Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mutant identification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed1980

Pathogenicity of wild-type and temperature-sensitive mutants of herpes simplex virus type 2 in guinea pigs.

The pathogenicity of herpes simplex virus type 2 strain 186, the wild-type (WT) strain, and four temperature-sensitive (ts) mutants was studied after genital inoculation of female guinea pigs. Infection with the WT virus was generally severe, with extensive skin lesions in 89% and mortality in 37% of inoculated animals. Guinea pigs inoculated with ts mutants manifest remarkably mild disease, with lesions occurring in only 16% of the guinea pits and a mortality rate of 7%. WT virus was recovered from nerve and non-nerve tissues of all acutely infected animals and from the majority of latently infected animals (71%). Virus was isolated from nerve or genital tissues from only 13% of ts mutant-inoculated animals during acute infection and from 7% during latent infection. Three of the seven isolates from mutant-infected animals appeared to be WT virus. Identification of WT and ts mutant isolates was done by biological characterization in selective cell cultures at permissive (33 degrees C) and nonpermissive (38 degrees C) temperatures. One month after initial infection with WT virus, guinea pigs were challenged with the same virus and were completely resistant to overt clinical disease. Animals inoculated with ts mutants A1b and C2b had mild manifestations of disease after challenge with WT virus; however, the capacity of WT virus to establish latent infection was conserved. Although complement-required neutralizing antibodies were detectable after challenge in animals previously inoculated with mutant virus A1b, C2b, or D6b, there was no significant protection against subsequent infection with WT virus. No complement-required neutralizing antibodies were detected in F3b animals after challenge. The present study of WT and ts mutants of herpes simplex virus type 2 in the guinea pig model provides a means for better understanding the mechanisms of pathogenesis and latency after genital infection.

Animals↗
PubMed1993

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1.

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Identification of residues of the epidermal growth factor receptor proximal to residue 45 of bound epidermal growth factor.

A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-alpha in their recognition by the receptor. Cyclization of Gln1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125I-Labeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/R45K is Lys465 of the receptor.

Amino Acid Sequence↗
PubMed1975

Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.

Adenosine Diphosphate Sugars↗
PubMed1999

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.

Amino Acid Sequence↗
PubMed2003

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.

Cell Death↗
PubMed2004

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis.

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.

Animals↗
PubMed1998

Identification and characterization of the Staphylococcus carnosus nitrate reductase operon.

Physiological and genetic characterization of Staphylococcus carnosus nitrate reductase-negative mutants led to the identification of the nitrate reductase operon, narGHJI. Transcription from the nar promoter was stimulated by anaerobiosis, nitrate, and nitrite. This is in accordance with the nitrate reductase activities determined with benzyl viologen as electron donor. However, in the presence of oxygen and nitrate, high transcriptional initiation but low nitrate reductase activity was observed. Since the alphabeta complex of the nitrate reductase formed during anaerobic growth was insensitive to oxygen, other oxygen-sensitive steps (e.g., post-transcriptional mechanisms, molybdenum cofactor biosynthesis) must be involved. The nitrate-reducing system in S. carnosus displays similarities to the dissimilatory nitrate reductases of Escherichia coli. However, in the S. carnosus nar promoter, no obvious Fnr and integration host factor recognition sites are present; only one site that is related to the E. coli NarL consensus sequence was found. Studies to determine whether the E. coli proteins NarL and Fnr are functional at the S. carnosus narGHJI promoter indicated that the promoter is not functional in E. coli.

Amino Acid Sequence↗
PubMed2005

Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon.

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.

Bacterial Proteins↗
PubMed2003

Identification and molecular characterization of a naturally occurring RNA virus mutant defective in the initiation of host recovery.

The host recovery response is characterized by the disappearance of disease symptoms and activation of the RNA silencing virus resistance in the new growth following an initial symptomatic infection. However, it is not clear what triggers the initiation of recovery, which occurs naturally only in some virus-host interactions. Here we report the identification and characterization of a spontaneous mutant of Tobacco streak virus (TSV) that became defective in triggering recovery in tobacco plants. Infectious full-length cDNA clones corresponding to the tripartite RNA genome were constructed from both the wild-type and the nonrecovery mutant of TSV (TSVnr), the first sets of infectious cDNA clones from an Ilarvirus. Genetic and molecular analyses identified an A --> G mutation in the TSVnr genome that was sufficient to confer nonrecovery when introduced into TSV. The mutation was located in the intergenic region of RNA 3 upstream of the mapped transcriptional start site of the coat protein mRNA. Intriguingly, induction of recovery by TSV was not accompanied by virus clearance and TSV consistently accumulated to significantly higher levels than TSVnr did even though TSVnr-infected plants displayed severe symptoms throughout the course of infection. Thus, our findings indicate that recovery of host can be initiated by minimal genetic changes in a viral genome and may occur in the absence of virus clearance. Mechanisms possibly involved in the initiation of host recovery are discussed.

