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D L Hartl

Publications and source records attributed to D L Hartl.

At least 145 records · Page 8Linked to original sources
PubMed1989

An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.

Alkaline Phosphatase↗
PubMed1989

IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations.

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.

Base Sequence↗
PubMed1989

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli.

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.

Blotting, Southern↗
PubMed1989

The physiology of weak selection.

Reaction rates in metabolic pathways typically exhibit a kind of diminishing returns in which small variations in the activities of the individual enzymes have very little effect on overall flux. These effects are measured by the control coefficients of the enzymes, and most systems are governed by the summation theorem stating that all control coefficients must sum to unity. One implication is that complex systems will not usually contain single rate limiting steps, but rather be controlled to a greater or lesser extent by many enzymes, each exerting relatively small control. Wright understood this principle in 1934 and used it for his physiological theory of dominance. With respect to small variations in enzyme activity, the principle implies that many small variations should have only mild effects on fitness. Analysis of nucleotide polymorphisms in the genes for glucose-6-phosphate dehydrogenase and alkaline phosphatase in Escherichia coli implies that most amino acid replacements are harmful, and that the average selection coefficient against amino acid replacements that are polymorphic in natural populations is 1 x 10(-7) to 5 x 10(-7). In experiments to determine the a priori distribution of selection coefficients among random amino acid replacements, 25 replacements in beta-galactosidase were created by genetic means, and 22 of these produced selective effects too small to be detected in chemostat competition experiments (s less than 0.004 per generation).

Amino Acids↗
PubMed1988

Maternally inherited transposon excision in Drosophila simulans.

A mutation in the white gene of Drosophila mauritiana resulting from insertion of the transposable element mariner exhibits genetic instability in germline and somatic cells. The instability is greatly enhanced in the presence of the trans-acting autosomal factor Mos, giving eye-color mosaics with pigmented sectors of tissue on an otherwise peach-colored background. The Mos factor, when introduced into the genome of the sibling species Drosophila simulans, exhibited a dramatic maternal effect on expression of the mosaic phenotype. When D. simulans mosaic females (heterozygous for Mos) were crossed with non-mosaic males, two distinct classes of mosaic offspring occurred, one resulting from a maternal effect in which the non-Mos offspring were nevertheless mosaic. The maternal effect was mediated by a product acting after fertilization, and was expressed to varying extents in different backcross strains.

Animals↗
PubMed1988

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.

Animals↗
PubMed1988

Comparative evolution: latent potentials for anagenetic advance.

One of the principles that has emerged from experimental evolutionary studies of microorganisms is that polymorphic alleles or new mutations can sometimes possess a latent potential to respond to selection in different environments, although the alleles may be functionally equivalent or disfavored under typical conditions. We suggest that such responses to selection in microorganisms serve as experimental models of evolutionary advances that occur over much longer periods of time in higher organisms. We propose as a general evolutionary principle that anagenic advances often come from capitalizing on preexisting latent selection potentials in the presence of novel ecological opportunity.

Animals↗
PubMed1988

Genetic exchange among natural isolates of bacteria: recombination within the phoA gene of Escherichia coli.

An 1871-nucleotide region including the phoA gene (the structural gene encoding alkaline phosphatase, EC 3.1.3.1) was cloned and sequenced from eight naturally occurring strains of Escherichia coli. Alignment with the sequence from E. coli K-12 made apparent that there were 87 polymorphic nucleotide sites, of which 42 were informative for phylogenetic analysis. Maximum parsimony analysis revealed six equally parsimonious trees with a consistency index of 0.80. Of the 42 informative sites, 22 were inconsistent with each of the maximum parsimony trees. The spatial distribution of the inconsistent sites was highly nonrandom in a manner implying that intragenic recombination has played a major role in determining the evolutionary history of the nine alleles. The implication is that different segments of the phoA gene have different phylogenetic histories.

Alkaline Phosphatase↗
PubMed1988

Why do unrelated insertion sequences occur together in the genome of Escherichia coli?

Natural isolates of Escherichia coli are polymorphic for the presence or absence of insertion sequences. Among the ECOR reference collection of 71 natural isolates studied for the number of copies of the insertion sequences IS1, IS2, IS3, IS4, IS5 and IS30, the number of strains containing no copies of the insertion sequences were 11, 28, 23, 43, 46 and 36, respectively. Significant correlations occur in the ECOR strains in the presence or absence of unrelated insertion sequences in the chromosome and plasmid complements. Strains containing any insertion sequence are more likely to contain additional, unrelated insertion sequences than would be expected by chance. We suggest that the positive correlations result from horizontal transfer mediated by plasmids. A branching-process model for the plasmid-mediated transmission of insertion sequences among hosts yields such a correlation, even in the absence of interactions affecting transposition or fitness. The predictions of the model are quantitatively in agreement with the observed correlations among insertion sequences.

