Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.     

The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Background

The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved.

Methodology/Principal Findings

We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3′ overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5′-GACGGGAAGACCCT-3′) and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA.

Conclusions/Significance

The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.     

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

Molecular Biology - Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only...     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

A Group I Intron in the SSUrDNA of Some Naegleria spp. Demonstrated by Polymerase Chain Reaction Amplification

ABSTRACT. The small subunit ribosomal DNA (SSUrDNA) of all described Naegleria spp. was amplified by polymerase chain reaction with universal primers. In all strains of N. andersoni andersoni, N. andersoni jamiesoni, N. australiensis italica and two related strains, and one out of four clusters of N. gruberi , a band of approximately 3.3 kb was obtained. All other strains displayed a band with the expected DNA length of 2.0 kb. This means the former have a 1.3 kb intron in the SSUrDNA. Restriction analysis demonstrated that the intron is between two conserved Pst I sites at the 5' end of the SSUrDNA It also suggested the introns might not be identical in each species or subspecies. The Pst I fragment of SSUrDNA containing the 1.3 kb insert in N. andersoni andersoni was cloned and sequence. The 1,296-nucleotide insert is situated in helix 19 of the SSUrDNA, which is an area of conserved primary and secondary structure. Sequence and secondary structure analyses of the insert revealed it is a group I intron. This group I intron is very large and contains an open reading frame that could serve to encode a polypeptide of 139 amino acids in size.     

A Mutational Analysis of DNA Binding and Cleavage by the Homing Endonuclease PI-SceI

We have carried out an extensive mutational analysis of PI-SceI, the best studied intein-like homing endonuclease of the LAGLIDADG family, to find out which amino acid residues are involved in substrate binding and processing. Our analysis was focused on domain I, in which two regions were shown to be in contact with DNA, and on domain II, in which the amino acid residues making up catalytic centers I and II were identified and their role in catalysis investigated. As a result of our comprehensive mutational analysis a model is presented for DNA binding and cleavage by PI-SceI.     

Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene

The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in Escherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.     

Novel Group I Introns Encoding a Putative Homing Endonuclease in the Mitochondrial <Emphasis Type="Italic">cox1</Emphasis> Gene of Scleractinian Corals

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 (cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans. [Reviewing Editor: Dr. Niles Lehman]     

A Group I Intron in the 18S Ribosomal DNA from the Parasitic Fungus Isaria japonica

The nucleotide sequence of the 18S rDNA coding gene in the ascomycetes parasitic fungus Isaria japonica contains a group I intron with a length of 379 nucleotides. The identification of the DNA sequence as a group I intron is based on its sequence homology to other fungal group I introns. Its group I intron contained the highly conserved sequence elements P, Q, R, and S found in other group I introns. Surprisingly, the intron sequence of I. japonica is more similar to that of Ustilago maydis than to the one found in Sclerotinia sclerotiorum. This is in contrast to the sequence identity found on the neighboring rDNA. This is an interesting finding and suggests a horizontal transfer of group I intron sequences. Received: 19 September 1997 / Accepted: 10 September 1998     

Genomic Analysis of Freshwater Cyanophage Pf-WMP3 Infecting Cyanobacterium <Emphasis Type="Italic">Phormidium foveolarum</Emphasis>: The Conserved Elements for a Phage

Cyanophages are ecologically abundant, genetically diverse in aquatic environments, and affect the population and evolutionary trajectories of their hosts. After reporting the cyanophage Pf-WMP4 genome (Liu et al. in Virology 366:28–39, 2007), we hereby present a related cyanophage, Pf-WMP3, which also infects the freshwater cyanobacterium Phormidium foveolarum. The Pf-WMP3 genome contains 43,249 bp with 234 bp direct terminal repeats. The overall genome organization and core genes of the two phages are comparable to those of the T7 supergroup phages. Compared with Pf-WMP4, cyanophage Pf-WMP3 has diverged extensively at the DNA level; however, they are closely related at the protein level and genome architecture. The left arm genes for the two phages, which mainly encode the DNA replication machinery, are not conserved in the gene order. Whereas the right arm genes of the two phages coding for structural proteins show high similarity in amino acid sequences and modular architecture, indicating that they have retained similar development strategies. The differences in similarity levels between the left and right arm genes suggest that the structural genes are the most conserved elements for a phage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Group I Intron in the Nuclear Small Subunit rRNA Gene of Cryptendoxyla hypophloia, an Ascomycetous Fungus: Evidence for a New Major Class of Group I Introns

The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns. Received: 11 January 1998 / Accepted: 12 October 1998     

Suppression of a Mutation in Gene 3 of Bacteriophage T7 (T7 Endonuclease I) by Mutations in Phage and Host Polynucleotide Ligase

Bacteriophage T7 bearing amber mutations in both gene 1.3 (T7 DNA ligase) and gene 3 (T7 endonuclease I) are viable when grown in suppressor-negative, ligase-negative hosts. This is evidenced by a high plating efficiency and a large burst size compared to the single mutants. These findings may be explained by a limited destruction of cellular DNA by the double mutant.     

The Peperomia Mitochondrial coxI Group I Intron: Timing of Horizontal Transfer and Subsequent Evolution of the Intron

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out. Received: 29 May 1997 / Accepted: 1 November 1997