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At least 613 records · Page 34Linked to original sources
PubMed1997

C/EBP-epsilon: chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia.

We and others have cloned a novel human gene CCAAT/enhancer-binding protein epsilon (C/EBP-epsilon) encoding a member of the C/EBP gene family. It is exclusively expressed in myeloid and T-lymphoid cells and appears to have an important role in inducing expression of several myeloid-specific genes. We used a polymerase chain reaction (PCR)-based technique to examine DNA from 93 hamster/human radiation hybrid clones in order chromosomally to map C/EBP-epsilon to 14q11.2 (between D14S264 and D14S275) which is telomeric to the T-cell receptor alpha and delta genes and centromeric to several other myeloid gene products including Cathepsin G (CTSG) and Chymase-1 (CMA1). To determine whether C/EBP-epsilon behaves as an altered tumor-suppressor gene, samples from patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) evolving to AML were studied for loss of heterozygosity (LOH) using microsatellite sequences that we identified within 0.2 kb of the amino-terminus of the human C/EBP-epsilon gene. Allelic loss of the C/EBP-epsilon gene was detected in four out of 20 (20%) evolving MDS cases and in none of the 17 AML and 17 T-cell leukemia cases. Mutational analysis of the gene was performed using PCR-SSCP on 37 AML and 40 MDS cases including those with LOH at the gene. No abnormalities were found suggesting that the altered gene in this region is not C/EBP-epsilon. Also, C/EBP-epsilon was examined by Southern blot analysis on DNA samples from 20 AML patients and 10 AML cell lines. No rearrangements or amplifications of the gene were detected. Taken together, we have mapped C/EBP-epsilon to 14q11.2, a region containing other myeloid and T-lymphoid specific genes. Furthermore, no structural alterations were detected in the C/EBP-epsilon gene.

Acute Disease↗
PubMed1998

Characterization of a novel gene, C21orf2, on human chromosome 21q22.3 and its exclusion as the APECED gene by mutation analysis.

Exon trapping was performed from a partial cosmid, PAC, and P1 clone contig from human chromosome 21 between MX1 and 21qter to identify genes that may be involved in the pathogenesis of Down syndrome or several of the genetic diseases that map to chromosome 21q22.3. One 19-bp exon showed identity to three ESTs. The complete sequence of the EST clones, RT-PCR, and cDNA library screening were used to determine the full-length cDNA sequence of 2.2 kb with an open reading frame of 256-amino-acids. The putative 256-amino-acid peptide has homology with a hypothetical Caehorhabditis elegans protein of unknown function. Northern blot analysis of this gene, termed C21orf2 (chromosome 21 open reading frame 2), revealed two ubiquitously expressed mRNAs of 2.2 and 1.2 kb produced by use of alternative polyadenylation sites. Hybridization of the EST clones to a cosmid contig in chromosome 21q22.3 mapped C21orf2 just distal to PFKL, a critical mapping region for several genetic diseases. Comparison to publicly available genomic sequence, and additional data, revealed that the gene is split into seven exons over 10.5 kb, further refining the mapping position to only 1.2 kb distal to PFKL with the direction of transcription toward the centromere. The 5'UTR is contiguous with D21S400, and intron 2 contains a 52-bp VNTR polymorphism. Given its mapping position, C21orf2 is a candidate for involvement in disorders including autoimmune polyglandular disease type I (also called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy or APECED) and the autosomal nonsyndromic deafness loci, DFNB8 and DFNB10. Mutation analysis using sequencing of RT-PCR and genomic DNA-derived PCR products, SSCP, and Southern and Northern blot analyses in APECED patients excluded C21orf2 as the gene for APECED.

Amino Acid Sequence↗
PubMed1999

Mutational analysis of the coding regions of the genes encoding protein kinase B-alpha and -beta, phosphoinositide-dependent protein kinase-1, phosphatase targeting to glycogen, protein phosphatase inhibitor-1, and glycogenin: lessons from a search for genetic variability of the insulin-stimulated glycogen synthesis pathway of skeletal muscle in NIDDM patients.

The finding of a reduced insulin-stimulated glucose uptake and glycogen synthesis in the skeletal muscle of glucose-tolerant first-degree relatives of patients with NIDDM, as well as in cultured fibroblasts and skeletal muscle cells isolated from NIDDM patients, has been interpreted as evidence for a genetic involvement in the disease. The mode of inheritance of the common forms of NIDDM is as yet unclear, but the prevailing hypothesis supports a polygenic model. In the present study, we tested the hypothesis that the putative inheritable defects of insulin-stimulated muscle glycogen synthesis might be caused by genetic variability in the genes encoding proteins shown by biochemical evidence to be involved in insulin-stimulated glycogen synthesis in skeletal muscle. In 70 insulin-resistant Danish NIDDM patients, mutational analysis by reverse transcription-polymerase chain reaction-single strand conformation polymorphism-heteroduplex analysis was performed on genomic DNA or skeletal muscle-derived cDNAs encoding glycogenin, protein phosphatase inhibitor-1, phophatase targeting to glycogen, protein kinase B-alpha and -beta, and the phosphoinositide-dependent protein kinase-1. Although a number of silent variants were identified in some of the examined genes, we found no evidence for the hypothesis that the defective insulin-stimulated glycogen synthesis in skeletal muscle in NIDDM is caused by structural changes in the genes encoding the known components of the insulin-sensitive glycogen synthesis pathway of skeletal muscle.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1996

Mutational analysis of the hsp70-interacting protein Hip.

