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PubMed1997

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti.

The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated. During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected. A glucuronan lyase was identified, this new bacterial lyase produces d.p. 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity.

Acetylation↗
PubMed2000

Immunohistochemical detection of mutant p53 protein in small-cell lung cancer: relationship to treatment outcome.

We investigated the expression of mutant p53 proteins in small-cell lung cancer (SCLC) immunohistochemically, by identification of stabilized mutant p53 proteins with a much longer half-life than the wild-type protein. Of 103 tumor specimens obtained by transbronchial tumor biopsy for histologic diagnosis, 52 (50%) showed positive staining for p53 protein with a p53 monoclonal antibody, DO-1. Positive staining for p53 protein was not correlated with age, sex, performance status, lifetime cigarette consumption, serum concentration of neuron-specific enolase and extent of disease. Complete response rates in patients with a mutant p53 protein-positive tumor were significantly lower than those in p53-negative patients (25% versus 59%; P=0.0005, by chi-square test). Similarly, survival periods in patients with a mutant p53 protein-positive tumor were significantly shorter than those in mutant p53-protein-negative patients (10.8 months versus 20.6 months; P=0.0001, by generalized Wilcoxon test). Multivariate analysis using Cox's proportional hazards model revealed that the presence of mutant p53 protein is an independent factor associated with differences in overall survival (hazards ratio=2.72; 95% confidence interval, 1.71-4.34; P=0.0001). These observations suggest that the expression of mutant p53 proteins in SCLC may be an important factor predicting poor prognosis.

Adult↗
PubMed2000

Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing.

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.

Actins↗
PubMed1980

Identification and biochemical analysis of mouse mutants deficient in cytoplasmic malic enzyme.

During the biochemical screening of mutant enzymes in mice, individuals with an apparent nonfunctional allele at the locus (Mod-l) responsible for cytoplasmic malic enzyme were identified by starch gel electrophoresis and by enzyme activity measurements. A series of matings and genetic analyses were made, and mice homozygous for the nonfunctional or null allele (Mod-ln) were produced. The mutation appeared to occur spontaneously in the C57BL/6J strain. By double-immunodiffusion and enzyme immunoinactivation assays, the null mutants were shown to express no proteins that cross-react with the antiserum to cytoplasmic malic enzyme (CRM-negative). In liver homogenates of homozygous null mutants, lack of protein components that form complexes with IgG from the cytoplasmic malic enzyme specific antiserum was further demonstrated by passage of the original serum through a mutant liver homogenate--Sepharose column, where the postadsorbed serum retained its titer and specificity. The residual malic enzyme activity (< 10% of the normal) observed in various tissue homogenates of the homozygous null mutants was attributed to that of mitochondrial isozyme of malic enzyme. Assays of enzymes from tissues of different genotypes revealed no significant differences in activities of six other enzymes in the related metabolic pathways. However, in liver from mutant mice, a lower NADPH/NADP+ ratio was consistently observed in comparison to that from control mice. Both the mutant and the control mice of the same age were found to have comparable body weight and lipid content.

Alleles↗
PubMed1998

Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles.

The structure of the gene encoding the apoprotein of phytochrome B (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225.

Alleles↗
PubMed1997

Identification of a tumor-associated mutant form of the NF-kappaB RelA gene with reduced DNA-binding and transactivating activities.

Alterations of NF-kappaB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for kappaB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-kappaB members, such as p50, cREL and Ikappa Balpha. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

Animals↗
PubMed2000

Identification and characterization of JunD missense mutants that lack menin binding.

Menin, the product of the MEN1 tumor suppressor gene, binds to the AP1 transcription factor JunD and represses JunD transcriptional activity. The effects of human or mouse JunD missense mutations upon menin interaction were studied by random and alanine scanning mutagenesis of the menin binding region of JunD (amino acids 1-70). JunD mutant proteins were tested for menin binding in a reverse yeast two-hybrid assay, and for transcriptional regulation by menin in AP1-reporter assays. Random mutagenesis identified two different mutations that disrupted menin interaction at mouse JunD amino acid 42 (G42E and G42R). Mutation G42A generated by alanine scanning did not affect menin binding, likely reflecting the conserved nature of this amino acid substitution. Furthermore, by size exclusion chromatography menin co-migrated with wild type JunD but not with the JunD mutant tested (G42E). Alanine scanning mutagenesis of residues 30-55 revealed two different amino acids, P41 and P44, of mouse JunD that were critical for interaction with menin. Mouse JunD missense mutants P41A, G42R, G42E and P44A failed to bind menin and also escaped menin's control over their transcriptional activity. At lower amounts of transfected menin, the transcriptional effect of menin on the mutants P41A, G42R and G42E was changed from repression to activation, similar to that with c-jun. In conclusion, a small N-terminal region of JunD mediates a key difference between JunD and c-jun, and a component of this difference is dependent on JunD binding to menin.

Amino Acid Sequence↗
PubMed2000

Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection.

