Mutagenic inverted repeat assisted genome engineering (MIRAGE)

Here we describe a one-step method to create precise modifications in the genome of Saccharomyces cerevisiae as a tool for synthetic biology, metabolic engineering, systems biology and genetic studies. Through homologous recombination, a mutagenesis cassette containing an inverted repeat of selection marker(s) is integrated into the genome. Due to its inherent instability in genomic DNA, the inverted repeat catalyzes spontaneous self-excision, resulting in precise genome modification. Since this excision occurs at very high frequencies, selection for the integration event can be followed immediately by counterselection, without the need for growth in permissive conditions. This is the first time a truly one-step method has been described for genome modification in any organism

Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential

BRG1 helps RNA polymerase II to overcome a nucleosomal barrier during elongation, in vivo

7 páginas, 5 figuras.RNA polymerase II (RNAPII) transcribes genes in a chromatin context. We have designed a system to investigate the role of chromatin remodelling during elongation in vivo, which involves inserting a strong nucleosome-positioning sequence between a promoter and a reporter gene. Our data indicate that a nucleosome positioned in the body of a transcription unit impairs RNAPII progression, provokes RNAPII accumulation upstream to the positioned nucleosome and reduces transcription. By using this system, we show that BRG1, the enzymatic motor of the SWI–SNF chromatin-remodelling complex, is recruited to the positioned nucleosome in a transcription elongation-dependent manner and facilitates traversal of the nucleosome by RNAPII.This study was supported by Ministerio de Educación y Ciencia (BFU2008-00238, CSD2006-00049) and by Junta de Andalucía (P06-CVI-01400).Peer reviewe

A two-step method for the introduction of single or multiple defined point mutations into the genome of <i>Saccharomyces cerevisiae</i>

The introduction of defined mutations into open reading frames (ORF) or non-translated regions of the genome is important to study of the structure-function relationship of amino acid residues in proteins or that of sequence motifs at the genome level. We describe a simple two-step method for the introduction of defined single or multiple point mutations into the genome of Saccharomyces cerevisiae. This method circumvents the need for plasmid-based mutagenesis and thus ensures homogenous expression of the gene of interest within the cell population. It is based on the introduction of a selectable marker downstream of the gene of interest. This marker is then amplified with a gene-specific primer that harbours the desired point mutation, creating a selectable marker-tagged mutant version of the gene of interest. The mutant fragment is then integrated into the genome of a wild-type strain through homologous recombination. Successive rounds of amplification of the mutant loci with primers that introduce additional point mutations upstream of existing mutations will generate multiple defined mutations within a single gene. As a proof of principle, we have employed this method to generate a temperature-sensitive mutant version of the plasma membrane ATPase, pma1-7, which bears two point mutations (Pro434Ala and Gly789Ser). Phenotypic analysis of a pma1-7 haploid strain indicates that this allele has the same characteristics as the original pma1-7 allele. It confers a temperature-sensitive growth phenotype and the newly synthesized Pma1-7 protein is unstable and rapidly degraded