Messenger RNA metabolism of animal cells: possible involvement of untranslated sequences and mRNA-associated proteins

The past several years have seen a virtual revolution in the study of eukaryotic mRNA. Among the notable recent achievements are the positive identification of mRNA precursors in HnRNA, the enumeration of the DNA sequences from which mRNA is transcribed, and the finding that mRNA in cultured cells is much more stable than was previously believed. One of most far-reaching discoveries has been the finding that mRNA in eukaryotes contains poly A. This discovery, aside from providing a powerful tool for mRNA isolation, has generated a large body of research into the properties and metabolism of poly A itself. In addition, the finding of a poly A-associated protein has given a renewed stimulus to the study of proteins associated with mRNA. This review is devoted to a discussion of these and related achievements, and some of their implications     

Mutants of Salmonella typhimurium deficient in an endoprotease.

Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.     

The Genetic Structure and Evolutionary Fate of Parthenogenetic Amphibian Populations as Determined by Markovian Analysis

SYNOPSIS. One-locus, two-allele models are presented which describethe genetic consequences of naturally occurring andexperimentallyinduced parthenogesis in triploid and diploid amphibians. Themodels may in general be used to investigate genetic changeresulting from apomictic (ameiotic) and automictic (meiotic)parthenogenetic reproduction. These models quantify the influence of mutation, segregation,and selection upon genetic variability in parthenogeneticpopulations.They also allow an estimate of the relative importance of stochasticforces in altering this variability. They thus provide a basisfor understanding evolution in these populations. Some of the conclusions derived from this study contradict previouspredictions regarding genetic variability in parthenogeneticpopulations. First, if mutation is the sole source of geneticchange (i.e., strict apomixis), parthenogenetic populationsshould not become completely heterozygous. Second, small amountsof segregation occurring in apomictic populations have enormouseffects upon the genetic variability of these populations, i.e.,they should lose much of their heterozygosity. In addition to these conclusions, the results of this studysuggest that studies of protein variability in parthenogeneticspecies should contribute toward answering the question: Howmuch of the genetic variability observed in nature is evolutionarilyrelevant?     

Dating of graptolite zones by sedimentation rates: implications for rates of evolution

Preliminary determinations of ancient pelagic sedimentation rates agree with modern rates at about 4 meters per million years. By combining data on the thickness of graptolite zones from the North American Cordillera with data from other parts of the world, we have refined the Early Silurian time scale and obtained much better resolution than is possible for radiometric dates. The new Early Silurian time scale allows estimation of true rates of change in graptolite diversity. The Llandoverian diversity explosion is twice as rapid as was previously thought. The brevity of diversity lows and rapidity of speciation support modern theories of quantum evolution.     

Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

Background

Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.

Methods/Principal Findings

An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries.

Conclusions/Significance

The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Parthenogenetic development of dwarf surf dam,Mulinia lateralis,oocytes treated with polar body suppressing agents

Summary

Parthenogenesis following oocyte activation has been observed in a number of marine invertebrates, but the fate of parthenogenesis in bivalve mollusc embryos is unclear. We used the dwarf surf clam, Mulinia lateralis, to examine parthenogenetic development of KC1-activated oocytes using the polar body suppressing agents caffeine and heat or cytochalasin B. Development was followed by epifluorescence microscopy and flow-cytometric analysis using the DNA-specific fluorochrome DAPI. All agents suppressed polar body formation to some degree, putatively increasing the ploidy level and retaining a meiotic centrosome in the zygote; but the zygotes failed to develop normally. Failure of the zygotes to develop suggests that the meiotic centrosome is incapable of participating in mitosis in bivalves.