In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

scholarly journals In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551 ◽  
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria

1988 ◽  
Vol 8 (6) ◽  
pp. 2562-2571
Author(s):  
S Partono ◽  
A S Lewin
Keyword(s):  
Group I Introns ◽  
Yeast Mitochondria ◽  
High Concentration ◽  
Functional Protein ◽  
Full Spectrum ◽  
Group I ◽  
Intron Boundary ◽  
Monovalent Ion

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.

scholarly journals Sensitivity of Bipolaris oryzae Isolates Pathogenic on Cultivated Wild Rice to the Quinone Outside Inhibitor Azoxystrobin

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 1910-1917 ◽  
Author(s):  
Claudia V. Castell-Miller ◽  
Deborah A. Samac
Keyword(s):  
Cytochrome B ◽  
Fungicide Resistance ◽  
Wild Rice ◽  
Brown Spot ◽  
Bipolaris Oryzae ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Group I ◽  
Mitochondrial Cytochrome B

The occurrence of fungal brown spot, caused by Bipolaris oryzae, has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of azoxystrobin-based fungicides. The active ingredient blocks electron transfer at the quinone outside inhibitor (QoI) site in the mitochondrial cytochrome b within the bc1 complex, thus obstructing respiration. The in vitro averaged EC50 of baseline isolates collected in 2007 before widespread fungicide use was estimated to be 0.394 µg/ml with PROBIT and 0.427 µg/ml with linear regression analyses. Isolates collected during 2008, 2015, and 2016 had a range of sensitivity as measured by relative spore germination (RG) at a discriminatory dose of 0.4 µg/ml azoxystrobin. Isolates with a higher (≥80%) and lower RG (≤40%) had the wild type nucleotides at amino acid positions F129, G137, and G143 of cytochrome b, sites known to be associated with QoI fungicide resistance. Two Group I introns were found in the QoI target area. The splicing site for the second intron was found immediately after the codon for G143. A mutation for fungicide resistance at this location would hinder splicing and severely reduce fitness. B. oryzae expresses an alternative oxidase in vitro, which allows the fungus to survive inhibition of respiration by azoxystrobin. This research indicates that B. oryzae has not developed resistance to QoI fungicides, although monitoring for changes in sensitivity should be continued. Judicious use of QoI fungicides within an integrated disease management system will promote an effective and environmentally sound control of the pathogen in wild rice paddies.

scholarly journals Rho5p Is Involved in Mediating the Osmotic Stress Response in Saccharomyces cerevisiae, and Its Activity Is Regulated via Msi1p and Npr1p by Phosphorylation and Ubiquitination

2008 ◽  
Vol 7 (9) ◽  
pp. 1441-1449 ◽  
Author(s):  
Robert B. Annan ◽  
Cunle Wu ◽  
Daniel D. Waller ◽  
Malcolm Whiteway ◽  
David Y. Thomas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Posttranslational Modifications ◽  
Bound State ◽  
Small Gtpases ◽  
Rho Proteins ◽  
Vitro Assay ◽  
Regulatory Circuit

ABSTRACT Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Δste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.

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 Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

1990 ◽  
Vol 10 (7) ◽  
pp. 3386-3396 ◽  
Author(s):  
D E Muscarella ◽  
E L Ellison ◽  
B M Ruoff ◽  
V M Vogt
Keyword(s):  
Ribosomal Dna ◽  
Physarum Polycephalum ◽  
Nuclear Dna ◽  
Expression System ◽  
Target Sequence ◽  
Group I Introns ◽  
Reading Frame ◽  
Group I

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

scholarly journals QoI Resistance in Fusicladium carpophilum Populations from Almond in California and Evaluation of Molecular Resistance Mechanisms

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1322-1330 ◽  
Author(s):  
Y. Luo ◽  
L. Hou ◽  
H. Förster ◽  
J. E. Adaskaveg
Keyword(s):  
Cytochrome B ◽  
High Resistance ◽  
Quantitative Polymerase Chain Reaction ◽  
Resistance Mechanisms ◽  
Cytochrome B Gene ◽  
Analysis Of Covariance ◽  
Mitochondrial Cytochrome B ◽  
Moderately Resistant ◽  
Qoi Resistance

Disease management failures have been reported in California for almond scab caused by Fusicladium carpophilum following quinone outside inhibitor (QoI) applications. Resistance in the pathogen populations was found to be common and at high incidence in the major almond-growing regions beginning in 2003, 4 years after registration of azoxystrobin on this crop. Two levels of azoxystrobin resistance, moderate and high, were identified with 50% effective concentration (EC50) values between 0.15 and 10 μg/ml or >40 μg/ml, respectively. Reference isolates collected before resistance was detected had EC50 values <0.05 μg/ml. High-resistance was associated with a G143A mutation in the mitochondrial cytochrome b gene. For the less commonly found moderately resistant isolates, no mutations in the gene were detected between codons 122 and 212. Using primers targeting the G143A mutation or the cytochrome b gene of all F. carpophilum isolates in quantitative polymerase chain reaction (qPCR) analyses, the frequency of highly resistant isolates was accurately determined in mixtures of conidia with selected ratios of sensitive and resistant isolates. The frequency of high resistance in bulked samples of scab lesions, however, was generally underestimated compared with in vitro testing of fungicide sensitivity of fungal isolates from the same lesions. Competition experiments using conidial suspensions demonstrated stability of the highly resistant genotype in the presence of different amounts of sensitive and moderately resistant genotypes. Analysis of covariance of linear regressions of cycle threshold values on DNA concentrations derived from qPCR amplifications using two primer pairs for cytochrome b alleles with and without the G143 mutation showed that several isolates differed in their slopes and midpoints. Thus, heteroplasmy of mitochondrial-inherited QoI resistance is suggested as a likely cause for incongruence in estimating resistance frequencies using the two methods.

scholarly journals Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

2008 ◽  
Vol 8 (3) ◽  
pp. 306-314 ◽  
Author(s):  
Régine Bosson ◽  
Isabelle Guillas ◽  
Christine Vionnet ◽  
Carole Roubaty ◽  
Andreas Conzelmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
In Vitro Assay ◽  
Gpi Anchor ◽  
Intact Cells ◽  
Vitro Assay ◽  
Gpi Proteins ◽  
Mutant Cells

ABSTRACT After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [3H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [3H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [3H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [3H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [3H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.

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