Robert S. Sikorski

Bio: Robert S. Sikorski is an academic researcher from Johns Hopkins University. The author has contributed to research in topics: Gene & Mutant. The author has an hindex of 4, co-authored 4 publications receiving 3414 citations.

Towards a proteome-scale map of the human protein–protein interaction network

Abstract: Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast.

Abstract: Publisher Summary This chapter describes the In Vitro mutagenesis and plasmid shuffling in yeast genes. method for generating mutant alleles uses replicating yeast episomes as a means of exchanging the wild-type gene for mutant copies. The basic scheme for the exchange, known as plasmid shuffling In the first step, one copy of the gene of interest is inactivated in a diploid, and a wild-type copy is propagated in the cell on an episome. This allows the generation of a haploid strain with a chromosomal null allele. Mutagenized copies of the gene are then introduced into this cell on a second episome and exchanged (or shuffled) with the wild-type version. Removal of the wild-type gene, YFG in our example, is the key step in any plasmid shuflting scheme. This can be accomplished by taking advantage of two factors. First, even relatively stable YCp episomes are lost from a cell by missegregation or misrepfication at a rate of 10 –2 per generation. Second, compounds are available that prevent the growth of cells carrying specific yeast genes, and in the presence of such compounds these genes act as counterselectable markers, allowing one to directly select for cells which have lost this marker. By including one of these counterselectable markers on the same plasmid that contains the wild-type YFG gene, an investigator can select for cells that have lost the entire plasmid.

Tissue-based map of the human proteome

Abstract: Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.

Network Medicine: A Network-Based Approach to Human Disease

Abstract: Given the functional interdependencies between the molecular components in a human cell, a disease is rarely a consequence of an abnormality in a single gene, but reflects the perturbations of the complex intracellular and intercellular network that links tissue and organ systems. The emerging tools of network medicine offer a platform to explore systematically not only the molecular complexity of a particular disease, leading to the identification of disease modules and pathways, but also the molecular relationships among apparently distinct (patho)phenotypes. Advances in this direction are essential for identifying new disease genes, for uncovering the biological significance of disease-associated mutations identified by genome-wide association studies and full-genome sequencing, and for identifying drug targets and biomarkers for complex diseases.

Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

Abstract: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.