High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow.

The very late Ag-4 (VLA-4) integrin supports both rolling and firm adhesion of leukocytes on VCAM-1 under shear flow. The molecular basis for the unique ability of a single adhesion molecule to mediate these versatile adhesive processes was investigated. VLA-4 occurs in multiple activation states, with different affinities to ligand. In this study we tested how these states regulate VLA-4 adhesiveness under shear flow in Jurkat T cells and PBL. VLA-4 on nonstimulated Jurkat cells supported rolling and spontaneous arrest on VCAM-1, whereas a Jurkat activation mutant with reduced VLA-4 affinity failed to spontaneously arrest after tethering to or during rolling on VCAM-1. The contribution of VLA-4 affinity for ligand to rolling and spontaneous arrests on immobilized VCAM-1 was dissected using soluble VLA-4 ligands, which selectively block high affinity states. VLA-4 saturation with ligand completely blocked spontaneous adhesion strengthening post-tethering to VCAM-1, but did not impair rolling on the endothelial ligand. High affinity VLA-4 was found to comprise a small subset of VLA-4 on resting Jurkat cells and PBL. This subset is essential for firm adhesion but not for tethering or rolling adhesions on VCAM-1. Interestingly, low and high affinity VLA-4 states were found to mediate similar initial tethering to ligand. High affinity VLA-4, constitutively expressed on circulating T cells, may control their early adhesion strengthening on VCAM-1-expressing endothelium before exposure to vascular chemokines and activation of additional integrins.

GlyCAM-1 supports leukocyte rolling in flow: evidence for a greater dynamic stability of L-selectin rolling of lymphocytes than of neutrophils.

L-selectin plays a major role in leukocyte traffic through lymph node high endothelial venules (HEV). We have investigated the role of GlyCAM-1, a major L-selectin ligand produced by HEV, in mediating leukocyte rolling under in vitro flow conditions. Purified GlyCAM-1 was found to support tethering and rolling in physiological shear flow of both human and murine L-selectin expressing leukocytes at an efficiency comparable to the HEV-derived L-selectin ligands termed peripheral node addressin (PNAd). Major dynamic differences between L-selectin rolling of peripheral blood T lymphocytes and neutrophils expressing similar L-selectin level were observed on GlyCAM-1. Lymphocytes established slower and more shear resistant rolling than neutrophils and could roll on GlyCAM-1 at shear stresses lower than the threshold values required for L-selectin-mediated neutrophil rolling. Notably, high stability of L-selectin rolling of lymphocytes requires intact cellular energy, although initial lymphocyte tethering to L-selectin ligands is energy-independent. By contrast, L-selectin mediated rolling of neutrophils is insensitive to energy depletion. The distinct dynamic behavior and energy-dependence of L-selectin rolling in different leukocytes suggest that L-selectin adhesiveness in shear flow is regulated in a cell-type specific manner. The greater stability of L-selectin rolling of lymphocytes on surface-adsorbed GlyCAM-1 may contribute to their selective recruitment at peripheral lymph nodes.

DNA unwinding in the CYC1 and DED1 yeast promoters.

The capacity of promoter DNA of two yeast genes to be unwound was studied. Both promoters, those of the CYC1 and DED1 genes, contain long oligopurine.oligopyrimidine (R.Y) tracts. The two promoters were cloned into negatively supercoiled plasmids, and their sensitivity to single-strand specific nuclease P1 was examined. Extensive P1 cleavage was located within the R.Y tracts, and cleavage sites were mapped. The extent of cleavage was only slightly dependent on P1 concentration, indicating a slow conversion of an intermediate form of DNA into the P1 reactive state. The cleavage required negative supercoiling and was suppressed by NaCl, MgCl2 and spermine. Two-dimensional topoisomer analysis showed that six superhelical turns were opened in the plasmids examined. The results indicate that at sufficient torsional stress, the R.Y tracts can intermittently undergo a transition into an unwound, ready-to-separate state. The oligopurine.oligopyrimidine tracts may thus serve as DNA unwinding centers in the gene promoters where they reside.

Unwound regions in yeast centromere IV DNA.

The DNA of the centromere of chromosome IV (CEN4) of Saccharomyces cerevisiae is found to be sensitive to single-strand specific nuclease P1 when inserted into a negatively supercoiled plasmid. Fine mapping identifies two P1-sensitive segments: one segment maps to essential centromere element CDEI and bordering CDEII bases, and the other segment is located in element CDEIII. The AT-rich element CDEII, which is expected to be early melting, is for the most part resistant to nuclease P1. Cleavage is inhibited by NaCl, MgCl2 and polyamines. The cleavage rate is only weakly dependent on P1 concentration in the range of 0.5 to 20 munits/microliters. The two P1-sensitive segments are also modified by the DNA-confirmation-specific reagent KMnO4. Negative superhelicity is required for all modifications. Two-dimensional topoisomer analysis indicates the unwinding of 80(+/- 10) bases within the negatively supercoiled CEN4-containing plasmid. The data best fit a model in which the DNA of the CEN4 region undergoes a transition into a paranemic intermediate in which each strand is folded into an RNA-like foldback structure.

Construction of nucleosome cores from defined sequence DNA of viral origin.

The de novo construction of defined nucleosomes from two DNA fragments of simian virus SV40 is described. One fragment spans the region containing the origin of replication of the virus from base -16 to base 161, a region which is nucleosome-free during virus replication. The other fragment, of 142 bp (1352 to 1493), is within the region coding for viral proteins VP2 and VP3, and serves for comparison. Both fragments form nucleosomes with similar efficiency when combined with histone cores as well as when exchanged with existing core particles. The DNase I digestion pattern and exonuclease III analysis both indicate that true nucleosome cores are formed, and that a prolonged tail is not protruding from the constructs. The efficient formation of a nucleosome core particle from the origin region of DNA implies that the absence of nucleosomes from this region during viral infection is not prescribed by the specific base sequence of origin DNA, and is therefore likely to be determined by non-histone nuclear factors associated with the SV40 replication process.

Construction of nucleosome cores from defined DNA sequences of prokaryotic origin.

A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described. Efficient de novo reconstitution without added carrier DNA is demonstrated. DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA. As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident. The possibility that a phosphate end group positions DNA fragments on the histone is considered. The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.