In vitro self-splicing reactions of chloroplast and mitochondrial group-I introns in Chlamydomonas eugametos and Chlamydomonas moewusii

1995 ◽  
Vol 27 (2) ◽  
pp. 177-183 ◽  
Author(s):  
Marie-Jos� C�t� ◽  
Monique Turmel
Keyword(s):  
Group I Introns ◽  
Group I ◽  
Chlamydomonas Eugametos ◽  
Chlamydomonas Moewusii ◽  
Self Splicing

scholarly journals A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant

2009 ◽  
Vol 191 (12) ◽  
pp. 4044-4046 ◽  
Author(s):  
Rahul Raghavan ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Group I Intron ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Group I ◽  
23S Rrna Gene ◽  
Self Splicing

ABSTRACT Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.

scholarly journals A Self-Splicing Group I Intron in DNA Polymerase Genes of T7-Like Bacteriophages

2004 ◽  
Vol 186 (23) ◽  
pp. 8153-8155 ◽  
Author(s):  
Richard P. Bonocora ◽  
David A. Shub
Keyword(s):  
Dna Polymerase ◽  
Group I Intron ◽  
Group I Introns ◽  
Homing Endonucleases ◽  
Structural Element ◽  
Group I ◽  
Close Relatives ◽  
Self Splicing

ABSTRACT 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 ΦI 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.

scholarly journals CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

1989 ◽  
Vol 9 (12) ◽  
pp. 5424-5433 ◽  
Author(s):  
A Gampel ◽  
M Nishikimi ◽  
A Tzagoloff
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Nuclear Gene ◽  
Group I Introns ◽  
Wild Type ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
High Concentrations ◽  
Self Splicing

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.

scholarly journals The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG

2007 ◽  
Vol 189 (18) ◽  
pp. 6572-6579 ◽  
Author(s):  
Rahul Raghavan ◽  
Scott R. Miller ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Acanthamoeba Castellanii ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Group I ◽  
23S Rrna Gene ◽  
Self Splicing

ABSTRACT We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.

scholarly journals Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris, Candida albicans, and Candida dubliniensis

2005 ◽  
Vol 49 (4) ◽  
pp. 1326-1330 ◽  
Author(s):  
Matthew D. Disney ◽  
Ruth Stephenson ◽  
Terry W. Wright ◽  
Constantine G. Haidaris ◽  
Douglas H. Turner ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Nucleic Acids ◽  
Mammalian Cells ◽  
Pneumocystis Carinii ◽  
Candida Dubliniensis ◽  
Group I Introns ◽  
Hoechst 33258 ◽  
Group I ◽  
Self Splicing

ABSTRACT Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC50)of 30 μM in 2 or 4 mM Mg2+, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s ranged from 1 to 9 μM for strains with group I introns and were 12 and 32 μM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron

1989 ◽  
Vol 9 (12) ◽  
pp. 5424-5433
Author(s):  
A Gampel ◽  
M Nishikimi ◽  
A Tzagoloff
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Nuclear Gene ◽  
Group I Introns ◽  
Wild Type ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
High Concentrations ◽  
Self Splicing

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.

scholarly journals Mobile Self-Splicing Group I Introns from thepsbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

2001 ◽  
Vol 21 (10) ◽  
pp. 3472-3481 ◽  
Author(s):  
Obed W. Odom ◽  
Stephen P. Holloway ◽  
Nita N. Deshpande ◽  
Jaesung Lee ◽  
David L. Herrin
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Open Reading Frames ◽  
Group I Introns ◽  
Resistance Marker ◽  
Free Standing ◽  
Spectinomycin Resistance ◽  
Highly Efficient ◽  
Group I ◽  
Short Exon ◽  
Self Splicing

ABSTRACT Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 andCr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16Srrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. TheCr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/μg of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp −88 and −194 (relative to the ORF) that stimulated homing and contained a possible bacterial (−10, −35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.

scholarly journals Toxic Introns and Parasitic Intein in Coxiella burnetii: Legacies of a Promiscuous Past

2008 ◽  
Vol 190 (17) ◽  
pp. 5934-5943 ◽  
Author(s):  
Rahul Raghavan ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Growth Cycle ◽  
Host Protein ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Bacterial Growth Rate ◽  
Group I ◽  
Halorhodospira Halophila

ABSTRACT The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.

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