Elbert Branscomb

The inevitable journey to being

Life is evolutionarily the most complex of the emergent symmetry-breaking, macroscopically organized dynamic structures in the Universe. Members of this cascading series of disequilibria-converting systems, or engines in Cottrells terminology, become ever more complicated—more chemical and less physical—as each engine extracts, exploits and generates ever lower grades of energy and resources in the service of entropy generation. Each one of these engines emerges spontaneously from order created by a particular mother engine or engines, as the disequilibrated potential daughter is driven beyond a critical point. Exothermic serpentinization of ocean crust is lifes mother engine. It drives alkaline hydrothermal convection and thereby the spontaneous production of precipitated submarine hydrothermal mounds. Here, the two chemical disequilibria directly causative in the emergence of life spontaneously arose across the mineral precipitate membranes separating the acidulous, nitrate-bearing CO2-rich, Hadean sea from the alkaline and CH4/H2-rich serpentinization-generated effluents. Essential redox gradients—involving hydrothermal CH4 and H2 as electron donors, CO2 and nitrate, nitrite, and ferric iron from the ambient ocean as acceptors—were imposed which functioned as the original ‘carbon-fixing engine’. At the same time, a post-critical-point (milli)voltage pH potential (proton concentration gradient) drove the condensation of orthophosphate to produce a high energy currency: ‘the pyrophosphatase engine’.

Enzymatic determinants of DNA polymerase accuracy. Theory of coliphage T4 polymerase mechanisms.

Abstract The specificity of base selection in DNA synthesis is affected by the DNA polymerase. This paper presents a detailed model of the enzymatic reaction steps involved in template-directed DNA synthesis by DNA polymerase possessing 3′ to 5′ exonuclease activity (the T4 coliphage enzyme and polymerases I, II and III of Escherichia coli are known examples). The central assumptions of the model imply that the strength of the hydrogen bonds formed between candidate bases and the template base provide the principal discrimination criteria for both the insertion and the “editing” excision of bases. We have performed a detailed analysis of this model, and of the stochastic, sequential, single-step process of insertion and excision, which it implies, and have compared the results with experimental data reported by Bessman et al. (1974a,b) for the DNA polymerase of T4. In the present version of the model, the presence of the editing exonuclease accounts for the enzymes contribution to the accuracy of polymerization and all binding and reaction sites on the enzyme are insensitive to differences between bases or base-pairs. The Bessman data, describing the competitive incorporation of adenine and 2-aminopurine by five allelic polymerases and spanning a spectrum of mutation rates, are well fit by this theory, and permit us to predict the effects of changes in reaction conditions and in basepairing free energy on the outcome of similar experiments.

The complete sequence of human chromosome 5

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

Estimating maximum limits to mutagenic potency from cytotoxic potency

RAPID and reliable screening methods are required for identifying environmental mutagens and estimating their mutagenic potency in preparation for use of more elaborate tests to assess the genetic risk to man. Many compounds are effectively screened by in vitro mutagenesis assays using either bacterial or mammalian cells. Although tests with mammalian cells are not as rapid and inexpensive as microbial assays, they are needed to confirm and extend the bacterial results, and probably provide a better assessment of potential risk to humans than do microbial tests. However, the screening of all potential mutagens with mammalian cell mutagenicity assays, although desirable, does not seem feasible unless, as we propose, test compounds are first pre-screened on the basis of their cytotoxic potency. On theoretical grounds, one expects a certain minimum cytotoxic potency to correlate with mutagenic potency, particularly when the latter is measured using forward mutations that result in inactive gene products. Specifically, cells cannot divide unless a substantial fraction of their genome is functionally intact; mutagenic agents should thus be obligatory cytotoxic agents, with a given mutagenicity conferring a certain irreducible cytotoxicity. We show here that the cytotoxic potency of 22 chemical mutagens is highly correlated with their mutagenic potency as assayed in five rodent and human in vitro cell systems. This relationship implies that the maximum potential mutagenic potency of such compounds may be reliably estimated from rapid and straightforward measurements of their cytotoxic potency, the latter defined as the failure of cultured cells to undergo continued cell division. The estimate is necessarily a maximum one, as an agent may exert cytotoxic effects by pathways independent of its mutagenic action.

Tandem Zinc-Finger Gene Families in Mammals: Insights and Unanswered Questions

Evidence for the remarkable conservation of mammalian genomes, in both content and organization of resident genes, is rapidly emerging from comparative mapping studies. The frequent occurrence of familial gene clustering, presumably reflecting a history of tandem in situ duplications starting from a single ancestral gene, is also apparent from these analyses. Genes encoding Kruppel-type zinc-finger (ZNF) proteins, including those containing Kruppel-associated box (KRAB) motifs, are particularly prone to such clustered organization. Existing data suggest that genes in KRAB-ZNF gene clusters have diverged in sequence and expression patterns, possibly yielding families of proteins with distinct, yet related, functions. Comparative mapping studies indicate that at least some of the genes within these clusters in mammals were elaborated prior to the divergence of mammalian orders and, subsequently, have been conserved. These data suggest a possible role for these tandem KRAB-ZNF gene families in mammalian evolution.