The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes. During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions. In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements. Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR). Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes. Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat. Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes. Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes. This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain. In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences.

Amino Acid Sequence↗
PubMed2001

Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2.

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.

Amino Acid Sequence↗
PubMed2005

Torovirus non-discontinuous transcription: mutational analysis of a subgenomic mRNA promoter.

Toroviruses (order Nidovirales) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3'-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5' end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3' end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5'-ACN3-4CUUUAGA-3', within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21:6571-6580, 2002). We have now studied the--for nidoviruses unusual--non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5'-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

Animals↗
PubMed2003

Interaction between vitamin D receptor and vitamin D ligands: two-dimensional alanine scanning mutational analysis.

We present a new method to investigate the details of interaction between vitamin D nuclear receptor (VDR) and various ligands, namely a two-dimensional alanine scanning mutational analysis. In this method, the transactivation of various ligands is studied in conjunction with a series of alanine scanning mutations of the residues lining the ligand binding pocket (LBP) of VDR, and the complete set of results is profiled in a patch table. We investigated examples from four structurally diverse groups of known VDR ligands: the native vitamin D hormone and two compounds with the same side chain configuration; four 20-epi compounds; three 19-nor compounds; and two nonsecosteroids. The patch table of the results indicates characteristics of each group in terms of its interaction with 18 LBP residues. We demonstrate the validity of this approach by application to docking studies of the two nonsecosteroids.

Alanine↗
PubMed1997

Structural and mutational analysis of a conserved gene (DGSI) from the minimal DiGeorge syndrome critical region.

The majority of patients with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face syndrome (CTAFS) and some individuals with familial or sporadic conotruncal cardiac defects have hemizygous deletions of chromosome 22. Most patients with these disorders share a common large deletion, spanning > 1.5 Mb within 22q11.21-q11.23. Recently, the smallest region of deletion overlap has been narrowed to a 250 kb area, the minimal DGS critical region (MDGCR), which includes the locus D22S75 (N25). We have isolated and characterized a novel, highly conserved gene, DGSI, within the MDGCR. DGSI has 10 exons and nine introns encompassing 1702 bp of cDNA sequence and 11 kb of genomic DNA. The encoded protein has 476 amino acids with a predicted mol. wt of 52.6 kDa. The intron-exon boundaries have been analyzed and conform to the consensus GT/AG motif. The corresponding murine Dgsi has been isolated and localized to proximal mouse chromosome 16. The mouse gene contains the same number of exons and introns, and the predicted protein has 479 amino acids with 93.2% identity to that of the human DGSI gene. By database searching, both genes have significant homology to a Caenorhabditis elegans hypothetical protein, F42H10.7. Further, mutation analysis has been performed in 16 patients, who have no detectable 22q11.2 deletion and some of the characteristic clinical features of DGS/VCFS. We have detected eight sequence variants in DGSI. These occurred in the 5'-untranslated region, the coding region and the intronic regions adjacent to the intron-exon boundaries of the gene. Seven of the eight variants were also present in normal controls or unaffected family members, suggesting they may not be of etiologic significance.

Amino Acid Sequence↗
PubMed1999

Fabry disease: comparison of enzymatic, linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG).

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). Affected males are reliably diagnosed by demonstration of deficient alpha-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma alpha-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the alpha-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease.

Base Sequence↗
PubMed2000

Mutation analysis of the entire PKD1 gene: genetic and diagnostic implications.

Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of >50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.

Alleles↗
PubMed1997

Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2.

A major peanut allergen, Ara h 2, is recognized by serum IgE from > 90% of patients with peanut hypersensitivity. Biochemical characterization of this allergen indicates that it is a glycoprotein of approximately 17.5 kDa. Using N-terminal amino acid sequence data from purified Ara h 2, oligonucleotide primers were synthesized and used to identify a clone (741 bp) from a peanut cDNA library. This clone was capable of encoding a 17.5-kDa protein with homology to the conglutin family of seed storage proteins. The major linear immunoglobulin E (IgE)-binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from 15 peanut-sensitive patients. Ten IgE-binding epitopes were identified, distributed throughout the length of the Ara h 2 protein. Sixty-three percent of the amino acids represented in the epitopes were either polar uncharged or apolar residues. In an effort to determine which, if any, of the 10 epitopes were recognized by the majority of patients with peanut hypersensitivity, each set of 10 peptides was probed individually with serum IgE from 10 different patients. All of the patient sera tested recognized multiple epitopes. Three epitopes (aa27-36, aa57-66, and aa65-74) were recognized by all patients tested. In addition, these three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 protein. Mutational analysis of the Ara h 2 epitopes indicate that single amino acid changes result in loss of IgE binding. Two epitopes in region aa57-74 contained the amino acid sequence DPYSP that appears to be necessary for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensitivity.