DNA Transposable Elements↗
PubMed1988

Genetic applications of an inverse polymerase chain reaction.

A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.

Base Sequence↗
PubMed1988

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli.

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey-lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.

Alleles↗
PubMed1988

Fitness effects of a deletion mutation increasing transcription of the 6-phosphogluconate dehydrogenase gene in Escherichia coli.

Directed evolution in microbial organisms provides an experimental approach to molecular evolution in which selective forces can be controlled and favorable mutations analyzed at the molecular level. Here we present an analysis of a mutation selected in Escherichia coli in response to growth in a chemostat in which the limiting nutrient was gluconate. The selectively favored mutation, designated gnd+ (862), occurred in the gene gnd coding for 6-phosphogluconate dehydrogenase, used in gluconate metabolism. Although the allele is strongly favored in chemostats in which the limiting nutrient is gluconate, the selective effects of gnd+ (862) are highly dependent on growth conditions. In chemostats in which growth is limited by a mixture of gluconate and either ribose, glucose, or succinate, the gnd+ (862) allele is favored, disfavored, or neutral according to the relative concentrations of the substrates. The gnd+ (862) allele results from a deletion of 385 nucleotide pairs in the region 5' to the promoter of gnd, and one endpoint of the deletion is contiguous with the terminus of an IS5 insertion sequence located near gnd in E. coli K12. The gnd+ (862) allele shows a marked increase in transcription that accounts for most or all of the increased enzyme activity.

Alleles↗
PubMed1987

Heritable somatic excision of a Drosophila transposon.

A mutation in the white gene of Drosophila mauritiana that results from insertion of the transposable element mariner is genetically unstable in both germ cells and somatic cells. Somatic instability is indicated by the occurrence of animals having mosaic eyes with patches of pigmented cells on a peach-colored background. Normally uncommon, the frequency of mosaicism is so greatly enhanced in a particular mutant strain that virtually every animal in the strain is an eye-color mosaic. The molecular basis of the mosaicism is the excision of the mariner element from its location in the DNA of the white gene in somatic cells. The phenomenon results from a single dominant genetic factor located in chromosome 3. Genetic control over the excision of transposable elements may play a role in determining the persistence of transposable elements in the genome.

Alleles↗
PubMed1987

Confidence interval for the number of selectively neutral amino acid polymorphisms.

A statistical approach to the analysis of DNA sequences has been developed, which provides a confidence interval estimate for the proportion of naturally occurring amino acid polymorphisms that are selectively neutral. When applied to the gnd gene coding for 6-phosphogluconate dehydrogenase in a sample of seven natural isolates of Escherichia coli, the method indicates that the proportion of observed amino acid polymorphisms that are selectively neutral is unlikely to be greater than 49% (upper 95% confidence limit). On the other hand, the observations are also consistent with a model in which all of the observed amino acid substitutions are mildly deleterious with an average selection coefficient approximating 1.6 X 10(-7). Various models for the distribution of configurations at silent sites are also investigated.

Amino Acids↗
PubMed1987

Metabolic flux and fitness.

Studies of Escherichia coli under competition for lactose in chemostat cultures have been used to determine the selective effects of variation in the level of the beta-galactoside permease and the beta-galactosidase enzyme. The results determine the adaptive topography of these gene products relative to growth in limiting lactose and enable predictions concerning the selective effects of genetic variants found in natural populations. In the terms of metabolic control theory, the beta-galactosidase enzyme at wild-type-induced levels has a small control coefficient with respect to fitness (C = 0.018), and hence genetic variants resulting in minor changes in enzyme activity have disproportionately small effects on fitness. However, the apparent control coefficient of the beta-galactoside permease at wild-type-induced levels is large (C = 0.551), and hence even minor changes in activity affect fitness. Therefore, we predict that genetic polymorphisms in the lacZ gene are subject to less effective selection in natural populations than are those in the lacY gene. The beta-galactoside permease is also less efficient than might be expected, and possible forces resulting in selection for an intermediate optimum level of permease activity are considered. The selective forces that maintain the lactose operon in a regulated state in natural populations are also discussed.

Bacterial Proteins↗
PubMed1987

Distribution and abundance of insertion sequences among natural isolates of Escherichia coli.

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.

Base Sequence↗
PubMed1986

The evolution of DNA sequences in Escherichia coli.

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.

Base Sequence↗