On the High Value of Low Standards

Is there a case to be made for draft sequencing? First, we need to get a fix on how much less it costs than complete genome sequencing, how much faster and/or easier it is to do, and how much and what types of scientific utility are sacrificed. But this is not a straightforward issue. No accepted standard for draft sequence data exists; in current practice it ranges from ∼3-fold coverage in short ( 600-bp), “paired-end” (PE) reads (sequencing reads are taken from both ends of the insert in a double-stranded vector and therefore come in oppositely directed pairs separated by an approximately known distance) of mixed separation lengths. Quality differences over that spectrum are relatively great, as are, though to a much smaller extent, cost differences. The “draft-or-finish” alternatives are hardly exclusive; mixed, staged, or context-dependent strategies may also make sense. All the parameters are evolving rapidly. And finally, there is as yet too little experience to support definitive answers, although clearly enough to get an argument going in the better genome bars. First, we address the production side of the question; consider the hypothetical case of sequencing factory X. This exemplary facility can produce over 30 Mb of high-quality (PE) bases per day at a fully loaded marginal cost of 0.3¢ base. Factory X has concluded that for most DNA, 8× PE coverage is usually optimal, both for producing draft data that are not intended for subsequent finishing and as a substrate for finishing. With this choice, finish-ready draft data have, at factory X, a current marginal cost of ∼2.5¢ base and can be produced at a rate of 3.6 Mb/day with a delay from time of DNA receipt to draft product on the order of 2 weeks. The quality of this sequence is discussed below, but the general nature of its coverage integrity should be noted here. In ∼8-fold PE draft data, the overall coverage is typically high (>95% of the sequence represented). Most importantly, and especially so if a judicious mix of large and small inserts is used in the sequencing, “almost all” points in the sequence—including gaps between the contigs (contigs are contiguous stretches of sequence produced by assembling overlapping individual reads)—are bridged, or spanned, by multiple plasmid clones. This permits the automatic production of relatively high-quality, internally verified assembly and makes it possible to order and orient most of the contigs relative to each other to form large “scaffolds,” or sequence islands of valid order and high coverage. In such data, the expected error rate across genes is often better than 1/104, and a good estimate of the accuracy of each base can be made available. Factory X can also finish such data to full “Bermuda” standards, i.e., an expected base-calling error rate of <1/104 and no gaps or other errors that mortal efforts could remove (these standards were established at meetings of the international Human Genome Project community), for an average additional cost of 7¢ base (and thus for a total cost of ∼10¢ base). Somewhat typically, however, factory Xs finishing capacity is manyfold below its drafting capacity. Furthermore, the time needed to finish a segment of draft sequence can average several months and is highly variable. In this landscape, “full Bermuda” data are about four times as expensive, and very much slower to produce, than “high-quality” draft data. For the extra cost of finishing a bacterial genome, three additional ones could be drafted. While factory X is finishing a bacterial genome, it could draft, in the sense described, upwards of a hundred more. To our necessarily imperfect knowledge, no sequencing facility is currently producing either PE raw data or “fully finished” sequence data for true costs significantly below those quoted. But the relative advantage in cost and project completion time of draft versus finished sequence data at factory X might well not be the same in other facilities. And of course, the differences in steady-state production capacity for draft versus finished sequence used in the example are in large measure merely an arbitrary matter of resource commitment. Also, there are some, at least potential, hidden costs in producing draft data that should be considered. (i) Draft sequence errors and imperfections may mislead users and thereby entail costs in wasted effort and delay. (ii) It may be substantially more expensive on average to finish draft sequence data later, should it prove desirable, than to do so at the start and in the same laboratory. (iii) Many have seen a risk that the will (at either the funding or bench level) to ever fully finish sequence data will be lost should we permit ourselves the cheap and easy pleasures of draft sequencing. We comment a little on these questions at the end. The next issue is the quality and utility of draft sequence data, focusing in particular on what we know about (i) sequence coverage, (ii) gene recovery and quality, and (iii) chromosome integrity and long-range order.

Fluorescence in Situ hybridization mapping of human chromosome 19: Mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

Somatic Mutations Detected by Immunofluorescence and Flow Cytometry

In order to detect the genotoxic effects of living in today’s complex industrialized society, we must devise sensitive ways to measure early subtle changes in human beings that may lead to more pathological effects in longer periods of time. The detection of mutational changes in somatic cells is a possible way of monitoring for effects that could ultimately lead to carcinogenic or heritable lesions. Depending on the lifespan of the somatic cells analyzed, the frequency of mutated somatic cells in an individual may be a measurement of the effects of recent toxic exposure or an indication of cumulative genetic insult.

Quantitative theory of in vivo lac regulation: significance of repressor packaging. I. Equilibrium considerations.

Abstract This paper presents a quantitative formulation of the repressor-operator interaction suitable for comparison with in vivo data. The theory predicts the average fraction of free operators in a population of exponentially growing cells whose content of repressors and operators is realistically represented. Both the smallness of the numbers of molecules involved in the reaction within each cell, and the variation in the number of repressors present from cell to cell are shown to effect substantially the free operator content of the population. The difference between this formulation and the simple “mass action” theory is presented both analytically and numerically. We are led to expect important quantitative differences between the results of certain in vivo experiments and their in vitro analogs.

Use of Fluorescence-Activated Cell Sorter for Screening Mutant Cells

Until recently, virtually all measurements of cellular mutation frequencies have been based on clonogenic assays. In fact, clonogenicity has been considered essential for the legitimate identification of mutant cells because it confirms the transmissbility of the variant phenotype. However, largely because of the apparent connection between somatic mutagenesis and carcinogenesis, it has become important to measure mutational injury in samples of normal somatic cells that generally have little or no replicative capacity. As a result, a number of efforts are underway to develop nonclonogenic mutation assays applicable to cells obtained from in vivo tissue samples and to find alternative means for authenticating the mutant pedigree of detected cells.