scholarly journals Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement

1995 ◽  
Vol 1232 (1-2) ◽  
pp. 67-74 ◽  
Author(s):  
Gaetano Villani ◽  
Maria Tattoli ◽  
Nazzareno Capitanio ◽  
Philippe Glaser ◽  
Sergio Papa ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Quinol Oxidase

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals In Vitro Mutagenesis of Bacillus subtilis by Using a Modified Tn7 Transposon with an Outward-Facing Inducible Promoter

2008 ◽  
Vol 74 (11) ◽  
pp. 3419-3425 ◽  
Author(s):  
Christophe Bordi ◽  
Bronwyn G. Butcher ◽  
Qiaojuan Shi ◽  
Anna-Barbara Hachmann ◽  
Joseph E. Peters ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Frequency ◽  
Inducible Promoter ◽  
In Vitro Mutagenesis ◽  
Donor Plasmid ◽  
Positive Bacterium ◽  
Expression Of Genes ◽  
Regulated Expression ◽  
Selection Of

ABSTRACT A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.

scholarly journals The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

2003 ◽  
Vol 23 (8) ◽  
pp. 2778-2789 ◽  
Author(s):  
Qinghu Ren ◽  
Martin A. Gorovsky
Keyword(s):  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Wild Type Protein ◽  
Lysine Residues ◽  
In The Wild ◽  
Essential Function

ABSTRACT Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up ∼80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

scholarly journals Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097 ◽  
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals In Vivo Analysis of Saccharomyces cerevisiae COX2 mRNA 5′-Untranslated Leader Functions in Mitochondrial Translation Initiation and Translational Activation

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Heather M Dunstan ◽  
Noelle S Green-Willms ◽  
Thomas D Fox
Keyword(s):  
Translation Initiation ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Mitochondrial Translation ◽  
Context Analysis ◽  
Stem Loop ◽  
Sequence Elements ◽  
Translational Activator

Abstract We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5′-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator, Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5′-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from −16 to −46, that contains several short sequence elements necessary for COX2 5′-UTL function in translation. The sequences from −16 to −47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/insertion alleles has refined the positions of several possible functional elements of the COX2 5′-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5′-UTLs that is a potential rRNA binding site.

scholarly journals Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

2012 ◽  
Vol 194 (18) ◽  
pp. 4933-4940 ◽  
Author(s):  
Lauren J. Rajakovich ◽  
John Tomlinson ◽  
Patricia C. Dos Santos
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Encoding ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
Sulfur Transfer ◽  
Essential Enzyme ◽  
Sulfur Trafficking

ABSTRACTThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. InEscherichia coliandSalmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, inBacillus subtilisand most species from theFirmicutesphylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis ofB. subtilisthiIand the adjacent gene,nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments inB. subtilisindicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine.In vitrosynthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain.In vivocomplementation studies inE. coliIscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal thatB. subtilisNifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

scholarly journals Mutational analyses reveal a novel function of the nucleotide-binding domain of γ-tubulin in the regulation of basal body biogenesis

2005 ◽  
Vol 171 (6) ◽  
pp. 1035-1044 ◽  
Author(s):  
Yuhua Shang ◽  
Che-Chia Tsao ◽  
Martin A. Gorovsky
Keyword(s):  
Basal Body ◽  
Point Mutations ◽  
Nucleotide Binding ◽  
Gene Replacement ◽  
Binding Domain ◽  
In Vitro Mutagenesis ◽  
Nucleotide Binding Domain ◽  
Spatial Regulation ◽  
Essential Function

We have used in vitro mutagenesis and gene replacement to study the function of the nucleotide-binding domain (NBD) of γ-tubulin in Tetrahymena thermophila. In this study, we show that the NBD has an essential function and that point mutations in two conserved residues lead to over-production and mislocalization of basal body (BB) assembly. These results, coupled with previous studies (Dammermann, A., T. Muller-Reichert, L. Pelletier, B. Habermann, A. Desai, and K. Oegema. 2004. Dev. Cell. 7:815–829; La Terra, S., C.N. English, P. Hergert, B.F. McEwen, G. Sluder, and A. Khodjakov. 2005. J. Cell Biol. 168:713–722), suggest that to achieve the precise temporal and spatial regulation of BB/centriole assembly, the initiation activity of γ-tubulin is normally suppressed by a negative regulatory mechanism that acts through its NBD.

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