The inconsistent distribution of introns in the T-even phages indicates recent genetic exchanges.

Group I self-splicing introns are present in the td, nrdB and sunY genes of bacteriophage T4. We previously reported that whereas the td intron is present in T2, T4 and T6, the nrdB intron is present in T4 only. These studies, which argue in favor of introns as mobile genetic elements, have been extended by defining the distribution of all three T4 introns in a more comprehensive collection of T2, T4 and T6 isolates. The three major findings are as follows: First, all three introns are inconsistently distributed throughout the T-even phage family. Second, different T2 isolates have different intron complements, with T2H and T2L having no detectable introns. Third, the intron open reading frames are inherited or lost as a unit with their respective flanking intron core elements. Furthermore, exon sequences flanking sites where introns are inserted in the T4 td, sunY and nrdB genes were determined for all the different T-even isolates studied. Six of eighteen residues surrounding the junction sequences are identical. In contrast, a comprehensive comparison of exon sequences in intron plus and intron minus variants of the sunY gene indicate that sequence changes are concentrated around the site of intron occurrence. This apparent paradox may be resolved by hypothesizing that the recombination events responsible for intron acquisition or loss require a consensus sequence, while these same events result in sequence heterogeneity around the site.     

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages

Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of PhiI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.     

Self-splicing of the bacteriophage T4 group I introns requires efficient translation of the pre-mRNA in vivo and correlates with the growth state of the infected bacterium

Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.     

Widespread distribution of a group I intron and its three deletion derivatives in the lysin gene of Streptococcus thermophilus bacteriophages

Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilus bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillus phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from Lactococcus, Lactobacillus, and Bacillus phages.     

Multiple self-splicing introns in bacteriophage T4: evidence from autocatalytic GTP labeling of RNA in vitro

RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions. One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe. These mapped in or near the unlinked genes nrdB and nrdC. A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro. Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4.     

Phylogenetic evidence for horizontal transmission of group I introns in the nuclear ribosomal DNA of mushroom-forming fungi

Group I introns were discovered inserted at the same position in the nuclear small-subunit ribosomal DNA (nuc-ssu-rDNA) in several species of homobasidiomycetes (mushroom-forming fungi). Based on conserved intron sequences, a pair of intron-specific primers was designed for PCR amplification and sequencing of intron-containing rDNA repeats. Using the intron-specific primers together with flanking rDNA primers, a PCR assay was conducted to determine presence or absence of introns in 39 species of homobasidiomycetes. Introns were confined to the genera Panellus, Clavicorona, and Lentinellus. Phylogenetic analyses of nuc-ssu-rDNA and mitochondrial ssu-rDNA sequences suggest that Clavicorona and Lentinellus are closely related, but that Panellus is not closely related to these. The simplest explanation for the distribution of the introns is that they have been twice independently gained via horizontal transmission, once on the lineage leading to Panellus, and once on the lineage leading to Lentinellus and Clavicorona. BLAST searches using the introns from Panellus and Lentinellus as query sequences retrieved 16 other similar group I introns of nuc-ssu-rDNA and nuclear large-subunit rDNA (nuc-lsu-rDNA) from fungal and green algal hosts. Phylogenetic analyses of intron sequences suggest that the mushroom introns are monophyletic, and are nested within a clade that contains four other introns that insert at the same position as the mushroom introns, two from different groups of fungi and two from green algae. The distribution of host lineages and insertion sites among the introns suggests that horizontal and vertical transmission, homing, and transposition have been factors in intron evolution. As distinctive, heritable features of nuclear rDNAs in certain lineages, group I introns have promise as phylogenetic markers. Nevertheless, the possibility of horizontal transmission and homing also suggest that their use poses certain pitfalls.      

A unique group of self-splicing introns in bacteriophage T4

We describe in this review, the salient splicing features of group I introns of bacteriophage T4 and propose, a hypothetical model to fit in the self-splicing of nrdB intron of T4 phage. Occurrence of non-coding sequences in prokaryotic cells is a rare event while it is common in eukaryotic cells, especially the higher eukaryotes. Therefore, T4 bacteriophage can serve as a good model system to study the evolutionary aspects of splicing of introns. Three genes of T4 phage were found to have stretches of non-coding sequences which belonged to the group IA type introns of self-splicing nature.     

Origin and evolution of chloroplast group I introns in lichen algae

The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free‐living Trebouxiophyceae compared to free‐living non‐Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non‐Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free‐living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free‐living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms.     

Nested Evolution of a tRNALeu(UAA) Group I Intron by both Horizontal Intron Transfer and Recombination of the Entire tRNA Locus

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.     

