Dataset Information

Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.

ABSTRACT: Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TF(ZF)s). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TF(ZF) encoding gene. Using this strategy, we successfully selected specific TF(ZF)s that upregulate the expression of the gamma-globin promoter. Selected transcription factors induced the expression of gamma-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This new retroviral approach might be used to select other TF(ZF)s but might also be generalized for the selection of other protein and small-molecule interactions.

SUBMITTER: Tschulena U

PROVIDER: S-EPMC2880176 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.

Tschulena Ulrich U Peterson Kenneth R KR Gonzalez Beatriz B Fedosyuk Halyna H Barbas Carlos F CF

Nature structural & molecular biology 20091018 11

Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TF(ZF)s). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TF(ZF) encoding gene. Using this strategy, ...[more]

PMID: 19838191

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Project description:Genome-wide scans for positively selected genes (PSGs) in mammals have provided insight into the dynamics of genome evolution, the genetic basis of differences between species, and the functions of individual genes. However, previous scans have been limited in power and accuracy owing to small numbers of available genomes. Here we present the most comprehensive examination of mammalian PSGs to date, using the six high-coverage genome assemblies now available for eutherian mammals. The increased phylogenetic depth of this dataset results in substantially improved statistical power, and permits several new lineage- and clade-specific tests to be applied. Of approximately 16,500 human genes with high-confidence orthologs in at least two other species, 400 genes showed significant evidence of positive selection (FDR<0.05), according to a standard likelihood ratio test. An additional 144 genes showed evidence of positive selection on particular lineages or clades. As in previous studies, the identified PSGs were enriched for roles in defense/immunity, chemosensory perception, and reproduction, but enrichments were also evident for more specific functions, such as complement-mediated immunity and taste perception. Several pathways were strongly enriched for PSGs, suggesting possible co-evolution of interacting genes. A novel Bayesian analysis of the possible "selection histories" of each gene indicated that most PSGs have switched multiple times between positive selection and nonselection, suggesting that positive selection is often episodic. A detailed analysis of Affymetrix exon array data indicated that PSGs are expressed at significantly lower levels, and in a more tissue-specific manner, than non-PSGs. Genes that are specifically expressed in the spleen, testes, liver, and breast are significantly enriched for PSGs, but no evidence was found for an enrichment for PSGs among brain-specific genes. This study provides additional evidence for widespread positive selection in mammalian evolution and new genome-wide insights into the functional implications of positive selection.

Project description:Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.

Dataset Information

Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.

Publications

Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production.

Similar Datasets

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