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SummaryWe have investigated whether sequence 67 to 74 shared by B chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRBI*0401, DRBI*0404, and the closely related, RA nonassociated DRBI*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRBI*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR ~ chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

A 5600-base-pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A poll temperature-sensitive mutation, encoding a DNA polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity. The DNA polymerase-primase complex from Saccharomyces cerevisiae has been purified to near homogeneity (2), and specific antibodies have been used to isolate the genes encoding DNA polymerase I (3-5) and the 48-kDa subunit of DNA primase (p48) (6). Both genes are unique in the haploid yeast genome and perform essential functions (4-6). The availability of the cloned DNA polymerase I gene has allowed the production, by in vitro mutagenesis, of temperature-sensitive mutants that will be extremely useful to establish the role ofthis enzyme in DNA metabolism (7,8).Moreover, direct analysis of POLl mRNA showed that the amount of DNA polymerase I mRNA fluctuates during the cell cycle and meiosis and that DNA polymerase I mRNA is induced after DNA damage (9). The finding that the expression of the POLI gene is under cell-cycle control leads to questions about the nature of the cis-and trans-acting genetic elements that regulate the expression of this essential gene.To meaningfully address such questions, the nucleotide sequence of the DNA polymerase I gene and of its putative upstream regulatory region is required. Moreover, the nucleotide sequence will permit calculation of the molecular weight of the POLI encoded protein and identification of possible functional domains as homologies with other DNA polymerase sequences are found and as poll mutants are characterized. We have determined the nucleotide sequence of the POLl genet and have identified regions of homology between the yeast DNA polymerase polypeptide and DNA polymerases from animal viruses and bacteriophages. We also report the sequence of one point mutation, poll-l (8), that confers a temperature-sensitive phenotype by altering the stability of the DNA polymerase-primase complex and that possibly defines a functional domain involved in protein-protein interactions. MATERIALS AND METHODSPlasmids and Strains. Plasmids, pAP70, pAP33, and pAP1, were constructed by cloning, respectively, the 3.4-kilobase (kb) Sal I segment, in both orientations, and the 2.2-kb Sal I-BamHI segment (Fig. 1) into pGEM4 (Promega Biotec, Madison, WI). The two restriction fragments, derived from plasmid pGL7-4 (4), span the entire POLl coding sequence plus 700 base pairs (bp) of the 5'-noncoding region that are sufficient for cell viability (8). Plasmid DNA was digested with Kpn I and BamHI (p...

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Murine macrophage clones were generated from thymus, spleen, brain, and bone marrow by in vitro immortalization with recombinant retroviruses carrying an avian v-myc oncogene. The cloned cell lines express F4/80 molecules, exert phagocytosis, have nonspecific esterase activ-