Localization and In Vitro Mutagenesis of the Active Site in the Saccharomyces cerevisiae mRNA Capping Enzyme1

1995 ◽  
Vol 118 (6) ◽  
pp. 1303-1309 ◽  
Author(s):  
Yoshio Shibagaki ◽  
Hideo Gotoh ◽  
Misako Kato ◽  
Kiyohisa Mizumoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
In Vitro Mutagenesis ◽  
Mrna Capping

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 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.

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7836-7849
Author(s):  
P Russo ◽  
W Z Li ◽  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
In Vitro Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Element ◽  
A Site ◽  
Derivatives Of ◽  
Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

In vitro Mutagenesis of Human Dihydropteridine Reductase at the Active Site and at Altered Sites Found in the Reductases of Deficient Children

Pteridines ◽  
1996 ◽  
Vol 7 (4) ◽  
pp. 123-136 ◽  
Author(s):  
Hong-Ping Zhang ◽  
Nan Yang ◽  
Wilfred L. F. Armarego
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
General Procedure ◽  
Maximum Velocity ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Dihydropteridine Reductase ◽  
Proton Source

Summary A general procedure for in vitro site-directed mutagenesis of the wild-type dihydropteridine reductase gene has been used successfully to make eight mutant proteins. Five mutations were at the active site, viz Tyrl50His, Tyrl50Ser, Tyrl50Phe, Tyr150Glu and Tyrl50Lys. The proteins were expressed as glutathione S-transferase fusion proteins from which the unconjugated reductases were obtained by thrombin cleavage. The kinetic parameters of the conjugated and unconjugated reductases were measured using natural quinonoid R-7,8(6H)-dihydrobiopterin and non-natural quinonoid RS-6-methyl-7,8(6H)-dihydropterin and NADH. The kcat (maximum velocity at saturating concentrations of substrates) and kcatl Km (first order rate constant at low concentration of substrates) values show that the phenolic OH of Tyr 150 was the most likely proton source to complete the hydride reduction of the quinonoid pterin cofactor. However in the absence of a proton source at residue 150, measurable enzyme activities were observed indicating that a proton was relayed via a water molecule(s) from some neighbouring acidic amino acid residue. Three mutant dihydropteridine reductases, which were found in defective children, have been similarly attempted, viz GlylSlSer, Gly23Asp and a threonine insertion at position 123. The enzyme activities of the first two mutant reductases were consistent with the severity of the disease. The unconjugated reductase from the third mutation could not be obtained due to proteolysis but the fusion protein was enzymically active.

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 Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (12) ◽  
pp. 7836-7849 ◽  
Author(s):  
P Russo ◽  
W Z Li ◽  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
In Vitro Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Element ◽  
A Site ◽  
Derivatives Of ◽  
Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

scholarly journals MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (2) ◽  
pp. 387-403 ◽  
Author(s):  
M T Brown ◽  
L Goetsch ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
In Vitro Mutagenesis ◽  
Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2396-2405
Author(s):  
R L Last ◽  
J B Stavenhagen ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mrna Accumulation ◽  
Isolation And Characterization ◽  
Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

scholarly journals FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 5968-5975 ◽  
Author(s):  
C M Alarcón ◽  
J Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Changes ◽  
Active Site ◽  
Feedback Inhibition ◽  
Immunosuppressive Drugs ◽  
Intermediate Step ◽  
Active Site Residues ◽  
Peptidyl Prolyl Isomerase

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

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