Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis

Yeo, Chew Chieng; Tham, Jill Maelan; Yap, Melvyn W.; Poh, Chit Laa

doi:10.1099/00221287-143-8-2833

Cited by 35 publications

(29 citation statements)

References 35 publications

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“…Much was made of the initial finding of group II introns in bacteria, as their discovery added fuel to the debate concerning the evolutionary origins of eukaryotic spliceosomal introns (22,70) which have both structural and functional similarities to group II introns (53,79,84). Yet, the number of group II introns in bacteria is small, many of which are inferred only from database matches to reverse transcriptases or maturases encoded within known introns (13,41,73,89), and only two have been shown to splice or be mobile in vivo (48,49,55,80) (Table 2). While group II intron homing is mechanistically distinct from group I intron homing, the principle is similar; group II introns home from intron-containing to intronless alleles.…”

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confidence: 99%

Barriers to Intron Promiscuity in Bacteria

2000

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2"The field of bacterial viruses is a fine playground for serious children who ask ambitious questions."Max DelbruckThe first bacterial intron, a self-splicing group I intron, was found to interrupt the thymidylate synthase (td) gene of the Escherichia coli phage T4 (11). The second and third bacterial group I introns were found to interrupt the aerobic (nrdB) and anaerobic (nrdD [initially named sunY]) ribonucleotide reductases of phage T4 (29,90), and another group I intron was soon discovered in the DNA polymerase gene of SPO1, a Bacillus phage (25). From this (admittedly) small sampling of phage genomes, one might have naively expected that group I introns would be abundant in phage or bacterial genomes, especially since subsequent laboratory experiments demonstrated that group I introns could propagate themselves (by a process called homing) throughout populations of intron-minus alleles with near 100% efficiency (5, 68). That a similar homing phenomenon had also been previously demonstrated for a group I intron in the large rRNA gene of yeast mitochondria (34) gave additional support to the notion that group I introns should be able to spread efficiently throughout populations. However, this expected outcome has never been realized in natural phage populations; some phage populations harbor many introns, while other related phage populations are strangely lacking in any introns whatsoever (Table 1). Why do group I introns have an unusual distribution in phage and bacterial genomes, and what potential barriers might exist to prevent their spread?A similar question might be asked of group II introns in bacteria. Much was made of the initial finding of group II introns in bacteria, as their discovery added fuel to the debate concerning the evolutionary origins of eukaryotic spliceosomal introns (22, 70) which have both structural and functional similarities to group II introns (53,79,84). Yet, the number of group II introns in bacteria is small, many of which are inferred only from database matches to reverse transcriptases or maturases encoded within known introns (13,41,73,89), and only two have been shown to splice or be mobile in vivo (48,49,55,80) (Table 2). While group II intron homing is mechanistically distinct from group I intron homing, the principle is similar; group II introns home from intron-containing to intronless alleles. Many elegant biochemical and genetic experiments have unraveled the complexities of bacterial group II intron homing (14,48,49), and based on these results, there seems no a priori reason why group II introns should not be able to spread efficiently through populations of intron-minus alleles. The paucity of bacterial group II introns becomes even more perplexing given the recent demonstration of group II intron transposition to novel chromosomal sites (15). That group II introns are abundant in mitochondrial and chloroplast genomes (52) and present in bacterial genomes but at lower levels (and absent in phages) only adds to the mystery surrounding the lack of group II intro...

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confidence: 99%

Barriers to Intron Promiscuity in Bacteria

2000

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“…This is an important observation as, although group II introns have recently been found in a number of different bacteria (5,6,8,10,13,15,26), only the Lactococcus lactis group II intron Ll.ltrB has previously been showed to be capable of in vivo splicing (13). The same group II intron has also been found independently in the putative relaxase gene of a conjugative element inserted in the chromosome of L. lactis 712 (22).…”

Section: Discussionmentioning

confidence: 77%

“…Over the past few years, group II introns have been found in a number of different bacteria (5,6,8,10,13,15,26). In most cases, however, splicing and mobility have not been reported to occur in vivo.…”

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Demonstration that the Group II Intron from the Clostridial Conjugative Transposon Tn 5397 Undergoes Splicing In Vivo

et al. 2001

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Previous work has identified the conjugative transposon Tn5397 from Clostridium difficile. This element was shown to contain a group II intron. Tn5397 can be conjugatively transferred from C. difficile to Bacillus subtilis. In this work we show that the intron is spliced in both these hosts and that nonspliced RNA is also present. We constructed a mutation in the open reading frame within the intron, and this prevented splicing but did not prevent the formation of the circular form of the conjugative transposon (the likely transposition intermediate) or decrease the frequency of intergeneric transfer of Tn5397. Therefore, the intron is spliced, but splicing is not required for conjugation of Tn5397.

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“…; accession no. U77945) (37). As observed in all RTs, the Trt sequence contains most of the highly conserved amino acids that fall into seven distinct domains (underlined sequences labeled I to VII).…”

Section: Methodsmentioning

confidence: 99%

A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus ( Geobacillus ) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

Vellore

Moretz

Lampson

2004

Appl Environ Microbiol

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The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75°C.Heat-stable DNA polymerases are an essential and widely used tool in molecular biology research. Such applications as cloning DNA via the PCR, determining the sequence of DNA, and generating random mutations in DNA all use these thermostable DNA polymerases. Of particular utility is the process of reverse transcriptase PCR (RT-PCR), where an RNA molecule is converted to a more stable cDNA copy by reverse transcription, followed by amplification of the cDNA via PCR (9). This is a powerful technique used to detect and analyze RNA molecules from many types of cells.In addition to a thermostable DNA polymerase, RT-PCR also usually requires a dedicated enzyme like an RT, whose RNA-dependent, DNA polymerase activity allows RNA to be reverse transcribed into cDNA. cDNA synthesis is most often done using RTs derived from animal viruses at temperatures below about 50°C. However, there are a number of advantages to synthesizing cDNA at hot temperatures (above 50°C). For example, higher temperatures will melt the secondary structures that can form in the RNA template and thus increase the length of cDNA product synthesized by the RT (7, 10). In addition, higher temperatures also increase the specificity and accuracy of DNA synthesis during reverse transcription (8,11,14). Thus, an RT that remains stable and active at high temperatures is especially useful for cDNA synthesis in RT-PCRs, as well as other applications.A search of the nearly completed genome sequence of the eubacterial, thermophile Bacillus stearothermophilus (now designated Geobacillus stearothermophilus) (19) indicated the presence of an open reading frame (ORF) that we have designated as trt for thermostable intron RT. This ORF potentially codes for a 420-a...

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Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis

Cited by 35 publications

References 35 publications

Barriers to Intron Promiscuity in Bacteria

Barriers to Intron Promiscuity in Bacteria

Demonstration that the Group II Intron from the Clostridial Conjugative Transposon Tn 5397 Undergoes Splicing In Vivo

A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus ( Geobacillus ) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

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