Superhelix organization by DNA curvature as measured through site-specific labeling.

For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix.

Sequence-specific labeling of superhelical DNA by triple helix formation and psoralen crosslinking.

Site-specific labeling of covalently closed circular DNA was achieved by using triple helix-forming oligonucleotides 10, 11 and 27 nt in length. The sequences consisted exclusively of pyrimidines (C and T) with a reactive psoralen at the 5'-end and a biotin at the 3'-end. The probes were directed to different target sites on the plasmids pUC18 (2686 bp), pUC18/4A (2799 bp) and pUC1 8/4A-H 1 (2530 bp). After triple helix formation at acid pH the oligonucleotides were photocrosslinked to the target DNAs via the psoralen moiety, endowing the covalent adduct with unconditional stability, e.g. under conditions unfavorable for preservation of the triplex, such as neutral pH. Complex formation was monitored after polyacrylamide gel electrophoresis by streptavidin-alkaline phosphatase (SAP)-induced chemiluminescence. The yield of triple helix increased with the molar ratio of oligonucleotide to target and the length of the probe sequence (27mer > 11mer). The covalent adduct DNA were visualized by scanning force microscopy (SFM) using avidin or streptavidin as protein tags for the biotin group on the oligonucleotide probes. We discuss the versatility of triple helix DNA complexes for studying the conformation of superhelical DNA.

Effect of gliadin and other food peptides on expression of MHC class II molecules by HT-29 cells.

Expression of major histocompatibility (MHC) class II molecules by enterocytes is known to be enhanced in coeliac disease and other disorders characterised by intestinal inflammation--an effect thought to be mediated via intestinal lymphocytes. To investigate if food peptides can exert direct effects on class II expression, the influence of gliadins, casein, and beta lactoglobulin on an intestinal epithelial cell line (HT-29) was examined in the absence of immune cells. Class II expression was determined by flow cytometry and immunofluorescence microscopy using antibodies against the beta chain of all products of the gene subregions DR, DQ, and DP. MHC expression was low in HT-29 cells but could be stimulated by interferon gamma. Tryptin digested gliadin had no effect on class II expression. In the presence of interferon gamma, however, it was able to amplify MHC class II expression to mean (SEM) 150 (4)%. Casein exerted a similar effect (160 (14)%), but undigested gliadin, tryptin digested casein, and beta lactoglobulin had no influence. The observations suggest that within the concert of cytokine mediated interactions between enterocytes and lymphocytes, some dietary peptides could upregulate the presentation of food antigens, leading to a more efficient stimulation of lymphocytes, which in the case of coeliac disease might result in damage to the enterocytes.