2S Albumins, Plant↗
PubMed2005

SALL1 mutation analysis in Townes-Brocks syndrome: twelve novel mutations and expansion of the phenotype.

Townes-Brocks syndrome is an autosomal dominantly inherited disorder, which comprises multiple birth defects including renal, ear, anal, and limb malformations. TBS has been shown to result from mutations in SALL1, a human gene related to the developmental regulator SAL of Drosophila melanogaster. The SALL1 gene product is a zinc finger protein thought to act as a transcription factor. It contains four highly conserved, evenly distributed C2H2 double zinc finger domains. A single C2H2 motif is attached to the second domain, and at the amino terminus SALL1 contains a C2HC motif. Most mutations causing TBS are clustered in the N-terminal third of the SALL1 coding region and result in the production of truncated proteins containing only one or none of the C2H2 domains and the N-terminal transcriptional repressor domain of SALL1. Twenty-three SALL1 mutations were reported prior to this work, 22 of which are located in exon 2, 5' of the second double zinc finger-encoding region. Here we present 12 novel mutations in SALL1 associated with Townes-Brocks syndrome in 13 unrelated families. These include three nonsense mutations, three short insertions and six short deletions. Thus the number of SALL1 mutations increases to 35. Rare phenotypical features among mutation positive patients include hypothyroidism, vaginal aplasia with bifid uterus, cryptorchidism, bifid scrotum without hypospadia scrotalis, unilateral chorioretinal coloboma with loss of vision, dorsal hypoplasia of the corpus callosum, and umbilical hernia.

Abnormalities, Multiple↗
PubMed2002

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours.

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers.

Acetyltransferases↗
PubMed1999

Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication.

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

Amino Acid Sequence↗
PubMed2005

Mutational analysis of human tumor necrosis factor-alpha.

To understand the structure-function relationship of human tumor necrosis factor-alpha (TNF-alpha), mutational analysis was carried out on the lower regions (regions 1-6) of the molecule. The muteins were prepared as a soluble form by using a chaperonin co-expression system and the cytotoxic activities of the purified muteins were evaluated on TNF-sensitive murine fibrosarcoma L929 cells. Three regions (regions 1, 2 & 4) were found where mutations significantly influenced the bioactivity. In region 1 (residues 1-10), the number of deleted residues and the positioning of positive charges are important to achieve a maximum activity and in region 4 (residues 84-88), introduction of charged residues in one of the positions 86-88 significantly increased the cytotoxic activity. On the other hand, any mutation introduced in region 2 (residues 37-41) had a deleterious effect. The present study provides a structural basis for the design of highly potent TNF-alpha as a therapeutic agent.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of BRAF in gallbladder carcinomas in association with K-ras and p53 mutations and microsatellite instability.

BACKGROUND: Little is known about the genetic changes involved in the pathogenesis of gallbladder cancer. The aim of this study was to examine the presence of mutations in exon 15 of the B-raf gene to investigate its role in gallbladder carcinogenesis. MATERIALS AND METHODS: We examined the mutational status in exon 15 of B-raf gene in 21 gallbladder carcinoma specimens and investigated its association with the presence of K-ras and p53 alterations, microsatellite instability and the clinicopathological features of tumors. RESULTS: B-raf mutations were observed in 7 of 21 (33%) gallbladder carcinomas examined, and all were located at the hot spot codon 599 of exon 15. K-ras and B-raf mutations were never in the same specimens. CONCLUSIONS: B-raf gene mutations seem to be a quite common event in gallbladder carcinomas, implying that B-raf may play an important role in the pathogenesis of this tumor.

Adult↗
PubMed1994

The use of shuttle vectors for mutation analysis in transgenic mice and rats.

The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.

Animals↗
PubMed1990

Mutational analysis of the VirG protein, a transcriptional activator of Agrobacterium tumefaciens virulence genes.

The VirG protein of Agrobacterium tumefaciens is required in conjunction with the VirA protein for transcriptional activation of the virulence (vir) genes in response to plant phenolic compounds. These proteins are members of a family of two component regulatory systems. vir genes are activated via a cascade of phosphorylation reactions involving a specific aspartic acid residue of the VirG protein. We have conducted a mutational analysis of the VirG protein. By mutating conserved and nonconserved aspartic acid residues in the N-terminal domain, we demonstrated that two of three conserved aspartic acid residues located in two different regions are important for the phosphorylation of VirG by VirA phosphate. A third conserved N-terminal region was also shown to be critical for the biological function of VirG as a transcriptional activator. The identification of phosphorylatable but biologically inactive mutated VirG proteins suggests that not only phosphorylation but also a conformational change is necessary for its activity. We further demonstrated that phosphorylation is not required for sequence-specific binding to a vir gene regulatory sequence (vir box) and that the C-terminal domain is sufficient for DNA binding. The data support the model of a two-domain structure for the VirG protein and demonstrate that the sequence homologies to other two-component regulatory systems reflect both functional and structural homologies.

Amino Acid Sequence↗