Dominant transformation by mutated human ras genes in vitro requires more than 100 times higher expression than is observed in cancers

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-ras, “play a dominant part in cancer,” because they are sufficient to transform transfected mouse cell lines in vitro [Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York)]. However, in cells transformed in vitro mutated human ras genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of ras transcription in cancers and the very high levels in cells transformed in vitro, we have investigated the minimal level of human ras expression for transformation in vitro. Using point-mutated human ras genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant in vitro transformation of the mouse C3H cell line only with ras genes linked to viral promoters. These ras genes were expressed more than 120-fold higher than are native ras genes of C3H cells. The copy number of transfected ras genes ranged from 2–6 in our system. In addition, nondominant transformation was observed in a small percentage (2–7%) of C3H cells transfected with ras genes that are expressed less than 20 times higher than native C3H ras genes. Because over 90% of cells expressing ras at this moderately enhanced level were untransformed, transformation must follow either a nondominant ras mechanism or a non-ras mechanism. We conclude that the mutated, but normally expressed, ras genes found in human and animal cancers are not likely to “play a dominant part in cancer.” The conclusion that mutated ras genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated ras in normal animals, and in benign human tissue, “which has little potential to progress” [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994) Cancer Res. 54, 5523–5526]. Even the view that mutated ras is necessary for cancer is hard to reconcile with (i) otherwise indistinguishable cancers with and without ras mutations, (ii) metastases of the same human cancers with and without ras mutations, (iii) retroviral ras genes that are oncogenic without point mutations, and (iv) human tumor cells having spontaneously lost ras mutation but not tumorigencity.

Late Quaternary extinction of a tree species in eastern North America

Widespread species- and genus-level extinctions of mammals in North America and Europe occurred during the last deglaciation [16,000–9,000 yr B.P. (by 14C)], a period of rapid and often abrupt climatic and vegetational change. These extinctions are variously ascribed to environmental change and overkill by human hunters. By contrast, plant extinctions since the Middle Pleistocene are undocumented, suggesting that plant species have been able to respond to environmental changes of the past several glacial/interglacial cycles by migration. We provide evidence from morphological studies of fossil cones and anatomical studies of fossil needles that a now-extinct species of spruce (Picea critchfieldii sp. nov.) was widespread in eastern North America during the Last Glacial Maximum. P. critchfieldii was dominant in vegetation of the Lower Mississippi Valley, and extended at least as far east as western Georgia. P. critchfieldii disappeared during the last deglaciation, and its extinction is not directly attributable to human activities. Similarly widespread plant species may be at risk of extinction in the face of future climate change.

Recurrent invasion and extinction of a selfish gene

Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the ω-homing endonuclease gene and associated group I intron. The status of ω could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of ω differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.

Determining divergence times with a protein clock: Update and reevaluation

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

The Malthusian parameter of ascents: What prevents the exponential increase of one’s ancestors?

The reason that the indefinite exponential increase in the number of one’s ancestors does not take place is found in the law of sibling interference, which can be expressed by the following simple equation:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\begin{matrix}{\mathit{N}}_{{\mathit{n}}} \enskip & \\ {\mathit{{\blacksquare}}} \enskip & \\ {\mathit{ASZ}} \enskip & \end{matrix} {\mathrm{\hspace{.167em}{\times}\hspace{.167em}2\hspace{.167em}=\hspace{.167em}}}{\mathit{N_{n+1},}}\end{equation*}\end{document} where Nn is the number of ancestors in the nth generation, ASZ is the average sibling size of these ancestors, and Nn+1 is the number of ancestors in the next older generation (n + 1). Accordingly, the exponential increase in the number of one’s ancestors is an initial anomaly that occurs while ASZ remains at 1. Once ASZ begins to exceed 1, the rate of increase in the number of ancestors is progressively curtailed, falling further and further behind the exponential increase rate. Eventually, ASZ reaches 2, and at that point, the number of ancestors stops increasing for two generations. These two generations, named AN SA and AN SA + 1, are the most critical in the ancestry, for one’s ancestors at that point come to represent all the progeny-produced adults of the entire ancestral population. Thereafter, the fate of one’s ancestors becomes the fate of the entire population. If the population to which one belongs is a successful, slowly expanding one, the number of ancestors would slowly decline as you move toward the remote past. This is because ABZ would exceed 2. Only when ABZ is less than 2 would the number of ancestors increase beyond the AN SA and AN SA + 1 generations. Since the above is an indication of a failing population on the way to extinction, there had to be the previous AN SA involving a far greater number of individuals for such a population. Simulations indicated that for a member of a continuously successful population, the AN SA ancestors might have numbered as many as 5.2 million, the AN SA generation being the 28th generation in the past. However, because of the law of increasingly irrelevant remote ancestors, only a very small fraction of the AN SA ancestors would have left genetic traces in the genome of each descendant of today.

