A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

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Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2989-2999 ◽

Cited By ~ 36

Author(s):

H M Traglia ◽

N S Atkinson ◽

A K Hopper

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Open Reading Frame ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Reading Frame ◽

Genomic Deletions ◽

Single Amino Acid Change ◽

Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

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A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

Journal of Virology ◽

10.1128/jvi.00510-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 3

Author(s):

Sujit K. Mohanty ◽

Bryan Donnelly ◽

Phylicia Dupree ◽

Inna Lobeck ◽

Sarah Mowery ◽

...

Keyword(s):

Amino Acid ◽

Biliary Atresia ◽

Amino Acid Change ◽

Reverse Genetics ◽

Single Amino Acid ◽

Wild Type ◽

Neonatal Mice ◽

Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

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A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

Journal of Virology ◽

10.1128/jvi.79.12.7327-7337.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7327-7337 ◽

Cited By ~ 60

Author(s):

Valery Z. Grdzelishvili ◽

Sherin Smallwood ◽

Dallas Tower ◽

Richard L. Hall ◽

D. Margaret Hunt ◽

...

Keyword(s):

Amino Acid ◽

Vesicular Stomatitis Virus ◽

In Vitro Transcription ◽

Single Amino Acid ◽

Wild Type ◽

L Protein ◽

Critical Residue ◽

Single Amino Acid Change ◽

Vesicular Stomatitis

ABSTRACT The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-N7 and 2′-O-adenosine positions (7mGpppAm). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5′-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.

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Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2989-2999.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2989-2999

Author(s):

H M Traglia ◽

N S Atkinson ◽

A K Hopper

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Open Reading Frame ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Reading Frame ◽

Genomic Deletions ◽

Single Amino Acid Change ◽

Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

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Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

Journal of General Virology ◽

10.1099/vir.0.009449-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1741-1747 ◽

Cited By ~ 11

Author(s):

Tahir H. Malik ◽

Candie Wolbert ◽

Laura Nerret ◽

Christian Sauder ◽

Steven Rubin

Keyword(s):

Amino Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Mumps Virus ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Supporting Evidence ◽

L Protein ◽

Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

Canadian Journal of Microbiology ◽

10.1139/w2012-033 ◽

2012 ◽

Vol 58 (5) ◽

pp. 589-595

Author(s):

Guy Lemay ◽

Martin Bisaillon

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Genetic Alterations ◽

Biological Properties ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Gene Encoding ◽

Viral Particles ◽

Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

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Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa162 ◽

2020 ◽

Vol 367 (19) ◽

Author(s):

Vanessa C Thompson ◽

Bailey E McGuire ◽

Mia S Frier ◽

Max S G Legg ◽

Tyler W Dyer ◽

...

Keyword(s):

Amino Acid ◽

Molecular Biology ◽

Temperature Stability ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Nickel Affinity Chromatography ◽

Fold Reduction ◽

Single Amino Acid Change ◽

Secreted Proteases ◽

Encoding Gene

ABSTRACT We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

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