The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.

Heritable, population-wide damage to cells as the driving force of neoplastic transformation.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.

Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development

DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ). However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency. However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs. ATM with respect to NHEJ. Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria

Speciation involves the establishment of genetic barriers between closely related organisms. The extent of genetic recombination is a key determinant and a measure of genetic isolation. The results reported here reveal that genetic barriers can be established, eliminated, or modified by manipulating two systems which control genetic recombination, SOS and mismatch repair. The extent of genetic isolation between enterobacteria is a simple mathematical function of DNA sequence divergence. The function does not depend on hybrid DNA stability, but rather on the number of blocks of sequences identical in the two mating partners and sufficiently large to allow the initiation of recombination. Further, there is no obvious discontinuity in the function that could be used to define a level of divergence for distinguishing species.

Rhizobium gone native: Unexpected plasmid stability of indigenous Rhizobium leguminosarum

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.