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R Milkman

Publications and source records attributed to R Milkman.

At least 19 recordsLinked to original sources
PubMed1999

Molecular evolution of the Escherichia coli chromosome. V. Recombination patterns among strains of diverse origin.

Incorporation patterns of donor DNA into recipient chromosomes following transduction or conjugation have been studied in the progeny of a variety of Escherichia coli crosses in which donor and recipient nucleotide sequences differ by 1-3%. Series of contiguous or variously spaced PCR fragments have been amplified from each recombinant chromosome and digested with a commercial restriction endonuclease previously shown to distinguish the respective parents in a given fragment. We conclude that entering donor DNA fragments are frequently abridged (cut and shortened) before incorporation, the cutting being due to restriction systems, and the shortening presumably due to exonuclease activity. Analysis of several backcrosses confirms, and extends to conjugation, the importance of restriction in E. coli recombination in nature. The transmission patterns in conjugation are similar to those of transduction, but (as expected) on a much larger scale. Asymmetric results of reciprocal crosses imply that mismatch frequency is not a major factor. Marked differences among the results of simple crosses according to parental strain combinations are consistent with observations that E. coli strains in nature vary dramatically in their restriction-modification systems.

Chromosomes, Bacterial↗
PubMed1995

Transduction, restriction and recombination patterns in Escherichia coli.

Chromosomal DNA from several Escherichia coli reference (ECOR) strains was transduced by bacteriophage P1 into E. coli strain K12 W3110 trpA33. Recombination patterns of the transductants were determined by restriction fragment length polymorphism over a 40-kb region centering on a single marker (trpA+) in the tryptophan operon. These experiments demonstrate that transduction between different strains of E. coli can result in recombinational replacements that are small in comparison to the entrant molecule (replacements average 8-14 kb, whereas P1 packages approximately 100 kb) often in a series of discrete segments. The transduction patterns generated resemble the natural mosaic sequence patterns of the ECOR strains described in previous work. Extensive polymorphisms in the restriction-modification systems of the ECOR strains are a possible explanation for the sequence patterns in nature. To test this possibility two transductants were back-transduced into strain K12 W3110 trpA33. The resulting patterns were strikingly different from the original transductions. The size of the replacements was greater, and no multiple replacements were observed, suggesting a role for restriction-modification systems in the transduction patterns and perhaps for the mosaic sequence patterns in nature.

Bacteriophage P1↗
PubMed1995

Identity of the Escherichia coli cls and nov genes.

cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.

Escherichia coli↗
PubMed1994

An Escherichia coli homologue of eukaryotic potassium channel proteins.

A DNA sequence in Escherichia coli K-12 contains an evident gene, kch, which predicts a protein 417 residues long with extensive similarity to a group of eukaryotic potassium channel proteins in amino acid sequence, in the presence of six apparent transmembrane (S) regions, and in the potassium-specific P (or H5) "pore" region found between S5 and S6. Most of the kch gene, including all of these regions and the 5' flanking region, have been sequenced in 38 wild reference (ECOR) strains as well; variation is conservative, indicating the protein's importance to the species, possibly as a defense against osmotic shock. Since the major family of eukaryotic potassium channel proteins is thought to have evolved from a common ancestor, the evolutionary position of this evident bacterial homologue is of interest, particularly since its function may have changed less than those of eukaryotic channels in the last billion years. While cases of probable importation of eukaryotic genes into bacteria are known, there is no evidence that kch has been imported. The relevant properties of the Kch protein and further ways to investigate its evolutionary position are discussed.

Amino Acid Sequence↗
PubMed1993

Molecular evolution of the Escherichia coli chromosome. IV. Sequence comparisons.

DNA sequences have been compared in a 4,400-bp region for Escherichia coli K12 and 36 ECOR strains. Discontinuities in degree of similarity, previously inferred, are confirmed in detail. Three clonal frames are described on the basis of the present local high-resolution data, as well as previous analyses of restriction fragment length polymorphism (RFLP) and of multilocus enzyme electrophoresis (MLEE) covering small regions more widely dispersed on the chromosome. These three approaches show important consistency. The data illustrate the fact that, in the limited context of intraspecific genomic sequence variation, clonality and homology are synonymous. Two estimable quantitative properties are defined: recency of common ancestry (the reciprocal of the log10 of the number of generations since the most recent common ancestor), and the number of nucleotide pairs over which a given recency of common ancestry applies. In principle, these parameters are measures of the degree and physical extent of homology. The small size of apparent recombinational replacements, together with the observation that they occasionally occur in discontinuous series, raises the question of whether they result from the superimposition of replacements of much larger size (as expected from an elementary interpretation of conjugation and transduction in experimental E. coli systems) or via an alternative mechanism. Length polymorphisms of several sorts are described.

Base Sequence↗
PubMed1990

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

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.

Base Sequence↗
PubMed1988

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

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.

Base Sequence↗
PubMed1988

Molecular evolution of the Escherichia coli chromosome. II. Clonal segments.

Remarkable sequence similarities in the trp region among Escherichia coli strains of diverse natural origins imply the existence of worldwide clones of very recent origin. This in turn implies a low rate of fixation of new universally favorable alleles, which carry adjacent stretches of chromosome to high frequency. These clonal segments begin as entire chromosomes; recombination shortens them progressively by substituting less closely related homologous DNA. The rate of this recombination, comprising the introduction of a homologous chromosomal fragment to a cell and the replacement of part of the original chromosome, is estimated from observations.

Biological Evolution↗
PubMed1983

Clustered third-base substitutions among wild strains of Escherichia coli.

Nucleotide sequences of translated regions of the trp operon in 12 wild strains of Escherichia coli reveal striking uniformity among eight strains (suggesting recent common ancestry and supporting the importance of periodic selection in natural populations) and clustered substitutions in four strains (implicating events affecting runs of nucleotides).

Base Sequence↗
PubMed1976

Further evidence of thermostability variation within electrophoretic mobility classes of enzymes.

Two Drosophila melanogaster strains, each heterozygous for "fast" and "slow" alleles at the Adh locus, and each having balanced second chromosomes, were found to differ in the apparent thermostability of the slow allozyme. The two strains were crossed, and f, heterozygotes were separated on the basis of the origin of the slow allele. After electrophoresis, the cellulose acetate strips were treated 1 1/2 min at 35 C. The putatively more sensitive allozyme showed a strikingly greater response to heat. These findings further support the conclusion that electrophoretically cryptic allelic differences exist which are expressed in thermostability differences. Further application of this approach has revealed one similar sensitive slow allozyme and three cases of a relatively resistant fast ADH allozyme in wild-caught flies.

Alcohol Oxidoreductases↗
PubMed1973

Electrophoretic variation in Escherichia coli from natural sources.

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.

Alcohol Oxidoreductases↗