Biomedical subjects

Andrew B Reams

Publications and source records attributed to Andrew B Reams.

6 recordsLinked to original sources

PubMed2006

Multiple pathways of selected gene amplification during adaptive mutation.

In a phenomenon referred to as "adaptive mutation," a population of bacterial cells with a mutation in the lac operon (lac-) accumulates Lac+ revertants during prolonged exposure to selective growth conditions (lactose). Evidence was provided that selective conditions do not increase the mutation rate but instead favor the growth of rare cells with a duplication of the leaky lac allele. A further increase in copy number (amplification) improves growth and increases the likelihood of a sequence change by adding more mutational targets to the clone (cells and lac copies per cell). These duplications and amplifications are described here. Before selection, cells with large (134-kb) lac duplications and long junction sequences (>1 kb) were common (0.2%). The same large repeats were found after selection in cells with a low-copy-number lac amplification. Surprisingly, smaller repeats (average, 34 kb) were found in high-copy-number amplifications. The small-repeat duplications form when deletions modify a preexisting large-repeat duplication. The shorter repeat size allowed higher lac amplification and better growth on lactose. Thus, selection favors a succession of gene-amplification types that make sequence changes more probable by adding targets. These findings are relevant to genetic adaptation in any biological systems in which fitness can be increased by adding gene copies (e.g., cancer and bacterial drug resistance).

Adaptation, Biological↗

PubMed2006

Origin of mutations under selection: the adaptive mutation controversy.

Growth under selection causes new genotypes to predominate in a population. It is difficult to determine whether selection stimulates formation of new mutations or merely allows faster growth of mutants that arise independent of selection. In the practice of microbial genetics, selection is used to detect and enumerate pre-existing mutants; stringent conditions prevent growth of the parent and allow only the pre-existing mutants to grow. Used in this way, selection detects rare mutations that cause large, easily observable phenotypic changes. In natural populations, selection is imposed on growing cells and can detect the more common mutations that cause small growth improvements. As slightly improved clones expand, they can acquire additional mutational improvements. Selected sequential clonal expansions have huge power to produce new genotypes and have been suggested to underlie tumor progression. We suggest that the adaptive mutation controversy has persisted because the distinction between these two uses of selection has not been appreciated.

Adaptation, Physiological↗

PubMed2004

Gene amplification involves site-specific short homology-independent illegitimate recombination in Acinetobacter sp. strain ADP1.

A system for studying gene amplification in the bacterium Acinetobacter sp. strain ADP1 was used to isolate 105 spontaneous mutants. The method selects for the elevated expression of neighboring transcriptional units in a parent strain lacking its normal transcriptional activators. Gene amplification can compensate for the activator loss by increasing the copy number of seven weakly expressed genes. Mutant colonies arose from the parent strain at a frequency of 10(-8) within three weeks. All but one of these mutants carried tandem head-to-tail repeats of a chromosomal segment (amplicon). These amplicons varied in size from approximately 12-290 kb and ranged in copy number from 3 to more than 30. Gene amplification involved a two-step process in which duplications formed independently of recA. Illegitimate recombination fused normally distant chromosomal regions to create novel DNA duplication junctions. These junctions were isolated from amplification mutants using an assay that exploits Acinetobacter natural transformability. Sequence analysis of 72 junctions revealed little identity in the recombining regions. Furthermore, multiple independently isolated mutants contained identical junctions. Six different junctions, each found in two to six mutants, revealed that some recombination events are site-specific. Several recurring junctions were studied using PCR. In each case, the identical duplication present in the mutant was estimated to have occurred in as many as one in a million cells in populations of strains never exposed to selective conditions. These duplications appeared to form spontaneously by a novel type of short homology-independent, site-specific process. However, in the absence of recA, mutant colonies were not selected from parent cells containing these duplications. Thus, the second gene amplification step most likely depends on homologous recombination to increase amplicon copy number. These studies support the theory that gene amplification is a driving force in the evolution of functionally related gene clusters.

Acinetobacter↗

PubMed2004

Selection for gene clustering by tandem duplication.

In prokaryotic genomes, related genes are frequently clustered in operons and higher-order arrangements that reflect functional context. Organization emerges despite rearrangements that constantly shuffle gene and operon order. Evidence is presented that the tandem duplication of related genes acts as a driving evolutionary force in the origin and maintenance of clusters. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes confer selective benefits via their ability to be coamplified. During evolution, rearrangements that bring together related genes can be selected if they increase the fitness of the organism in which they reside. Similarly, the benefits of gene amplification can prevent the dispersal of existing clusters. Examples of frequent and spontaneous amplification of large genomic fragments are provided. The possibility is raised that tandem gene duplication works in concert with horizontal gene transfer as interrelated evolutionary forces for gene clustering.

Benzoates↗

PubMed2003

Genome plasticity in Acinetobacter: new degradative capabilities acquired by the spontaneous amplification of large chromosomal segments.

In Acinetobacter sp. ADP1, growth on benzoate requires regulation of the cat genes by two transcriptional activators. Here, mutants were obtained from a strain lacking both activators by selecting for growth on benzoate medium. The mutants, which arose within 3 weeks at a frequency of approximately 10-8, carried amplified chromosomal regions (amplicons) encompassing the cat genes. Multiple occurrences of low-level expression of catA and the catBCIJFD operon provided sufficient transcription for growth. The amplicons of four independently isolated mutants varied in size from approximately 30-100 kbp of the normally 3.8 Mbp chromosome. Mutants had approximately 10-20 copies of an amplicon in adjacent head-to-tail orientations. At the amplicon's chromosomal endpoint, an atypical junction juxtaposed normally distant DNA regions from opposite sides of the cat genes. The sequences of these junctions revealed the precise recombination sites underlying amplification. Additionally, amplicon stability was evaluated in the absence of selective pressure. The natural competence of Acinetobacter for transformation by linear DNA has allowed the development of a powerful new model system for investigating chromosomal rearrangements and for engineering DNA amplifications for wide-ranging applications. The frequent spontaneous amplification of these large chromosomal segments demonstrated the importance of supra-operonic gene clustering in the evolution of catabolic pathways.

Acinetobacter↗

PubMed2003

Transcriptional cross-regulation of the catechol and protocatechuate branches of the beta-ketoadipate pathway contributes to carbon source-dependent expression of the Acinetobacter sp. strain ADP1 pobA gene.

Transcriptional control of carbon source preferences by Acinetobacter sp. strain ADP1 was assessed with a pobA::lacZ fusion during growth on alternative substrates. The pobA-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates, pobA was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate pobA transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of pobA expression. In each case, pobA expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the beta-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent pobA expression indirectly during growth on benzoate. These regulators may affect pobA expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several pca genes, including pcaK. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the beta-ketoadipate pathway.

Acinetobacter↗