Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

scholarly journals Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551 ◽  
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

scholarly journals Suppressor Analysis of the Saccharomyces cerevisiae Gene REC104 Reveals a Genetic Interaction With REC102

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1261-1272 ◽  
Author(s):  
Laura Salem ◽  
Natalie Walter ◽  
Robert Malone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Null Allele ◽  
Genetic Interaction ◽  
In Vitro Mutagenesis ◽  
Conditional Mutations ◽  
Suppressor Analysis

Abstract REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for highcopy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not “bypass” suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115 ◽  
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

scholarly journals The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Philipp K Zuber ◽  
Irina Artsimovitch ◽  
Monali NandyMazumdar ◽  
Zhaokun Liu ◽  
Yuri Nedialkov ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Complex Structure ◽  
Transcription Regulator ◽  
Transcription Bubble ◽  
Template Strand ◽  
Domains Of Life ◽  
Dna Strand ◽  
Functional Analyses ◽  
Template Dna

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

1984 ◽  
Vol 26 (3) ◽  
pp. 386-389 ◽  
Author(s):  
Linda J. Reha-Krantz ◽  
Sükran Parmaksizoglu
Keyword(s):  
Dna Polymerase ◽  
Low Temperatures ◽  
Bacteriophage T4 ◽  
Effect Of Temperature ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

scholarly journals RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (10) ◽  
pp. 4433-4440 ◽  
Author(s):  
N Chiannilkulchai ◽  
R Stalder ◽  
M Riva ◽  
C Carles ◽  
M Werner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Stop Codon ◽  
Sequence Similarity ◽  
Start Codon ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Multicopy Plasmid

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

The Role of Microbiology in Models of Dental Caries: Reaction Paper

1995 ◽  
Vol 9 (3) ◽  
pp. 255-269 ◽  
Author(s):  
G.H. Bowden
Keyword(s):  
Process Selection ◽  
Advantages And Disadvantages ◽  
Test Models ◽  
Plaque Development ◽  
In Vitro Systems ◽  
Selection Of

Models of the caries process have made significant contributions toward defining the roles of bacteria in caries. Microbiologists use a variety of in vitro systems to model aspects of the caries process. Also, in situ models in humans provide information on the microbiology of caries in vivo. These models do not involve the entire process leading to natural caries; consequently, the results from such studies are used to deduce the roles of bacteria in natural caries. Therefore, they can be described as Inferential Caries Models. In contrast, animal models and some clinical trials in humans involve natural caries and can be described as Complete Caries Models. Furthermore, these models are used in two distinct ways. They can be used as Exploratory Models to explore different aspects of the caries process, or as Test Models to determine the effects of anticaries agents. This dichotomy in approach to the use of caries models results in modification of the models to suit a particular role. For example, if we consider Exploratory Models, the in situ appliance in humans is superior to others for analyzing the microbiology of plaque development and demineralization in vivo. The chemostat and biofilm models are excellent for exploring factors influencing bacterial interactions. Both models can also be used as Test Models. The in situ model has been used to test the effects of fluoride on the microflora and demineralization, while the chemostat and biofilm models allow for the testing of antibacterial agents. Each model has its advantages and disadvantages and role in analysis of the caries process. Selection of the model depends on the scientific question posed and the limitations imposed by the conditions available for the study.

scholarly journals Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

2009 ◽  
Vol 53 (6) ◽  
pp. 2463-2468 ◽  
Author(s):  
Patrizia Spigaglia ◽  
Fabrizio Barbanti ◽  
Thomas Louie ◽  
Frédéric Barbut ◽  
Paola Mastrantonio
Keyword(s):  
Clostridium Difficile ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Toxin B ◽  
Relevant Role ◽  
Vitro Development ◽  
Selection Of

ABSTRACT Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10−6 to 6.6 × 10−5 for MX and from 1.0 × 10−6 to 2.4 × 10−5 for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.

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