The DIVa Maturase Binding Site in the Yeast Group II Intron aI2 Is Essential for Intron Homing but Not for In Vivo Splicing

ABSTRACT Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.

Download Full-text

Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽

10.1186/s13100-021-00237-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Justin M. Waldern ◽

Dorie Smith ◽

Carol Lyn Piazza ◽

E. Jake Bailey ◽

Nicholas J. Schiraldi ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Fold Increase ◽

Group Ii Intron ◽

In Vivo Experiments ◽

Cellular Factors ◽

Group Ii ◽

Self Splicing ◽

Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

Download Full-text

Group II intron domain 5 facilitates a trans-splicing reaction.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2361 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2361-2366 ◽

Cited By ~ 83

Author(s):

K A Jarrell ◽

R C Dietrich ◽

P S Perlman

Keyword(s):

Mitochondrial Dna ◽

Group Ii Intron ◽

Intron Sequence ◽

Trans Splicing ◽

Group Ii ◽

Self Splicing ◽

Yeast Mitochondrial Dna ◽

Domain 4

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Download Full-text

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster

Anesthesiology ◽

10.1097/00000542-200310000-00019 ◽

2003 ◽

Vol 99 (4) ◽

pp. 867-875 ◽

Cited By ~ 16

Author(s):

Sumiko Gamo ◽

Junya Tomida ◽

Katsuyuki Dodo ◽

Dai Keyakidani ◽

Hitoshi Matakatsu ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Double Mutant ◽

Developmental Stages ◽

Northern Blotting ◽

General Anesthetics ◽

Wild Type ◽

Mutant Strains ◽

Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

Download Full-text

Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

Infection and Immunity ◽

10.1128/iai.00452-10 ◽

2010 ◽

Vol 78 (12) ◽

pp. 5324-5331 ◽

Cited By ~ 24

Author(s):

Nicolas Bertrand ◽

Sébastien Houle ◽

Guillaume LeBihan ◽

Édith Poirier ◽

Charles M. Dozois ◽

...

Keyword(s):

Escherichia Coli ◽

Regulatory System ◽

Rabbit Serum ◽

Infection Model ◽

Pho Regulon ◽

Wild Type ◽

Mutant Strains ◽

Pst System

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

Download Full-text

The Alternative Sigma Factor RpoN Is Required for hrpActivity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

Journal of Bacteriology ◽

10.1128/jb.182.12.3508-3516.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3508-3516 ◽

Cited By ~ 59

Author(s):

Erik L. Hendrickson ◽

Pablo Guevera ◽

Frederick M. Ausubel

Keyword(s):

Pseudomonas Syringae ◽

Transcriptional Activity ◽

Constitutive Expression ◽

Wild Type ◽

In Planta ◽

Disease Symptoms ◽

Nicotiana Tobacum ◽

Mutant Strains

ABSTRACT β-Glucuronidase (uidA) reporter gene fusions were constructed for the hrpZ, hrpL, andhrpS genes from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. These reporters, as well as an avrRpt2-uidA fusion, were used to measure transcriptional activity in ES4326 and a ES4326 rpoNmutant. rpoN was required for the expression ofavrRpt2, hrpZ, and hrpL in vitro in minimal media and in vivo when infiltrated into Arabidopsis thaliana leaves. In contrast, the expression of hrpSwas essentially the same in wild-type and rpoN mutant strains. Constitutive expression of hrpL in anrpoN mutant restored hrpZ transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (Nicotiana tobacum), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into Arabidopsis leaves. These data indicate that rpoN-mediated control of hrp gene expression acts at the level of hrpL and that in planta growth of P. syringae is not required for the elicitation of disease symptoms.

Download Full-text

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster

International Journal of Biochemistry ◽

10.1016/0020-711x(84)90248-9 ◽

1984 ◽

Vol 16 (12) ◽

pp. 1401-1408 ◽

Cited By ~ 13

Author(s):

Piroska Dévay ◽

Magda Solti ◽

István Kiss ◽

Viktor Dombrádi ◽

Peter Friedrich

Keyword(s):

Drosophila Melanogaster ◽

Protein Phosphorylation ◽

Wild Type ◽

Mutant Strains

Download Full-text

In vivo and in vitro assays for pathogenicity of wild-type and mutant strains of Metarhizium anisopliae for three insect species

Journal of Invertebrate Pathology ◽

10.1016/0022-2011(89)90001-3 ◽

1989 ◽

Vol 53 (2) ◽

pp. 143-151 ◽

Cited By ~ 15

Author(s):

I.M. Huxham ◽

K.D.Z. Samuels ◽

J.B. Heale ◽

N.J. McCorkindale

Keyword(s):

Metarhizium Anisopliae ◽

Insect Species ◽

In Vitro Assays ◽

Wild Type ◽

Mutant Strains

Download Full-text

TFIIA Plays a Role in the Response to Oxidative Stress

Eukaryotic Cell ◽

10.1128/ec.00071-06 ◽

2006 ◽

Vol 5 (7) ◽

pp. 1081-1090 ◽

Cited By ~ 11

Author(s):

Susan M. Kraemer ◽

David A. Goldstrohm ◽

Ann Berger ◽

Susan Hankey ◽

Sherry A. Rovinsky ◽

...

Keyword(s):

Oxidative Stress ◽

Transcription Factor ◽

Rna Polymerase Ii ◽

Expression Profiles ◽

Intracellular Localization ◽

Regulation Of Gene Expression ◽

Wild Type ◽

Mutant Strains

ABSTRACT To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.

Download Full-text

Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

Infection and Immunity ◽

10.1128/iai.66.5.2180-2185.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2180-2185 ◽

Cited By ~ 35

Author(s):

Karsten R. O. Hazlett ◽

Suzanne M. Michalek ◽

Jeffrey A. Banas

Keyword(s):

Streptococcus Mutans ◽

Mutant Strain ◽

Rat Model ◽

Binding Protein ◽

Protein A ◽

Wild Type ◽

Mutant Strains

ABSTRACT Glucan-binding protein A (GbpA) of Streptococcus mutanshas been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenicgbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutanswas grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. ThegbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB andgtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

Download Full-text