An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.
ABSTRACT: The thymidylate synthase gene (thy) (EC 220.127.116.11) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.
Project description:A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.
Project description:A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1.
Project description:Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 18.104.22.168), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.
Project description: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.
Project description:The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.
Project description:Catalytic RNAs are often regarded as molecular fossils from the RNA World, yet it is usually difficult to get more specific information about their evolution. Here we have investigated the coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases (RTs). Unlike group I introns, there has been no obvious reshuffling between intron RNA structures and ORFs. Of the six classes of intron structures that encode ORFs, three are conventional forms of group II A1, B1, and B2 secondary structures, whereas the remaining classes are bacterial, are possibly associated with the most primitive ORFs, and have unusual features and hybrid features of group IIA and group IIB intron structures. Based on these data, we propose a new model for the evolution of group II introns, designated the retroelement ancestor hypothesis, which predicts that the major RNA structural forms of group II introns developed through coevolution with the intron-encoded protein rather than as independent catalytic RNAs, and that most ORF-less introns are derivatives of ORF-containing introns. The model is supported by the distribution of ORF-containing and ORF-less introns, and by numerous examples of ORF-less introns that contain ORF remnants.
Project description:Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.
Project description:BACKGROUND: Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. METHODS: Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. RESULTS: Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. CONCLUSIONS: Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.
Project description:It is well understood how mobile introns home to allelic sites, but how they are stimulated to transpose to ectopic locations on an evolutionary timescale is unclear. Here we show that a group I intron can move to degenerate sites under oxidizing conditions. The phage T4 td intron endonuclease, I-TevI, is responsible for this infidelity. We demonstrate that I-TevI, which promotes mobility and is subject to autorepression and translational control, is also regulated posttranslationally by a redox mechanism. Redox regulation is exercised by a zinc finger (ZF) in a linker that connects the catalytic domain of I-TevI to the DNA binding domain. Four cysteines coordinate Zn(2+) in the ZF, which ensures that I-TevI cleaves its DNA substrate at a fixed distance, 23-25 nucleotides upstream of the intron insertion site. We show that the fidelity of I-TevI cleavage is controlled by redox-responsive Zn(2+) cycling. When the ZF is mutated, or after exposure of the wild-type I-TevI to H(2)O(2), intron homing to degenerate sites is increased, likely because of indiscriminate DNA cleavage. These results suggest a mechanism for rapid intron dispersal, joining recent descriptions of the activation of biomolecular processes by oxidative stress through cysteine chemistry.
Project description: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.