseudomonas. The group I intron from this isolate is the first to be reported for gamma-proteobacteria, and the first instance of a tRNA(Leu)(UAA) group I intron to be found in a group of bacteria other than cyanobacteria. The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10. The D0715 intron possesses the 11-nt motif CCUACG. UAUGG in its P8 region, a feature not common in bacterial introns. To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements.     

Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley

Protoplasts of a barley ( Hordeum vulgare L. cv. Golden Promise) suspension cell line were used for PEG-mediated gene transfer. Transient gene expression in barley protoplasts was studied using a chimeric CaMV 35S cat construct, which was only poorly expressed in barley cells. However, insertion of exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene in the 5'-untranslated leader of the construct strongly stimulated gene expression. By using the optimized chimeric cat construction the amount of CAT protein that was reached 19 hours after DNA uptake was 0.5% of total protein, which was calculated from western blot data.
As an alternative marker gene for expression studies, we also tested the firefly luciferase gene in barley protoplasts. Low level expression of chimeric CaMV 35S luciferase genes could be highly stimulated when Sh1 exon1 and intron1 were inserted in the 5'-untranslated leader of the constructs. Enhanced luciferase gene expression by Shrunken-1 intronic sequences enabled us to monitor gene integration events early after DNA uptake using a promoterless luciferase marker gene, which could only be expressed after integration behind an endogenous promoter.     

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.     

Two group I introns with long internal open reading frames in the chloroplast psbA gene of Chlamydomonas moewusii.

We report the nucleotide sequence of the chloroplast psbA gene encoding the 32 kilodalton protein of photosystem II from Chlamydomonas moewusii. Like its land plant homologues, this green algal protein consists of 353 amino acids. The C. moewusii psbA gene is composed of three exons containing 252, 11 and 90 codons and of two group I introns containing 2363 and 1807 nucleotides. Each of the introns features an internal open reading frame (ORF) that potentially encodes a basic protein of more than 300 residues. The primary sequences of the putative intron-encoded proteins are unrelated and none of them shares conserved elements with any of the proteins predicted from the group I intron sequences published so far. The first C. moewusii intron is inserted at the same position as the fourth intron of the psbA gene from Chlamydomonas reinhardtii; the second intron lies at a novel site downstream of this position. On the basis of their RNA secondary structures, the C. moewusii introns 1 and 2 can be assigned to subgroups IA and IB, respectively. However, intron 1 is not typical of subgroup IA introns, its most unusual feature being the location of the ORF in the "loop L5" region. To our knowledge, this is the first time that an ORF is located in this region of the group I intron structure.     

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.     

Mobility of Two Optional G + C-Rich Clusters of the Var1 Gene of Yeast Mitochondrial DNA

Yeast mtDNA contains two different kinds of mobile optional sequences, two group I introns and a short G + C-rich insertion to some var1 genes. Movement of each element in crosses has been called gene conversion though little is known about the mechanism of G + C cluster conversion. A new allele of the var1 gene found in mtDNA of Saccharomyces capensis is described that permitted a more detailed comparison between intron mobility and G + C cluster conversion. The S. capensis var1 gene lacks the cc+ element present in all S. cerevisiae var 1 genes and the previously described optional a+ element. In crosses with cc+ a- and cc+ a+ S. cerevisiae strains, both clusters were found to be mobile and, in the latter cross, appear to convert independently and only to homologous insertion sites. No evidence for flanking marker coconversion (a hallmark feature of intron conversion) was obtained despite the availability of nearby physical markers on both sides of cluster conversion sites. These data indicate that G + C cluster conversion has only a superficial resemblance to intron mobility; analogies to procaryotic transposition mechanisms are considered.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Genetic Insertions and Diversification of the PolB-Type DNA Polymerase (gp43) of T4-Related Phages

In Escherichia coli phage T4 and many of its phylogenetic relatives, gene 43 consists of a single cistron that encodes a PolB family (PolB-type) DNA polymerase. We describe the divergence of this phage gene and its protein product (gp43) (gene product 43) among 26 phylogenetic relatives of T4 and discuss our observations in the context of diversity among the widely distributed PolB enzymes in nature. In two T4 relatives that grow in Aeromonas salmonicida phages 44RR and 25, gene 43 is fragmented by different combinations of three distinct types of DNA insertion elements: (a) a short intercistronic untranslated sequence (IC-UTS) that splits the polymerase gene into two cistrons, 43A and 43B, corresponding to N-terminal (gp43A) and C-terminal (gp43B) protein products; (b) a freestanding homing endonuclease gene (HEG) inserted between the IC-UTS and the 43B cistron; and (c) a group I intron in the 43B cistron. Phage 25 has all three elements, whereas phage 44RR has only the IC-UTS. We present evidence that (a) the split gene of phage 44RR encodes a split DNA polymerase consisting of a complex between gp43A and gp43B subunits; (b) the putative HEG encodes a double-stranded DNA endonuclease that specifically cleaves intron-free homologues of the intron-bearing 43B site; and (c) the group I intron is a self-splicing RNA. Our results suggest that some freestanding HEGs can mediate the homing of introns that do not encode their own homing enzymes. The results also suggest that different insertion elements can converge on a polB gene and evolve into a single integrated system for lateral transfer of polB genetic material. We discuss the possible pathways for the importation of such insertion elements into the genomes of T4-related phages.     

DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.

The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-PorI. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-PorI, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-PorI cleaves optimally at 80 degrees C, with kcat and Km values of about 2 min-1 and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within a 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with those of eucaryotic homing endonucleases.     

Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.     

CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.     

The sporadic occurrence of a group I intron-like element in the mtDNA rnl gene of Ophiostoma novo-ulmi subsp. americana

The presence of group I intron-like elements within the U7 region of the mtDNA large ribosomal subunit RNA gene (rnl) was investigated in strains of Ophiostoma novo-ulmi subsp. americana from Canada, Europe and Eurasia, and in selected strains of O. ips, O. minus, O. piceae, O. ulmi, and O. himal-ulmi. This insertion is of interest as it has been linked previously to the generation of plasmid-like mtDNA elements in diseased strains of O. novo-ulmi. Among 197 O. novo-ulmi subsp. americana strains tested, 61 contained a 1.6 kb insertion within the rnl-U7 region and DNA sequence analysis suggests the presence of a group I intron (IA1 type) that encodes a potential double motif LAGLIDADG homing endonuclease-like gene (HEG). Phylogenetic analysis of rnl-U7 intron encoded HEG-like elements supports the view that double motif HEGs originated from a duplication event of a single-motif HEG followed by a fusion event that combined the two copies into one open reading frame (ORF). The data also show that rnl-U7 intron encoded ORFs belong to a clade that includes ORFs inserted into different types of group I introns, e.g. IB, ID, IC3, IA1, present within a variety of different mtDNA genes, such as the small ribosomal subunit RNA gene (rns), apo-cytochrome b gene (cob), NADH dehydrogenase subunit 5 (nad5), cytochrome oxidase subunit 1 gene (coxI), and ATPase subunit 9 gene (atp9).

We also compared the occurrence of the rnl-U7 intron in our collection of 227 strains with the presence of the rnl-U11 group I intron and concluded that the U7 intron appears to be an optional element and the U11 intron is probably essential among the strains tested.