Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).

Fingerprinting genomes using PCR with arbitrary primers

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing-over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.

Slipped-strand mispairing: a major mechanism for DNA sequence evolution.

Summary Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types (enterohaemorrhagic E. coli , enteropathogenic E. coli , enteroinvasive E. coli , K1 and Shigella ) have arisen independently and repeat- edly in several lineages, whereas other lineages con- tain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homolo- gous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed through- out the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.

http://web.biosci.utexas.edu/ochman/Papers/Wirth_MolMicro2006.pdf

Sex and virulence in Escherichia coli: an evolutionary perspective

Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci. Some species (e.g., Salmonella) were found to be clonal at all levels of analysis. At the other extreme, Neisseria gonorrhoeae is panmictic, with random association between loci. Two intermediate types of population structure were also found. Neisseria meningitidis displays what we have called an "epidemic" structure. There is significant association between loci, but this arises only because of the recent, explosive, increase in particular electrophoretic types; when this effect is eliminated the population is found to be effectively panmictic. In contrast, linkage disequilibrium in a population of Rhizobium meliloti exists because the sample consisted of two genetically isolated divisions, often fixed for different alleles: within each division association between loci was almost random. The method of analysis is appropriate whenever there is doubt about the extent of genetic recombination between members of a population. To illustrate this we analyzed data on protozoan parasites and again found panmictic, epidemic, and clonal population structures.

https://www.pnas.org/content/pnas/90/10/4384.full.pdf

How clonal are bacteria

The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates' recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins, implemented in Python and C and supported on Linux and Mac OS X.

/pdf/rapid-phylogenetic-analysis-of-large-samples-of-recombinant-28bzcbqwh5.pdf

Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins.

Mytilus edulis is a prominent and widely distributed littoral mussel, whose life cycle is typical of intertidal marine invertebrates with an extended larval dispersal period and sedentary adulthood. The larvae appear capable of moving great distances. The present study is an attempt to begin the characterization of the genetic structure of this important species in both its static and dynamic aspects. The distributions of alleles and genotypes over a broad, environmentally diverse geographic range have been determined by electrophoretic analysis, and the possible contributions of selection and migration to these distributions have been investigated. As a result, we here report in detail a number of observations, including the following: the uniformity of genotypic frequencies over large portions of the species range; abrupt and gradual genotypic transitions over great, moderate and very small distances; certain discordances among patterns of variation of different loci; a widespread and striking deficiency of heterozygotes at two loci; and clear evidence of the local selection of certain genotypes at the Leucine aminopeptidase locus. We have sought to interpret our observations in terms of the classical influences on the structure of genetic variation: nonrandom mating, selection, and migration. Given the spawning behavior of mussels. any non-random mating is likely to be inbreeding. Selection might in some cases favor certain alleles or genotypes (heterozygotes, for example) over the entire species range. In other cases, the direction and intensity of selection might vary with habitat, creating a tendency towards genetic differentiation of the species. Dispersal would of course have the cumulative tendency to make the species more uniform, but there could be two characteristic shortterm effects in addition. First, in each generation a deficiency, or less likely, an excess (Li, 1969; Milkman, 1975) of heterozygotes could result from the mixing of individuals from genetically different populations; this deficiency or excess would of course last only for the generation in which it appeared, and another mixing would be required for a similar deviation in the next generation. A second short-term effect is linkage disequilibrium, which (unlike a deviant frequency of heterozygotes) dissipates with first-order kinetics and therefore takes a number of generations, depending on recombination frequency, to become indistinct. When the genotypic frequencies at a polymorphic locus are similar throughout a particular range, then, we can conclude that cumulative dispersal (together with any selection that operates uniformly) is sufficient to counterbalance inbreeding (together with any selection that operates non-uniformly). A deficiency or excess of heterozygotes can indicate selection or dispersal within the period of one life cycle. Linkage disequilibrium can indicate selection or dispersal within the period of a few life cycles. A variety of genetic differences, over distances and times of any magnitude, can be attributed to selection. While 1 This paper is dedicated to the memory of Prof. Ove Frydenberg. 2 Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794. 'Department of Zoology, University of Iowa, Iowa City, Iowa 52242. 'Present Address: Department of Biology-EPO, University of Colorado, Boulder, Colorado 80302.

https://www.jstor.org/stable/pdfplus/2407669.pdf

Population genetics of marine pelecypods. iv. selection, migration and genetic differentiation in the blue mussel mytilus edulis.

PCR fragments, 1500-bp, from 15 previously sequenced regions in the Escherichia coli chromosome have been compared by restriction analysis in a large set of wild (ECOR) strains. Prior published observations of segmental clonality are confirmed: each of several sequence types is shared by a number of strains. The rate of recombinational replacement and the average size of the replacements are estimated in a set of closely related strains in which a clonal frame is dotted with occasional stretches of DNA belonging to other clones. A clonal hierarchy is described. Some new comparative sequencing data are presented.

https://www.genetics.org/content/genetics/126/3/505.full.pdf

Molecular evolution of the Escherichia coli chromosome. III. Clonal frames.

At each of five loci in 829 Escherichia coli clones from 156 samples from diverse natural sources, electrophoretic analysis reveals a prominent mobility class (frequency over 0.70) and 2 to 11 distinct mobility classes at lower frequencies. The frequency distribution of the classes argues against the importance of neutral mutations in allozymic variation. Heterosis is not the universal cause of genic polymorphism.

https://science.sciencemag.org/content/sci/182/4116/1024.full.pdf

Electrophoretic Variation in Escherichia coli from Natural Sources

1. Ecdysone is in a highly dynamic state after its injection or its secretion by the ring-gland of Sarcophaga peregrina. Hormonal activity is rapidly destroyed by an inactivating mechanism which is present in the tissues but not in the blood.2. Inactivation is blocked by low temperatures or anaerobic conditions—a finding that implicates chemical and, more particularly, oxidative reactions. The mechanism in question could be demonstrated in larval fragments but not in crude or fractionated homogenates.3. When injected into mature larvae, 1 µg of α-ecdysone loses 50% of its activity in 1 hour and 98% in 8 hours. Lower doses show even briefer "half-lives."4. The rapid inactivation of ecdysone can account for its low titer in both the blood and tissues. Thus at the "critical period" for puparium formation, the entire larva contains only 2.5 nanograms, corresponding to only 7% of a Sarcophaga unit.5. The evidence points to the accumulation, not of the hormone itself, but the covert biochemical and biophysical ...

Dynamics of ecdysone secretion and action in the fleshfly sarcophaga peregrina

We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene. Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins. The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA. The sequences of several wild strains of E. coli have been determined for a large segment of the region described. Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination. In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions. However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways. The effects of genetic recombination between strains, recently found to be significant on a large scale in E. coli, are also apparent in the region between trp and tonB.

https://www.genetics.org/content/genetics/120/2/345.full.pdf

Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.

Roger Milkman

Papers

Population genetics of marine pelecypods. iv. selection, migration and genetic differentiation in the blue mussel mytilus edulis.

Molecular evolution of the Escherichia coli chromosome. III. Clonal frames.

Electrophoretic Variation in Escherichia coli from Natural Sources

Dynamics of ecdysone secretion and action in the fleshfly sarcophaga peregrina

Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.