Distribution and evolution of introns in drosophila amylase genes

While the two amylase genes of Drosophila melanogaster are intronless, the three genes of D. pseudoobscura harbor a short intron. This raises the question of the common structure of the Amy gene in Drosophila species. We have investigated the presence or absence of an intron in the amylase genes of 150 species of Drosophilids. Using polymerase chain reaction (PCR), we have amplified a region that surrounds the intron site reported in D. pseudoobscura and a few other species. The results revealed that most species contain an intron, with a variable size ranging from 50 to 750 bp, although the very majoritary size was around 60–80 bp. Several species belonging to different lineages were found to lack an intron. This loss of intervening sequence was likely due to evolutionarily independent and rather frequent events. Some other species had both types of genes: In the obscura group, and to a lesser extent in the ananassae subgroup, intronless copies had much diverged from intron-containing genes. Base composition of short introns was found to be variable and correlated with that of the surrounding exons, whereas long introns were all A-T rich. We have extended our study to non-Drosophilid insects. In species from other orders of Holometaboles, Lepidoptera and Hymenoptera, an intron was found at an identical position in the Amy gene, suggesting that the intron was ancestral. Received: 23 October 1995 / Accepted: 5 March 1996     

Group I introns as mobile genetic elements: facts and mechanistic speculations--a review

Group I introns form a structural and functional group of introns with widespread but irregular distribution among very diverse organisms and genetic systems. Evidence is now accumulating that several group I introns are mobile genetic elements with properties similar to those originally described for the omega system of Saccharomyces cerevisiae: mobile group I introns encode sequence-specific double-strand (ds) endoDNases, which recognize and cleave intronless genes to insert a copy of the intron by a ds-break repair mechanism. This mechanism results in: the efficient propagation of group I introns into their cognate sites; their maintenance at the site against spontaneous loss; and, perhaps, their transposition to different sites. The spontaneous loss of group I introns occurs with low frequency by an RNA-mediated mechanism. This mechanism eliminates introns defective for mobility and/or for RNA splicing. Mechanisms of intron acquisition and intron loss must create an equilibrium, which explains the irregular distribution of group I introns in various genetic systems. Furthermore, the observed distribution also predicts that horizontal transfer of intron sequences must occur between unrelated species, using vectors yet to be discovered.     

The recent transfer of a homing endonuclease gene

The myxomycete Didymium iridis (isolate Panama 2) contains a mobile group I intron named Dir.S956-1 after position 956 in the nuclear small subunit (SSU) rRNA gene. The intron is efficiently spread through homing by the intron-encoded homing endonuclease I-DirI. Homing endonuclease genes (HEGs) usually spread with their associated introns as a unit, but infrequently also spread independent of introns (or inteins). Clear examples of HEG mobility are however sparse. Here, we provide evidence for the transfer of a HEG into a group I intron named Dir.S956-2 that is inserted into the SSU rDNA of the Costa Rica 8 isolate of D.iridis. Similarities between intron sequences that flank the HEG and rDNA sequences that flank the intron (the homing endonuclease recognition sequence) suggest that the HEG invaded the intron during the recent evolution in a homing-like event. Dir.S956-2 is inserted into the same SSU site as Dir.S956-1. Remarkably, the two group I introns encode distantly related splicing ribozymes with phylogenetically related HEGs inserted on the opposite strands of different peripheral loop regions. The HEGs are both interrupted by small spliceosomal introns that must be removed during RNA maturation.     

The molecular evolution and structural organization of self-splicing group I introns at position 516 in nuclear SSU rDNA of myxomycetes

Group I introns are relatively common within nuclear ribosomal DNA of eukaryotic microorganisms, especially in myxomycetes. Introns at position S516 in the small subunit ribosomal RNA gene are particularly common, but have a sporadic occurrence in myxomycetes. Fuligo septica, Badhamia gracilis, and Physarum flavicomum, all members of the family Physaraceae, contain related group IC1 introns at this site. The F. septica intron was studied at the molecular level and found to self-splice as naked RNA and to generate full-length intron RNA circles during incubation. Group I introns at position S516 appear to have a particularly widespread distribution among protists and fungi. Secondary structural analysis of more than 140 S516 group I introns available in the database revealed five different types of organization, including IC1 introns with and without His-Cys homing endonuclease genes, complex twin-ribozyme introns, IE introns, and degenerate group I-like introns. Both intron structural and phylogenetic analyses indicate a multiple origin of the S516 introns during evolution. The myxomycete introns are related to S516 introns in the more distantly related brown algae and Acanthamoeba species. Possible mechanisms of intron transfer both at the RNA- and DNA-levels are discussed in order to explain the observed widespread, but scattered, phylogenetic distribution.     

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.     

Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.

A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.