BACKGROUND AND AIMS: The expression of hepatitis B viral (HBV) antigens in liver tissue reflects the replicative status of chronic HBV infection. We have previously recognized a novel marginal pattern of hepatitis B surface antigen (HBsAg) in hepatocytes, which usually clusters in groups and emerges at the late non-replicative phase. This study was designed to investigate whether the marginal-type HBsAg represented the gene product of a specific HBV-surface mutant. METHODS: Microdissection of cirrhotic nodules homogeneously expressing marginal HBsAg was performed on two of 12 resected livers from HBsAg-seropositive patients with hepatocellular carcinoma. The gene presumably encoding marginal HBsAg was polymerase chain reaction (PCR)-cloned, sequenced and analysed. In vitro transfection and expression of the cloned surface mutant plasmids were performed on the Huh7 cell line to illustrate intrahepatic HBsAg expression. RESULTS: Immunohistochemical staining revealed that the marginal HBsAg was positive for pre-S1 and thus contained large surface proteins. The PCR cloning and sequencing of the genes presumably encoding marginal-type HBsAg in both cases revealed the same deletion at the 5' terminus (nt 2-55) of pre-S2. A point mutation on the small-surface (S) antigen was also found in one case. The pre-S2 deletion sequence and the mutation sites of the S gene coincide with human lymphocyte antigen-restricted T- and/or B-cell epitopes. In vitro transfection of the mutant plasmid revealed a blot-like retention or accumulation of HBsAg in the cytoplasm or at the periphery of hepatocytes, accompanied by a decreased secretion of HBsAg in the culture supernatant, mimicking intrahepatic expression. CONCLUSION: A natural pre-S2 deletion mutant was identified in hepatocytes expressing a novel marginal pattern of HBsAg, which probably contains mutant, large, surface proteins. The biological significance of the pre-S2 deletion mutant should be interesting in view of the clustering proliferation of hepatocytes expressing marginal HBsAg.

Female↗
PubMed2006

The apoptosis inhibitor ARC undergoes ubiquitin-proteasomal-mediated degradation in response to death stimuli: identification of a degradation-resistant mutant.

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death.

Animals↗
PubMed2004

Identification of a transcriptionally inactive p53 mutant by functional analysis of separated alleles in yeasts (FASAY) in a child osteosarcoma tumor: a case report.

Tumor suppressor gene p53 is one of the most specific genetic alterations occurring in osteosarcoma pathogenesis. It is thought to be an early and key step in the tumorigenesis of osteosarcoma. However, whether the p53 status is a marker predictive of response to therapy and a marker of prognostic value remains controversial. The choice of p53 status detection method certainly account for discrepancies. The authors used a simple functional assay (functional analysis of separated alleles in yeast) on the tumor sample of an 8-year-old girl presenting with an osteosarcoma of the tibia. While making it possible to exclude the presence of a germline mutation, FASAY indicated the presence of a somatic p53 mutation lacking transcriptional activity on p21 and bax target genes. FASAY also strongly suggested a loss of heterozygosity p53, which was confirmed by cytogenetic analysis. Sequencing of cDNA extracted from yeast colonies containing mutated p53 identified a 213 stop mutation in exon 6. Despite these p53 alterations, the child is still in complete remission after a follow-up of 48 months.

Child↗
PubMed2003

GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana.

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/

Arabidopsis↗
PubMed1989

A female sterile screen of the Drosophila melanogaster X chromosome using hybrid dysgenesis: identification and characterization of egg morphology mutants.

We have conducted a hybrid dysgenic screen of the X chromosome for mutations affecting female fertility, with particular attention to those causing abnormal egg and eggshell morphology. In a screen of 4017 dysgenic strains, 398 mutants derived from 168 different germ lines were isolated and assigned to eight classes according to their diverse phenotypes. One interesting class consists of mutants that block oogenesis at specific stages. Our analysis has focused on mutations affecting eggshell formation, including mutants that lay morphologically abnormal sterile eggs as well as those that lay no eggs but exhibit blocks in the late stages of oogenesis. A subset of 48 mutants was assorted into 30 allelic groups by inter se complementation and genetically localized by interval mapping. Two multiallele complementation groups, de1 (7 alleles) and ne1 (8 alleles), were identified as well as five two-allele complementation groups. A search for alleles among mutants generated in other female sterile screens was unsuccessful, pointing to the distinctive nature of the dysgenic mutant collection. The single case of allelism determined in this study was one with a lethal allele of the Broad-Complex, l(1)npr, suggesting a possible involvement of ecdysone in choriogenesis. A subset of 18 dysgenic strains was analyzed for P element hybridization and 16 of these were found to have hybridization signals in the appropriate cytogenetic interval. By examining these signals in two or more alleles of the same complementation group, we have been able to tentatively localize two mutations. Light and electron microscopy of the eggshell in 43 different strains has revealed a variety of effects. The respiratory appendages were defective in 27 of these mutants. Effects on the ultrastructure of the main body of the endochorion were not strongly correlated with the appendage defects, and could be classified as minor (14 mutants) or major (16 mutants). Although 13 mutants showed no ultrastructural chorion defects, six of these had defective respiratory appendages.

Animals↗
PubMed1994

Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis.

Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokines mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.

Alleles↗
PubMed2002

Identification and analysis of a hyperactive mutant form of Drosophila P-element transposase.

Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI(3)-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

Amino Acid Sequence↗
PubMed2005

Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy.

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression.

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma.

In previous studies, we established two camptothecin (CPT)-resistant sublines, HT-29 / CPT and St-4 / CPT, from the human colon cancer cell line HT-29 and the human stomach cancer cell line St-4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight-fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT-29 / CPT. The longer mRNA (4.0 kb) is the wild-type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58 - 729 or 59 - 730) that caused the in-frame deletion of amino acids 20 - 243 of human topo I. The deleted region is identical to exons 3 - 9 of the TOP1 gene. The expression level of the 3.3-kb mRNA was similar to that of the wild-type mRNA in HT-29 / CPT. St-4 / CPT expressed only the wild-type TOP1 mRNA in lesser amounts than did St-4. Mouse NIH3T3 cells transfected with the wild-type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT-sensitive topo I in the resistant cells.

Amino Acid Sequence↗