Showing papers on "Mutant published in 1992"

Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours

Abstract: Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumorigenesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.

Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice

Abstract: Although long-term potentiation (LTP) has been studied as the mechanism for hippocampus-dependent learning and memory, evidence for this hypothesis is still incomplete. The mice with a mutation in the alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII), a synaptic protein enriched in the hippocampus, are appropriate for addressing this issue because the hippocampus of these mice is deficient in LTP but maintains intact postsynaptic mechanisms. These mutant mice exhibit specific learning impairments, an indication that alpha-CaMKII has a prominent role in spatial learning, but that it is not essential for some types of non-spatial learning. The data considerably strengthen the contention that the synaptic changes exhibited in LTP are the basis for spatial memory.

Randomization of genes by PCR mutagenesis.

Abstract: A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 066% +/- 013% (95% CI) per position per PCR, as determined by sequence analysis There are no strong preferneces with respect to the type of base substituion The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence

Requirement for a functional Rb-1 gene in murine development

Abstract: Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Abstract: Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.

Abstract: We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.

Oncogenic forms of p53 inhibit p53-regulated gene expression

Abstract: Mutant forms of the gene encoding the tumor suppressor p53 are found in numerous human malignancies, but the physiologic function of p53 and the effects of mutations on this function are unknown. The p53 protein binds DNA in a sequence-specific manner and thus may regulate gene transcription. Cotransfection experiments showed that wild-type p53 activated the expression of genes adjacent to a p53 DNA binding site. The level of activation correlated with DNA binding in vitro. Oncogenic forms of p53 lost this activity. Moreover, all mutants inhibited the activity of coexpressed wild-type p53, providing a basis for the selection of such mutants during tumorigenesis.

Structural alterations of the epidermal growth factor receptor gene in human gliomas

Abstract: The epidermal growth factor receptor (EGFR) gene is amplified in 40% of malignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations associated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.

Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

Abstract: The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunof...

Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant

Abstract: Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), are plant signaling molecules that affect plant growth and gene expression. Primary root growth of wild-type Arabidopsis thaliana seedlings was inhibited 50% when seedlings were grown on agar medium containing 0.1 M MeJA. An ethyl methanesulfonate mutant (jar1) with decreased sensitivity to MeJA inhibition of root elongation was isolated and characterized. Genetic data indicated the trait was recessive and controlled by a single Mendelian factor. MeJA-induced polypeptides were detected in Arabidopsis leaves by antiserum to a MeJA-inducible vegetative storage protein from soybean. The induction of these proteins by MeJA in the mutant was at least 4-fold less in jar1 compared to wild type. In contrast, seeds of jar1 plants were more sensitive than wild type to inhibition of germination by abscisic acid. These results suggest that the defect in jar1 affects a general jasmonate response pathway, which may regulate multiple genes in different plant organs.

Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62.

Abstract: A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.

A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli.

Abstract: A starvation-inducible DNA-binding protein was discovered as a result of the analysis of proteins synthesized in 3-day-old cultures of Escherichia coli. This 19-kD protein, designated Dps, is abundant in starved cells. In vitro, Dps forms extremely stable complexes with DNA, without apparent sequence specificity. When complexed with Dps, DNA is rendered DNase resistant. Mutant cells lacking Dps show dramatic changes in the pattern of proteins synthesized during starvation. The mutants also fail to develop starvation-induced resistance to hydrogen peroxide, an agent that can cause oxidative damage to DNA in vivo. These results have prompted us to postulate that Dps plays an important role both in gene expression and DNA protection during stationary phase. The existence of similar proteins, heretofore with no known function, in bacterial species distantly related to Escherichia coli suggests that Dps may define a novel class of widely conserved DNA-binding proteins.

Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.

Abstract: Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene

Abstract: Waardenburg's syndrome (WS) is an autosomal dominant combination of deafness and pigmentary disturbances, probably caused by defective function of the embryonic neural crest. We have mapped one gene for WS to the distal part of chromosome 2. On the basis of their homologous chromosomal location, their close linkage to an alkaline phosphatase gene, and their related phenotype, we suggested that WS and the mouse mutant Splotch might be homologous. Splotch is caused by mutation in the mouse Pax-3 gene. This gene is one of a family of eight Pax genes known in mice which are involved in regulating embryonic development; each contains a highly conserved transcription control sequence, the paired box. Here we show that some families with WS have mutations in the human homologue of Pax-3. Mutations in a related gene, Pax-6, which, like Pax-3, has both a paired box and a paired-type homeobox sequence, cause the Small-eye mutation in mice and aniridia in man. Thus mutations in the Pax genes are important causes of human developmental defects.

Wild-type p53 activates transcription in vitro

Abstract: The p53 protein is an important determinant in human cancer and regulates the growth of cells in culture. It is known to be a sequence-specific DNA-binding protein with a powerful activation domain, but it has not been established whether it regulates transcription directly. Here we show that intact purified wild-type human and murine p53 proteins strongly activate transcription in vitro. This activation depends on the ability of p53 to bind to a template bearing a p53-binding sequence. By contrast, tumour-derived mutant p53 proteins cannot activate transcription from the template at all, and when complexed to wild-type p53, these mutants block transcriptional activation by the wild-type protein. Moreover, the simian virus 40 large T antigen inhibits wild-type p53 from activating transcription. Our results support a model in which p53 directly activates transcription but this activity can be inhibited by mutant p53 and SV40 large T antigen through interaction with wild-type p53.

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans.

Abstract: The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.

Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis

Abstract: A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation.

Abstract: The yeast RAP1 protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAP1 is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(C1-3A) sequences. Previous studies have indicated that the regulatory function of RAP1 is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAP1-interacting factor (RIF1). Strains carrying gene disruptions of RIF1 grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rap1s mutants. Furthermore, hybrid proteins containing rap1s missense mutations are defective in an interaction with RIF1 in the two-hybrid system. Taken together, these data support the idea that the rap1s phenotypes are attributable to a failure to recruit RIF1 to silencers and telomeres and suggest that RIF1 is a cofactor or mediator for RAP1 in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIF1 that partially restores the interaction with rap1s mutant proteins.

Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin

Abstract: Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required > 13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the nontoxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.

MELAS mutation in mtDNA binding site for transcription termination factor causes defects in protein synthesis and in respiration but no change in levels of upstream and downstream mature transcripts.

Abstract: The pathogenetic mechanism of the mitochondrial tRNA(LeuUUR) gene mutation responsible for the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) syndrome was investigated in transformants obtained by transfer of mitochondria from three genetically unrelated MELAS patients into human mitochondrial DNA (mtDNA)-less (rho 0) cells. Marked defects in mitochondrial protein synthesis and respiratory activity were observed in transformants containing virtually pure mutant mtDNA, as compared to the parent of the rho 0 cells (the 143B cell line) or to transformants containing exclusively wild-type mtDNA, derived from one of the patients or a maternally related asymptomatic individual. A striking protective effect against the mutation was exerted in the transformants by levels of residual wild-type mtDNA above 6%. The MELAS mutation occurs within the mtDNA binding site for a protein factor (mTERF) that promotes termination of transcription at the 16S rRNA/tRNA(LeuUUR) gene boundary. A marked decrease in affinity of purified mTERF for the mutant target sequence was observed in in vitro assays. By contrast, RNA transfer hybridization experiments failed to show any significant change in the steady-state amounts of the two rRNA species, encoded upstream of the termination site, and of the mRNAs encoded downstream, in the transformants carrying the MELAS mutation.