The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40‐K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3‐K intervals and a limit digest containing 3‐K fragments. The native particle is shown to contain 7‐11 zinc atoms. These data suggest that the protein contains repetitive zinc‐binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the

No abstract

No abstract

SummaryMajor histocompatibility complex class II-associated invariant chain (Ii) provides several important functions that regulate class II expression and function. One of these is the ability to inhibit class II peptide loading early in biosynthesis. This allows for efficient class II folding and egress from the endoplasmic reticulum, and protects the class II peptide binding site from loading with peptides before entry into endosomal compartments. The ability of Ii to interact with class II and interfere with peptide loading has been mapped to Ii exon 3, which encodes amino acids 82-107. This same region of Ii has been described as a nested set of class II-associated Ii peptides (CLIPs) that are transiently associated with class II in normal cells and accumulate in human histocompatibility leukocyte antigen-DM-negative cell lines. Currently it is not clear how CLIP and the CLIP region of Ii blocks peptide binding. CLIP may bind directly to the class II peptide binding site, or may bind elsewhere on class II and modulate class II peptide binding a11osterically. In this report, we show that CLIP can interact with many different murine and human class II molecules, but that the affinity of this interaction is controlled by polymorphic residues in the class II chains. Likewise, structural changes in CLIP also modulate class II binding in an allele-dependent manner. Finally, the specificity and kinetics of CLIP binding to class II molecule is similar to antigenic peptide binding to class II. These data indicate that CLIP binds to class II in an analogous fashion as conventional antigenic peptides, suggesting that the CLIP segment of Ii may actually occupy the class II peptide binding site.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

During biosynthesis, class H major histocompatibility complex molecules are intimately associated with invariant chain (Ii). The I-class H association has been shown to block peptide-class H binding and to affect the ultimate conformation of class H expressed on the cell surface. To assess the biochemical basis for the effects of Ii on class H, we have analyzed the biosynthesis of class H in EL4 cells tranfected with I-Ad with and without Ii. In these studies, we found that Ii had a profound effect on the biosynthesis of I-Ad. In the absence of Ii, class H could form dimers efficiently, but these dimers appeared to be misfolded and this altered conformation resulted in the loss of some monoclonal antibody epitopes and inefficient transport from the endoplasmic reticulum to the Golgi. In addition, class H that was transported through the Golgi accumulated an abnormally increased molecular mass associated with N-linked glycosylation. Class II molecules function in the immune response by presenting foreign antigens to the CD4-positive subpopulation of T cells. These T cells recognize small processed peptide fragments of foreign antigens complexed with the class II molecule on the surface of an antigen presenting cell (6). Of particular interest, over recent years, has been defining both the mechanism(s) and the relevant intracellular compartments in which antigen presenting cells process and degrade antigens and then subsequently allow for class II association with the resultant antigenic peptide. Recent studies indicate that class II preferentially associates with exogenous antigens in an acidified late intracellular compartment, or endosome, where antigens are degraded into peptide fragments (6,7). The association of class II with these exogenous antigens is thought to occur through a unique transport route for class II. This route involves an apparent delay in the intracellular transport of class II to the cell surface in a post-Golgi compartment that intersects the endocytic pathway (8). This intersection has also been detected in an electron micrograph study that colocalized class II and antigen in an endosomal compartment (9). The transport delay of class II in this endosomal compartment indicates that newly synthesized class II molecules may be critical for proper antigen loading in the antigen presenting cell.Given the intimate intracellular association of newly synthesized class II with Ii, it has become ofinterest to determine the role of Ii in class II biosynthesis and transport and, ultimately, its role in class II-restricted antigen processing and presentation. Ii is not required for cell-surface transport of class II; however, in the absence of Ii, class II has an altered conformation at the cell surface (10). The goal of this study is to clarify where this conformational change occurs intracellularly and to determine any potential effects on class II transport caused by this conformational change. Here we show that synthesis of class II in the absence of Ii allows for efficient ad dimer forma...