Biomedical subjects

Steven Zimmerly

Publications and source records attributed to Steven Zimmerly.

11 recordsLinked to original sources

PubMed2006

Self-splicing of a group IIC intron: 5' exon recognition and alternative 5' splicing events implicate the stem-loop motif of a transcriptional terminator.

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5' junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem-loop and IBS1 motifs, suggesting that the 5' exon is recognized by both elements. These results provide the first example of a group II intron that uses 5' splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5' exon definition, and that the terminator stem-loop substitutes in part for the missing IBS2-EBS2 (intron and exon binding sites 2) interaction.

Alternative Splicing↗

PubMed2005

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures.

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.

Base Sequence↗

PubMed2004

Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements.

Bordetella bacteriophages generate diversity in a gene that specifies host tropism. This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR). Here we describe the genetic basis for diversity generation. The directionality of information transfer is determined by a 21-base-pair sequence present at the 3' end of VR. On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence. This allows the diversity-generating system to minimize variability to the subset of bases under selection. Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.

Adaptation, Physiological↗

PubMed2004

Mobile group II introns.

Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.

Bacteria↗

PubMed2004

Principles of 3' splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis.

We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis. One intron (B.a.I1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.I2) splices well while having apparently degenerated features. The spliced exons of B.a.I2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame. Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-EBS3 pairings, and can be as far away as four nucleotides from the wild-type site. Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.I2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF. The finding suggests that the structural irregularities of B.a.I2 may be an adaptation to facilitate gene expression in vivo.

Alternative Splicing↗

PubMed2003

Database for mobile group II introns.

Group II introns are self-splicing RNAs and retroelements found in bacteria and lower eukaryotic organelles. During the past several years, they have been uncovered in surprising numbers in bacteria due to the genome sequencing projects; however, most of the newly sequenced introns are not correctly identified. We have initiated an ongoing web site database for mobile group II introns in order to provide correct information on the introns, particularly in bacteria. Information in the web site includes: (1) introductory information on group II introns; (2) detailed information on subfamilies of intron RNA structures and intron-encoded proteins; (3) a listing of identified introns with correct boundaries, RNA secondary structures and other detailed information; and (4) phylogenetic and evolutionary information. The comparative data should facilitate study of the function, spread and evolution of group II introns. The database can be accessed at http://www.fp.ucalgary.ca/group2introns/.

Databases, Nucleic Acid↗

PubMed2003

ORF-less and reverse-transcriptase-encoding group II introns in archaebacteria, with a pattern of homing into related group II intron ORFs.

Although group II intron retroelements are prevalent in eubacteria, they have not been identified in archaebacteria in the first 10 genomes sequenced. However, the recently sequenced archael genome of Methanosarcina acetivorans contains 21 group II introns, including 7 introns that do not encode reverse transcriptase ORFs. To our knowledge, these are the first retroelements identified in archaebacteria, and the first ORF-less group II introns in bacteria. Furthermore, the insertion pattern of the introns is highly unusual. The introns appear to insert site-specifically into ORFs of other group II introns, forming nested clusters of up to four introns, but there are no flanking exons that could encode a functional protein after the introns have been spliced out.

Amino Acid Sequence↗

PubMed2002

Compilation and analysis of group II intron insertions in bacterial genomes: evidence for retroelement behavior.

Group II introns are novel genetic elements that have properties of both catalytic RNAs and retroelements. Initially identified in organellar genomes of plants and lower eukaryotes, group II introns are now being discovered in increasing numbers in bacterial genomes. Few of the newly sequenced bacterial introns are correctly identified or annotated by those who sequenced them. Here we have compiled and thoroughly analyzed group II introns and their fragments in bacterial DNA sequences reported to GenBank. Intron distribution in bacterial genomes differs markedly from the distribution in organellar genomes. Bacterial introns are not inserted into conserved genes, are often inserted outside of genes altogether and are frequently fragmented, suggesting a high rate of intron gain and loss. Some introns have multiple natural homing sites while others insert after transcriptional terminators. All bacterial group II introns identified to date encode reverse transcriptase open reading frames and are either active retroelements or derivatives of retroelements. Together, these observations suggest that group II introns in bacteria behave primarily as retroelements rather than as introns, and that the strategy for group II intron survival in bacteria is fundamentally different from intron survival in organelles.

Base Sequence↗

PubMed2002

Non-cognate template usage and alternative priming by a group II intron-encoded reverse transcriptase.

Group II introns are retroelements that site-specifically insert into DNA through homing. They are also implicated in related phenomena such as ectopic site insertions and precise intron deletions, but little is known about how group II intron reverse transcriptases (RTs) interact with non-cognate substrates. Here we show that wild-type aI2 RT readily reverse transcribes non-cognate RNAs in mitochondrial RNP particles when the aI2 intron structure is misfolded. In two closely related priming mutants, 1degree2(DeltaD5) and 1(+)2(DeltaD5), which contain wild-type RT but a disrupted intron structure, the RT has substantially lost specificity for aI2 RNA and copies multiple RNAs present in the RNP particles, using an alternative priming mechanism. The RT in 1degree2(DeltaD5) RNP particles can also copy exogenous RNAs but unlike the endogenous templates, a complementary primer is required, suggesting that the alternative priming event is specific to RT-RNA interactions formed in vivo. Alternatively primed cDNAs from strains 1degree2(DeltaD5), 1(+)2(DeltaD5) and 1degree2(P714T) (containing the mutation P714T in the RT) do not use homing site DNA as a primer, but appear to utilize a non-complementary DNA primer of approximately ten nucleotides. The alternative priming mechanism and reverse transcription of non-cognate templates has implications for in vivo reverse transcription of non-intronic RNAs, which is expected to occur during intron deletions and other retroprocessing events.

DNA Primers↗

PubMed2002

The dispersal of five group II introns among natural populations of Escherichia coli.

Group II introns are self-splicing RNAs that also act as retroelements in bacteria, mitochondria, and chloroplasts. Group II introns were identified in Escherichia coli in 1994, but have not been characterized since, and, instead, other bacterial group II introns have been studied for splicing and mobility properties. Despite their apparent intractability, at least five distinct group II introns exist naturally in E. coli strains. To illuminate their function and learn how the introns have dispersed in their natural host, we have investigated their distribution in the ECOR reference collection. Two introns were cloned and sequenced to complete their partial sequences. Unexpectedly, southern blots showed all ECOR strains to contain fragments and/or full-length copies of group II introns, with some strains containing up to 15 intron copies. One intron, E.c.14, has two natural homing sites in IS629 and IS911 elements, and the intron can be present in one, both, or neither homing site in a given strain. Nearly all strains that contain full-length introns also contain unfilled homing sites, suggesting either that mobility is highly inefficient or that most full-length copies are nonfunctional. The data indicate independent mobility of the introns, as well as mobility via the host DNA elements, and overall, the pattern of intron distribution resembles that of IS elements.

Base Sequence↗

PubMed2002

Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns.

Group I and group II introns are unrelated classes of introns that each encode proteins that facilitate intron splicing and intron mobility. Here we describe a new subfamily of nine introns in fungi that are group II introns but encode LAGLIDADG ORFs typical of group I introns. The introns have fairly standard group IIB1 RNA structures and are inserted into three different sites in SSU and LSU rRNA genes. Therefore, introns should not be assumed to be group I introns based solely on the presence of a LAGLIDADG ORF.

Base Sequence↗