Search PubMed⌕ Search

Biomedical subjects

I G Serebriiskii

Publications and source records attributed to I G Serebriiskii.

10 recordsLinked to original sources
PubMed2001

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42.

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.

Animals↗
PubMed2001

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene.

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.

Alternative Splicing↗
PubMed2001

Redefinition of the yeast two-hybrid system in dialogue with changing priorities in biological research.

Examination of the pattern of reagent creation and application in the two-hybrid system since 1989 reveals the expansion of a simple core technology to address increasingly sophisticated problems in protein interaction. As the technology has matured, its clear suitability for large-scale proteomic projects has made a major focus of its application the generation of global organismal protein interaction networks. In an inversion of emphasis, the increasing availability of such information now provides a master plan with the potential to specify the most promising directions for biological investigations (i.e., by directing the physiological validation of predicted critical protein-protein interactions). Recent derivatives of the two-hybrid system enable the targeting of such key interactions by facilitating the identification of essential amino acids conferring protein interaction specificity and of small molecules that selectively disrupt defined interaction pairs. Finally, the creation of mammalian expression systems based on two-hybrid principles became a new tool to create and probe novel biological systems. Taken in sum, this trajectory emphasizes the point that the creation of tools and the evolution of the idea of what is an interesting biological problem are in intimate dialogue.

Pharmacogenetics↗
PubMed2000

Streamlined yeast colorimetric reporter activity assays using scanners and plate readers.

Two-hybrid systems have become favored tools for detection and analysis of protein interactions because of their low cost and ease of use compared to biochemical or biophysical interaction technologies. It is possible to augment the utility of two-hybrid systems and derivative systems such as dual-bait two-hybrid systems by adapting strategies that speed the analysis of the relative strength of a series of protein-protein associations. This report describes two simple techniques that employ either a flatbed scanner or a plate reader to quantitate the activity of colorimetric reporters such as LacZ or GusA commonly used in two-hybrid approaches.

Automation↗
PubMed1996

Two new members of the bio B superfamily: cloning, sequencing and expression of bio B genes of Methylobacillus flagellatum and Corynebacterium glutamicum.

Cloning, characterization and expression of the bio B gene of the obligate methylotrophic bacterium, Methylobacillus flagellatum, are reported. A chromosomal fragment containing bio B has been isolated by complementation of a bio B- mutant of M. flagellatum. Nucleotide (nt) sequence analysis of this fragment revealed the presence of an open reading frame of 966 nt identified as bio B, which is the first gene of the M. flagellatum bio cluster. Gene bio B was expressed in Escherichia coli and M. flagellatum, resulting in efficient conversion of dethiobiotin to biotin. The Corynebacterium glutamicum bio B has also been cloned and sequenced. Comparison of the amino acid sequences derived from known bio B genes allowed us to identify four cysteines participating as putative ligands forming the [2Fe-2S] cluster. Genomic organization of the bio biosynthetic genes shows wide diversity in various bacteria. The results of the database screening suggested that bio B proteins belong to a superfamily of proteins, including biotin and lipoate synthases and some proteins with unidentified functions.

Bacterial Proteins↗