Cloning, Molecular↗
PubMed2001

Identification of chromosomal deficiencies that modify Drosophila mastermind mutant phenotypes.

Mastermind (Mam) is a component of Notch pathway signaling. In combination with the intracellular domain of Notch and Suppressor of Hairless, Mam forms a transcriptional activation complex. We have initiated a genetic approach to identify other loci involved in Mam function. The screen utilizes engineered mutations in Mam that derive from GAL4-UAS-directed expression of dominant negative constructs. When driven at the wing margin, truncated versions of Mam phenocopy Notch pathway mutations. Correlated with these phenotypes is depression of Notch pathway target expression. Strains expressing truncated versions of Mam were tested for genetic interactions with a large collection of chromosomal deficiencies. Genomic segments that enhanced and suppressed the dominant wing phenotype were identified. These regions may contain uncharacterized loci involved in Notch pathway function.

Animals↗
PubMed1978

Identification of eight-cell tw32 homozygous lethal mutants by aberrant compaction.

Early cleavage stage (4- and uncompacted 8-cell) embryos from experimental (+/tw32 X +/t32) and control (male +/tw32 X female +/T) matings were recovered at 56 hours post coitum, cultured, separated into uncompacted and compacted 8-cell embryo groups, and either analyzed histologically for cytoplasmic lipids or cultured through the morula-to-blastocyst transformation. Abnormal compaction was associated with excess cytoplasmic lipids at the 8-cell stage (P less than 0.01). Also, 89.2% (33/37) of the original uncompacted experimental embryos underwent developmental arrest characteristic of tw32 homozygous lethals. Thus, aberrant compaction, a newly reported symptom of the tw32/tw21 syndrome, affords a visual means to identify tw32 homozygotes at 8-cell stages.

Animals↗
PubMed1999

A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro.

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Animals↗
PubMed1993

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2.

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wild-type shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nia1-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.

Alleles↗
PubMed1988

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor).

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in wo brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T----G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5'-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.

Base Sequence↗
PubMed1992

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The cholorate-resistant mutant of Arabidopsis thaliana, chl2, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained. chl2 plants have low levels of NR activity, yet the map position of the chl2 mutation is clearly distinct from that of the two NR structural genes that have been identified in Arabidopsis. In addition, chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest that chl2 may be a NR regulatory mutant. We have examined chl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels in chl2 plants using a sensitive, specific assay for MoCo: complementation of Neurospora MoCo mutant extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed2003

Differential expression of metallothionein 1 and 2 isoforms in breast cancer lines with different invasive potential: identification of a novel nonsilent metallothionein-1H mutant variant.

Metallothionein (MT), a low-molecular weight protein with pleiotropic functions, is believed to play an important role in tumorigenesis. The aim of this study was to compare the expression of functional MT-1 and MT-2 mRNA isoforms in five breast cancer cell lines ranging from noninvasive MCF7 breast cancer cells to highly aggressive MDA-MB-231 breast cancer cells together with breast myoepithelial cells in vitro by conventional semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The MT-2A isoform was observed to be differentially upregulated in the invasive phenotype. The MT-1E isoform was found to be present in estrogen receptor-negative breast cancer cell lines (MDA-MB-231 and Hs578T) but not detectable in the estrogen receptor-positive cell lines (T47D, MCF7, and ZR75-1 cells). Only the myoepithelial cells exhibited the presence of the MT-1G transcript. Direct sequencing of the RT-PCR products revealed the occurrence of a variant MT-1H isoform with changes in amino acid residues in the protein sequence and notable differences in the predicted secondary protein structure. The observations in this study are relevant to the development of novel approaches to metastatic breast cancer disease, and may herald the search for novel MT mutants and the elucidation of their biological roles.

Breast Neoplasms↗