Rare group I intron with insertion sequence element in a bacterial ribonucleotide reductase gene

A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.     

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.     

Ribonucleotide reductase genes of Bacillus prophages: a refuge to introns and intein coding sequences

The ribonucleotide reductase gene tandem bnrdE/bnrdF in SPβ-related prophages of different Bacillus spp. isolates presents different configurations of intervening sequences, comprising one to three of six non-homologous splicing elements. Insertion sites of group I introns and intein DNA are clustered in three relatively short segments encoding functionally important domains of the ribonucleotide reductase. Comparison of the bnrdE homologs reveals mutual exclusion of a group I intron and an intein coding sequence flanking the codon that specifies a conserved cysteine. In vivo splicing was demonstrated for all introns. However, for two of them a part of the mRNA precursor molecules remains unspliced. Intergenic bnrdE–bnrdF regions are unexpectedly long, comprising between 238 and 541 nt. The longest encodes a putative polypeptide related to HNH homing endonucleases.     

Group I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, contains thirty-two introns. Twenty-five of these introns possess the characteristic secondary structures and consensus sequences of group II introns. The remaining seven are group I introns, six of which happen to interrupt the gene coding for subunit 1 of cytochrome oxidase (cox1). Interestingly, the insertion sites of one group II and four group I introns in the cox1 gene coincide with those of the respective fungal mitochondrial interns. Moreover, comparison of the four group I introns with their fungal counterparts shows that group I introns inserted at identical genomic sites in different organisms are indeed related to one another, in terms of the peptide sequences generated from the complete or fragmental ORFs encoded by these introns. At the same time, the liverwort introns turned out to be more divergent from their fungal cognates than the latter are from one another. We therefore conclude that vertical transmission from a common ancestor organism is the simplest explanation for the presence of cognate introns in liverwort and fungal mitochondrial genomes.     

Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences.

The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome. As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data. By this approach we identified a locus on a plasmid in the bacterium Escherichia coli. Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns. An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases. A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA. With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.     

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.

We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction. The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.     

Lineage-specific diversification of killer cell Ig-like receptors in the owl monkey,a New World primate

Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of natural killer cells. In primates, the KIRs are highly diverse as a consequence of variation in gene content, alternative domain composition, and loci polymorphism. We analyzed a bacterial artificial chromosome (BAC) clone draft sequence spanning the owl monkey KIR cluster. The draft sequence had seven ordered yet unconnected contigs containing six full-length and two partial gene models, flanked by the LILRB and FcAR framework genes. Gene models were predicted to encode KIRs with inhibitory, activating, or dual functionality. Four gene models encoded three Ig domain receptors, while three others encoded molecules with four Ig domains. The additional domain resulted from an insertion in tandem of a 2,101 bp fragment containing the last 289 bp of intron 2, exon 3, and intron 3, resulting in molecules with two D0 domains. Re-screening of the owl monkey BAC library and sequencing of partial cDNAs from an owl monkey yielded five additional KIRs, four of which encoded receptors with short cytoplasmic domains with premature stop codons due to either a single nucleotide substitution or deletion or the absence of exon 8. Phylogenetic analysis by domains showed that owl monkey KIRs were monophyletic, clustering independently from other primate KIR lineages. Retroelements found in introns, however, were shared by KIRs from different primate lineages. This suggests that the owl monkey inherited a KIR cluster with a rich history of exon shuffling upon which positive selection for ligand binding operated to diversify the receptors in a lineage-specific fashion. Nucleotide sequence data reported are available in the GenBank database under accession numbers FJ154791–5.     

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.     

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.     

An intron-encoded protein assists RNA splicing of multiple similar introns of different bacterial genes

Four group II introns were found in an unusually intron-rich dnaN gene (encoding the beta subunit of DNA polymerase III) of the cyanobacterium Trichodesmium erythraeum, and they have strong similarities to two introns of the RIR gene (encoding ribonucleotide reductase) of the same organism. Of these six introns, only the RIR-3 intron encodes a maturase protein and showed efficient RNA splicing when expressed in Escherichia coli cells. The other five introns do not encode a maturase protein and did not show RNA splicing in E. coli. But these maturase-less introns showed efficient RNA splicing when the RIR-3 intron-encoded maturase protein was co-expressed from a freestanding gene in the same cell. These findings demonstrated that an intron-encoded protein could function as a general maturase for multiple introns of different genes. Major implications may include an intron-mediated co-regulation of the different genes and a resemblance of the evolutionary origin of spliceosomal introns.     

Ⅱ组内含子(Group Ⅱ Intron)的分布及多样性

Ⅱ组内含子(group Ⅱ intron)存在于原生生物、真菌、藻类、植物细胞器以及细菌和古细菌基因组中.在体内,Ⅱ组内含子可通过两步连续的转酯反应从前体RNA中自剪接,并连接两 侧外显子.许多Ⅱ组内含子的剪接反应是由蛋白质辅助完成的,这种蛋白质有的是由内含子编码,有的是由宿主基因编码.Ⅱ组内含子能够有效地归巢进入无内含子的等位基因,也能 够以低频率逆转座进入非等位基因.转座过程依赖内含子RNA和内含子编码的蛋白质（内切核酸酶活性和逆转录酶活性）.本论文在总结Ⅱ组内含子最新研究成果的基础上,分析Ⅱ组内含子可能的起源和进化途径     

Characterization of a major histocompatibility class II A gene (Clha-DAA) with an embedded microsatellite marker in Atlantic herring (Clupea harengus L.)

An Atlantic herring major histocompatibility class II A ( Clha-DAA ) cDNA sequence has been characterized and was shown to encode a leader peptide, alpha-1 domain, alpha-2 domain, connecting peptide, transmembrane and cytoplasmic region. The Clha-DAA protein sequence has all the characteristics of a teleost class II A protein with conserved cysteines in both the alpha-1 and the alpha-2 domains and two potential N-linked glycosylation sites. Exon 2 sequences encoding the polymorphic alpha-1 domain from different individuals were analysed and revealed the presence of at least two loci. The Clha-DAA gene consists of four exons and three short introns. Four unique intron 3 sequences from multiple individuals were obtained and were shown to contain a (TG)_n microsatellite sequence. Primers were optimized such that only a single microsatellite locus designated Clha-DAA-INTR3 was amplified. Four herring populations from the North Sea and the Baltic Sea were genotyped for Clha-DAA-INTR3 . In total, 16 Clha-DAA-INTR3 alleles were detected; the distribution of the alleles showed no deviation from Hardy–Weinberg expectation. Levels of genetic differentiation among samples were of similar magnitude as have been reported earlier for neutral microsatellite loci between northern North Sea and Baltic Sea herring populations.