Intramolecular vibrational dephasing obeys a power law at intermediate times

Experimental intramolecular vibrational dephasing transients for several large organic molecules are reanalyzed. Fits to the experimental data, as well as full numerical quantum calculations with a factorized potential surface for all active degrees of freedom of fluorene indicate that power law decays, not exponentials, occur at intermediate times. The results support a proposal that power law decays describe vibrational dephasing dynamics in large molecules at intermediate times because of the local nature of energy flow.

Using rare mutations to estimate population divergence times: A maximum likelihood approach

In this paper we propose a method to estimate by maximum likelihood the divergence time between two populations, specifically designed for the analysis of nonrecurrent rare mutations. Given the rapidly growing amount of data, rare disease mutations affecting humans seem the most suitable candidates for this method. The estimator RD, and its conditional version RDc, were derived, assuming that the population dynamics of rare alleles can be described by using a birth–death process approximation and that each mutation arose before the split of a common ancestral population into the two diverging populations. The RD estimator seems more suitable for large sample sizes and few alleles, whose age can be approximated, whereas the RDc estimator appears preferable when this is not the case. When applied to three cystic fibrosis mutations, the estimator RD could not exclude a very recent time of divergence among three Mediterranean populations. On the other hand, the divergence time between these populations and the Danish population was estimated to be, on the average, 4,500 or 15,000 years, assuming or not a selective advantage for cystic fibrosis carriers, respectively. Confidence intervals are large, however, and can probably be reduced only by analyzing more alleles or loci.

Call to needle times after acute myocardial infarction in urban and rural areas in northeast Scotland: prospective observational study

Objective: To determine call to needle times and consider how best to provide timely thrombolytic treatment for patients with acute myocardial infarction.

Monophyly and extensive extinction of advanced eusocial bees: Insights from an unexpected Eocene diversity

Advanced eusociality sometimes is given credit for the ecological success of termites, ants, some wasps, and some bees. Comprehensive study of bees fossilized in Baltic amber has revealed an unsuspected middle Eocene (ca. 45 million years ago) diversity of eusocial bee lineages. Advanced eusociality arose once in the bees with significant post-Eocene losses in diversity, leaving today only two advanced eusocial tribes comprising less than 2% of the total bee diversity, a trend analogous to that of hominid evolution. This pattern of changing diversity contradicts notions concerning the role of eusociality for evolutionary success in insects.

Estimation of divergence times from multiprotein sequences for a few mammalian species and several distantly related organisms

When many protein sequences are available for estimating the time of divergence between two species, it is customary to estimate the time for each protein separately and then use the average for all proteins as the final estimate. However, it can be shown that this estimate generally has an upward bias, and that an unbiased estimate is obtained by using distances based on concatenated sequences. We have shown that two concatenation-based distances, i.e., average gamma distance weighted with sequence length (d2) and multiprotein gamma distance (d3), generally give more satisfactory results than other concatenation-based distances. Using these two distance measures for 104 protein sequences, we estimated the time of divergence between mice and rats to be approximately 33 million years ago. Similarly, the time of divergence between humans and rodents was estimated to be approximately 96 million years ago. We also investigated the dependency of time estimates on statistical methods and various assumptions made by using sequence data from eubacteria, protists, plants, fungi, and animals. Our best estimates of the times of divergence between eubacteria and eukaryotes, between protists and other eukaryotes, and between plants, fungi, and animals were 3, 1.7, and 1.3 billion years ago, respectively. However, estimates of ancient divergence times are subject to a substantial amount of error caused by uncertainty of the molecular clock, horizontal gene transfer, errors in sequence alignments, etc.

Metapopulation extinction caused by mutation accumulation

Theory suggests that the risk of extinction by mutation accumulation can be comparable to that by environmental stochasticity for an isolated population smaller than a few thousand individuals. Here we show that metapopulation structure, habitat loss or fragmentation, and environmental stochasticity can be expected to greatly accelerate the accumulation of mildly deleterious mutations, lowering the genetic effective size to such a degree that even large metapopulations may be at risk of extinction. Because of mutation accumulation, viable metapopulations may need to be far larger and better connected than would be required under just stochastic demography.