A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.

K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca 2+ -loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. KeywordsK-Ras4B; calmodulin; hypervariable region; catalytic domain Members of the Ras family of proto-oncogenes are mutated in up to a third of human malignancies(1,2). These are small p21 GTPases that cycle between the GDP-bound inactive and the GTP-bound active states to transmit intracellular signals. How Ras proteins contribute to cancer development is not fully understood, in spite of much study. They have many signaling partners and regulate a variety of cellular processes including proliferation, transformation, differentiation, metastasis, and apoptosis. There are four isoforms in the Ras family, and of these K-Ras, almost exclusively, is mutated in common epithelial cancers including those of the pancreas, colon and lung. Two forms of K-Ras are generated by alternate mRNA splicing, namely K-Ras4A and K-Ras4B. K-Ras4B is more abundant in most tissues, and has been demonstrated to cause tumor formation in studies with genetically engineered mice(3-5). Noonan syndrome, a developmental disorder, is caused by a mutation specifically in K-Ras4B(6).*To whom correspondence should be addressed: Vadim Gaponenko, Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, vadimg@uic Thus, K-Ras4B has a particularly important role in human cancer as well as human development. It differs from the other highly homologous Ras isoforms in the C-terminal region where the alternate 4B exon provides a polylysine region in addition to posttranslational farnesylation. The other Ras isoforms lack the polylysine tail and are modified with a palmitoyl group in addition to the farnesyl moiety. Some of the unique properties of K-Ras4B have been revealed in studies of comparative physiology. These include induced Raf-...

Alterations in gastrointestinal mucin induced by dietary fiber may affect nutrient bioavailability, cytoprotection of the mucosa or other aspects of gastrointestinal function. To allow quantitative study of gastrointestinal mucin, a polyclonal antibody to the mucin of the rat small intestine was produced by injecting rabbits with a high-molecular-weight subfraction (MW 2 x 10(6)) of purified mucin glycoprotein derived from rat intestinal mucin. An enzyme-linked immunosorbent assay was developed and used for the mucin assay. Three groups of male Wistar rats consumed 5% guar gum, 5% citrus fiber or a fiber-free control diet ad libitum for 4 wk. After an overnight fast, luminal and tissue mucin antibody reactivities were determined in the rat stomach, colon and small intestine. In all groups, total (luminal and tissue) mucin reactivity was greater in the small intestine than in the colon or stomach. The group fed 5% citrus fiber had significantly greater mucin reactivity in luminal samples from stomach and intestine than did the fiber-free control group. Fiber-induced increments in gastrointestinal mucin production or availability may be responsible for several reported consequences of fiber feeding, such as more rapid transit times and delayed or impaired nutrient absorption.

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