Induction,ultrastructure, isolation,and tissue culture of chlorophyll mutants in carrot

Summary Seeds ofDaucus carota “Danvers” were treated with ethyl methanesulfonate (EMS) for 6-hr periods at concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0% to induce plastid mutants. In these treatments, there was a gradual decrease in percent germination from control up to 1.0% EMS and no germinations at 3.0%. The number of chlorophyll mutants increased with dose of mutagen. One mutant plant was isolated from the 0.1% treatment and it had leaf sections of green, white layered on green and pure white; and white and green striped petioles. Histogenic analysis of this mutant showed it to be a GGW chimera, the “all-white” sectors being GWW, arising from displacement of L-II by L-III. Electron micrographs of the white sections showed plastids that had dilated thylakoids typical of PS-I mutants. So called “mixed cells” of normal and mutant plastids were found, suggesting a plastome mutation. Leaf and petiole sections have been successfully cultured through the development of callus, and both green and white plants have been regenerated. Regenerated white plantlets were insensitive to 10 mM methyl viologen (paraquat), whereas green tissues were killed by the herbicide. Support for some of this work was provided by the Miami University Faculty Research Committee and the Comprehensive Employment and Training Act (CETA). This paper will be included as part of the dissertation work of P. D. Miller.     

Thermo-inducible expression of cloned early genes of bacteriophage Mu.

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28 degrees C at high copy number, but at 42 degrees C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42 degrees C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.     

Two-nucleotide codon change in a hemoglobin polymorphism of the Celebes black ape (Macaca nigra)

A hemoglobin polymorphism involving variant -chains was demonstrated in the Celebes black ape, Macaca nigra. Fingerprinting and amino acid analysis of the tryptic peptides from the two chain types have shown that they differ by a single amino acid substitution, between lysine and aspartic acid, which requires a two-nucleotide change in the corresponding codon. Another substitution in the same codon is found as a species difference between the black ape -chain and that of other macaques.     

Extensive size homoplasy at a microsatellite locus in the Japanese bumblebee, Bombus diversus

An extensive size homoplasy was found at microsatellite locus B11 of the bumblebee, Bombus diversus, in northern to central Honshu, Japan. A total of 16 alleles of different nucleotide sequences in five length morphs was obtained at B11 for this species. Of these alleles, five were 141 base pairs (bp) in length, five were 137 bp and four were 133 bp. Allele diversity in each length morph was high compared with previous studies. It is noteworthy that this extensive size homoplasy was found in a relatively small geographic area, in contrast to results from previous studies. Reconstruction of a median‐joining network revealed the complicated evolutionary process of the locus, involving insertion/deletion and point mutations. Preliminary estimation of the mutation rate of the B11 locus in B. diversus gives a value comparable to those estimated from experimental Drosophila populations. Effects of the extensive size homoplasy in population genetic studies is discussed.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Systematic analysis of multigene predictors in gastric cancer exploiting gene expression signature

Gastric cancer (GC) is the second most common cause of cancer death worldwide but could be more curable if diagnosed at an earlier stage. At present, the capability to predict the efficaciousness of molecular diagnosis for GC for each patient remains elusive. The purpose of this study was to identify tumor biomarkers through systems analysis of multigene predictors exploiting the available data resource. In this study, we investigated the top 10% overexpressed genes in GC from five data sets of the Oncomine platform, with 265 GC samples versus 174 normal gastric mucosa samples. Sixteen candidate genes were identified as predictors of GC, of which 14 genes were verified through the comparison of expression levels in specimens from normal (chronic gastritis, 21 samples) and GC groups (38 samples). In addition, unique molecular portraits of diffuse adenocarcinoma (DA), intestinal adenocarcinoma (IA), and mixed adenocarcinoma (MA) were studied through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, where DA showed higher extracellular matrix alteration while IA and MA showed higher cell-cycle alteration than other types. We also found that the elevated expressions of genes during GC progression were independent of gene mutations, and high core-binding factor subunit β expression is correlated with a high overall survival rate in GC patients. Our research may provide an efficient clinical diagnosis of GC at an early stage with high accuracy and thus help improve the overall survival rate through early therapeutic interventions.     

Lethal mutagenesis and evolutionary epidemiology

The lethal mutagenesis hypothesis states that within-host populations of pathogens can be driven to extinction when the load of deleterious mutations is artificially increased with a mutagen, and becomes too high for the population to be maintained. Although chemical mutagens have been shown to lead to important reductions in viral titres for a wide variety of RNA viruses, the theoretical underpinnings of this process are still not clearly established. A few recent models sought to describe lethal mutagenesis but they often relied on restrictive assumptions. We extend this earlier work in two novel directions. First, we derive the dynamics of the genetic load in a multivariate Gaussian fitness landscape akin to classical quantitative genetics models. This fitness landscape yields a continuous distribution of mutation effects on fitness, ranging from deleterious to beneficial (i.e. compensatory) mutations. We also include an additional class of lethal mutations. Second, we couple this evolutionary model with an epidemiological model accounting for the within-host dynamics of the pathogen. We derive the epidemiological and evolutionary equilibrium of the system. At this equilibrium, the density of the pathogen is expected to decrease linearly with the genomic mutation rate U. We also provide a simple expression for the critical mutation rate leading to extinction. Stochastic simulations show that these predictions are accurate for a broad range of parameter values. As they depend on a small set of measurable epidemiological and evolutionary parameters, we used available information on several viruses to make quantitative and testable predictions on critical mutation rates. In the light of this model, we discuss the feasibility of lethal mutagenesis as an efficient therapeutic strategy.     

POLYOL DEHYDROGENASE: PURIFICATION. EVIDENCE FOR MULTIPLE FORMS AND SOME PROPERTIES OF THE DOMINANT VARIANT OF THE HORSE LIVER ENZYME

Purification of horse-liver polyoi dehydrogenase (PDH) on DE52 anion-exchange cellulose reveals the presence of three fractions with enzyme activity. These appear in the breakthrough volume (PDH-3) and the salt gradient (PDH-1, -2) respectively. The major band of activity (< 90%) is found in the PDH-2 fraction. A reexamination of sheep-liver polyol dehydrogenase also reveals the presence of three bands of activity, with the dominant fraction (PDH-3) corresponding to the preparation described by Smith (Biochem. J., 83, 135–144, (1962))³. The interaction between horse-liver (and sheep-liver) PDH and Blue Sepharose CL-6B is found to be endothermic. This property is utilized in the final purification step. Horse-liver PDH-2 has a molecular/subunit weight of 85, 000/28, 000, a Stokes' radius of 3.8 nm, and an isoelectric point of 7.4.     

A novel ABCB11 mutation in an Iranian girl with progressive familial intrahepatic cholestasis

Progressive familial intrahepatic cholestasis is an autosomal recessive liver disorder caused by (biallelic) mutations in the ATP8B1 of ABCB11 gene. A nine-year-old girl with cholestasis was referred for genetic counseling. She had a family history of cholestasis in two previous expired siblings. Genetic analysis of the ABCB11 gene led to the identification of a novel homozygous mutation in exon 25. The mutation 3593- A > G lead to a missense mutation at the amino acid level (His1198Arg). This mutation caused PFIC2 due to abnormal function in the bile salt export pump protein (BSEP).