Number Of Mutant Alleles Research Articles

Is K-ras Point Mutation an Early Event in Lung Tumorigenesis of Mice?

To investigate if K-ras point mutations could occur at an early stage of urethane-induced lung tumorigenesis in mice, we applied a sensitive mutation specific PCR assay technique (MSPA) to DNA from lung at the early stage of this condition. At 7 or 14 d after urethane injection (750 mg/kg, i. p.), pulmonary DNA was isolated and the existence of activated K-ras gene with 61st codon A→T mutation was checked according to the MSPA. This technique can detect mutant alleles in the presence of a very large excess of normal ras alleles ; however, the K-ras point mutation could not be detected by the present MSPA. These results suggest that the K-ras point mutations did not occur at the stage of lung tumorigenesis in mice or that the number of mutant alleles was below the detectable range of the technique.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.

Selection and Characterization of Ricin Toxin A-Chain Mutations in Saccharomyces cerevisiae

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

Studies on <italic>Fused</italic>, a Mutant Gene Producing Ovarian Tumors in <italic>Drosophila melanogaster</italic><xref ref-type="fn" rid="FN1">2</xref>

Flies homozygous for the mutant gene fused (fu) are characterized by fused wing veins, a maternal effect upon embryogenesis, and ovarian tumors. A number of mutant alleles have been studied and all exhibit this same syndrome. The rate at which ovarian tumors develop is characteristic for each allele. This rate was shown to be independent of modifying genes and unaffected by the maternal cytoplasm. The rate is also temperature-sensitive, tumors appearing more slowly at 18 than at 25 degrees C. Females heterozygous for all possible combinations of these alleles were produced, and, in all 21, tumors were observed in the compound females. Cases of dominance, complementarity, and interference were observed with reference to certain allelic combinations. A model is proposed to explain the diverse effects on development of the fused gene and also the interaction of the alleles in ovarian development. Females homozygous for fused and either heterozygous or homozygous for the second chromosomal recessive gene female-sterile (fes) were also studied. The fes gene was shown to be epistatic to fu in fu/fu; fes/fes females, and the rate of tumor production was greater in fu/fu; fes/+ females than in fu/fu; +/+ females. A mechanism for the action of fu and fes in controlling cell division is presented.