Biomedical subjects

Mihail Sarov

Publications and source records attributed to Mihail Sarov.

5 recordsLinked to original sources

PubMed2006

A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Animals↗

PubMed2006

An improved recombineering approach by adding RecA to lambda Red recombination.

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.

Animals↗

PubMed2005

The best control for the specificity of RNAi.

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

Animals↗

PubMed2004

In vitro acetylation of HMGB-1 and -2 proteins by CBP: the role of the acidic tail.

Histone acetyltransferases CBP, PCAF, and Tip60 have been tested for their ability to in vitro acetylate HMGB-1 and -2 proteins and their truncated forms lacking the C-terminal tail. It was found that these proteins were substrates for CBP only. Analyses of modified proteins by electrophoresis, amino acid sequencing, and mass spectrometry showed that full-length HMGB-1 and -2 were monoacetylated at Lys2. Removal of the C terminus resulted in (i) an increased incorporation of radiolabeled acetate within the proteins to a level close to that observed with histones H3/H4 and (ii) creation of a novel target site at Lys81. Acetylated and nonmodified HMGB-1 and -2 protein lacking the acidic tail were compared relative to their binding affinity to distorted DNA and the ability to bend linear DNA. Both proteins showed similar affinities to cisplatin-damaged DNA; the acetylated protein, however, was 3-fold more effective in inducing ligase-mediated circularization of a 111-bp DNA fragment. The alterations in the acetylation pattern of HMGB-1 and -2 upon removal of the C-terminal tail are regarded as a means by which the acidic domain modulates some properties of these proteins.

Acetylation↗

PubMed2003

The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation.

Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor. Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation. First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2. Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II. Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3. Fourth, in the absence of ssn3, mRNA production from gal1 required SET2. Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription. Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions. Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation.

Binding Sites↗