scholarly journals Mutational Analysis of the Helicobacter pylori Vacuolating Toxin Amino Terminus: Identification of Amino Acids Essential for Cellular Vacuolation

2000 ◽  
Vol 68 (7) ◽  
pp. 4354-4357 ◽  
Author(s):  
Dan Ye ◽  
Steven R. Blanke
Keyword(s):  
Helicobacter Pylori ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Wild Type ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Vacuolating Cytotoxin ◽  
Intracellular Activity ◽  
Mutant Forms

ABSTRACT The functional importance of the amino terminus of theHelicobacter pylori vacuolating cytotoxin (VacA) was investigated by analyzing the relative levels of vacuolation of HeLa cells transfected with plasmids encoding wild-type and mutant forms of the toxin. Notably, VacA's intracellular activity was found to be sensitive to small truncations and internal deletions at the toxin's amino terminus. Moreover, alanine-scanning mutagenesis revealed the first VacA point mutations (at proline 9 or glycine 14) that completely abolish the toxin's intracellular activity.

scholarly journals Fluorescence Resonance Energy Transfer Microscopy of the Helicobacter pylori Vacuolating Cytotoxin within Mammalian Cells

2002 ◽  
Vol 70 (7) ◽  
pp. 3824-3832 ◽  
Author(s):  
David C. Willhite ◽  
Dan Ye ◽  
Steven R. Blanke
Keyword(s):  
Helicobacter Pylori ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dominant Negative ◽  
Wild Type ◽  
Vacuolating Cytotoxin ◽  
Vacuolated Cells ◽  
Mutant Forms

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.

scholarly journals Mutational Analysis of Cdc19p, a Schizosaccharomyces pombe MCM Protein

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1025-1041 ◽  
Author(s):  
Susan L Forsburg ◽  
Daniel A Sherman ◽  
Sabine Ottilie ◽  
J Randy Yasuda ◽  
Jeffrey A Hodson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Protein Function ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Wild Type ◽  
Genes Encoding ◽  
Mcm Proteins ◽  
And Function ◽  
Mutant Forms ◽  
Replication Initiator

The cdc19  + gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19  + and genes encoding subunits of DNA polymerase δ and the replication initiator cdc18  +. We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342

1989 ◽  
Vol 9 (4) ◽  
pp. 1406-1414
Author(s):  
A A McCracken ◽  
K B Kruse ◽  
J L Brown
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitor ◽  
Conformational Stability ◽  
Point Mutations ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Importance ◽  
Bridge Formation ◽  
Cos Cells

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

scholarly journals A Hypervariable Region within the 3′ cis-Acting Element of the Murine Coronavirus Genome Is Nonessential for RNA Synthesis but Affects Pathogenesis

2006 ◽  
Vol 81 (3) ◽  
pp. 1274-1287 ◽  
Author(s):  
Scott J. Goebel ◽  
Timothy B. Miller ◽  
Corey J. Bennett ◽  
Kristen A. Bernard ◽  
Paul S. Masters
Keyword(s):  
Tissue Culture ◽  
Deletion Mutant ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Hepatitis Virus ◽  
Point Mutations ◽  
Hypervariable Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Group 2

ABSTRACT The 3′ cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3′ untranslated region (3′ UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3′ UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5′-GGAAGAGC-3′, which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.

scholarly journals Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1355-1365 ◽  
Author(s):  
C Smith ◽  
H Giordano ◽  
R DeLotto
Keyword(s):  
Serine Proteases ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Normal Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Rna Transcripts ◽  
Intron 1 ◽  
Regulation Of Activity

Abstract Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.

scholarly journals Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

1989 ◽  
Vol 9 (4) ◽  
pp. 1406-1414 ◽  
Author(s):  
A A McCracken ◽  
K B Kruse ◽  
J L Brown
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitor ◽  
Conformational Stability ◽  
Point Mutations ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Importance ◽  
Bridge Formation ◽  
Cos Cells

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

scholarly journals Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

1992 ◽  
Vol 12 (10) ◽  
pp. 4634-4642 ◽  
Author(s):  
B Rubinfeld ◽  
W J Crosier ◽  
I Albert ◽  
L Conroy ◽  
R Clark ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Gtpase Activating Protein ◽  
Mutant Proteins ◽  
Carboxy Terminal

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

scholarly journals Mutational Analysis of the Yeast DEAH-Box Splicing Factor Prp16

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 807-815 ◽  
Author(s):  
Hans-Rudolf Hotz ◽  
Beate Schwer
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Sequence Motifs ◽  
Wild Type ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scan ◽  
Hydrolysis Of

Abstract Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a Δprp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373–377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

scholarly journals Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535 ◽  
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Sign in / Sign up

Export Citation Format

Share Document