Patr-A and B, the orthologues of HLA-A and B, present hepatitis C virus epitopes to CD8+ cytotoxic T cells from two chronically infected chimpanzees.

Common chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) show a disease progression similar to that observed for human patients. Although most infected animals develop a chronic hepatitis, virus persistence is associated with an ongoing immune response, for which the beneficial or detrimental effects are uncertain. Lines of virus-specific cytotoxic CD8+ T lymphocytes (CTL) have been previously established from liver biopsies of two common chimpanzees chronically infected with HCV-1. The viral epitopes recognized by six lines of CTL have been defined using synthetic peptides and shown to consist of 8 to 9-residue peptides derived from various viral proteins. Five of the epitopes derive from sequences that vary among strains of HCV. The majority of the corresponding variant epitopes from different HCV strains were either recognized less efficiently or not at all by the CTL, suggesting their response may have limited potential for controlling replication of HCV variants. Complementary DNAs encoding class I alleles of the two common chimpanzees, Patr-A, -B, and -C were cloned, sequenced, and transfected individually into a class I-deficient human cell line. Analysis of peptide presentation by the class I transfectants to CTL identified the Patr class I allotypes that present the six epitopes defined here and an additional epitope defined previously. The assignment of epitopes to class I allotypes based upon analysis of the transfected cells correlates precisely with the segregation of antigen-presenting function within a panel of common chimpanzee cell lines and the expression of class I heavy chains as defined by isoelectric focusing. Five of the HCV-1 epitopes are presented by Patr-B allotypes, two epitopes are presented by a Patr-A allotype, and none is presented by Patr-C allotypes.

A small test of a sequence-based typing method: definition of the B*1520 allele.

Santamaria et al. (Human Immunology 1993 37: 39-50) describe a method of sequence-based typing (SBT) for HLA-A, B and C alleles said to give "unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information". From SBT analysis, which involves determination of partial sequences of mixed alleles, these investigators reported that cell lines KT17 (HLA-B35,62) and OLGA (HLA-B62) from the reference panel of the 10th International Histocompatibility Workshop express novel variants of HLA-B15 (B1501-MN6) and HLA-B35 (B3501-MN7) respectively. To study further the novel alleles, we cloned and sequenced full-length HLA-B cDNA clones isolated from the KT17 and OLGA cell lines. We find that KT17 expresses B*3501, as assigned by SBT, and B*1501, the common allele encoding the B62 antigen. We were unable to confirm that KT17 expresses the novel B1501-MN6 variant identified by SBT. For OLGA our analysis confirms the partial sequences obtained by SBT. Thus OLGA expresses B*1501 and a novel HLA-B allele. The complete sequence of the latter shows it is a hybrid having exons 1 and 2 in common with B*1501 and other B15 subtypes and exons 3-7 in common with B*3501 and related molecules including B*5301 and B*5801. The novel allele has been designated B*1520 because of its sequence similarity with the B15 group; furthermore, serological analysis shows that the B*1520 product does not express epitopes in common with either B35, B53 or B58. The B*1520 heavy chain has a similar isoelectric point to A*3101; B*1520 was undetected by previous applications of isoelectric focusing because B*1520 and A31 are both expressed by OLGA. In conclusion, HLA-B typing of two cell lines by cDNA cloning and sequencing gives concordant results with SBT for three of the four alleles. The cause of the discrepancy for the fourth allele is unknown, however, this finding indicates that the novel HLA-A, B and C sequences emerging from SBT studies need independent verification.

The HLA-B73 antigen has a most unusual structure that defines a second lineage of HLA-B alleles.

The nucleotide sequence of cDNA encoding the HLA-B73 antigen was determined; it is unusually divergent, differing from other HLA-B alleles by 44-77 nucleotide substitutions. Features that distinguish the B*7301 heavy chain from other HLA-B heavy chains include multiple substitutions in the alpha 3 domain and a duplication-deletion within the transmembrane region that increases the length of B*7301 compared to other HLA-B heavy chains. The duplication-deletion is shared with subsets of B alleles from the homologous gorilla (Gogo-B) and chimpanzee (Patr-B) loci. Other unusual features of B*7301 are individually shared with certain alleles of the HLA-A, HLA-C, HLA-F, Gogo-B and Patr-B loci. The B*7301 molecules has sequence elements in common with members of the B7 crossreacting group in the alpha 1 domain and is shown to possess the ME1 epitope, which is held in common with the B7, B22, B27, B42 and B67 antigens. B*7301 has a unique cysteine at position 270 of the alpha 3 domain which appears accessible but probably does not form disulphide-bonded B*7301 dimers in cell membranes. B*7301 represents a newly discovered but ancient lineage of HLA-B alleles that appears poorly represented in the modern human population.

HLA-B15: a widespread and diverse family of HLA-B alleles.

HLA-B15 embraces a multiplicity of antigenic specificities which vary in their distribution amongst human populations. To correlate B15 molecular structure with the serological picture we have sequenced alleles encoding the various subspecificities of the B15 antigen: B62, B63, B75, B76 and B77, and a number of "variants" of these antigens including the 8w66 split of B63. HLA-B63 (B*1517) and 8w66 (B*1516) heavy chains have sequence identity to B17 in the alpha 1 helix correlating with the antigenic crossreactivity of these molecules. HLA-B77(B*1513) and B75 (B*1502) heavy chains differ solely in segments determining the Bw4 and Bw6 public epitopes, consistent with the serological description of the B77 and B75 antigens. One allele encoding the B76 antigen (B*1512) appears to be the product of gene conversion between the HLA-A and -B loci and differs from B*1501 in codons 166 and 167. In contrast, a second allele encoding the B76 antigen (B*1514) differs from B*1501 by an unrelated substitution in codon 167 which confers similarily with B45, an antigen crossreactive with B76. A third allele encoding B76, B*1519, differs from B*1512 by a unique point substitution in exon 4. Three alleles encoding variant B15 and B62 antigens (B*1508, B*1511 and B*1515) differ from B*1501 by localized clusters of substitutions that probably result from interallelic conversion. The B15 sequences described in this paper, in combination with those previously determined, define a family of 22 alleles, including those encoding the B46 and B70 antigens. Within this family the patterns of allelic substitution are analogous to those of other HLA-A and -B families, in that pairwise differences almost always involve functional positions of the antigen recognition site and recombination is the major agent of diversification.

HLA-B67: a member of the HLA-B16 family that expresses the ME1 epitope.

HLA-B67 is an uncommon antigen that has been defined by serological crossreactivity with the HLA-B7 and HLA-B16 (B38 and B39) antigens. It is found at highest frequency in certain Oriental populations and has been best defined in the Japanese. Nucleotide sequencing of cDNA encoding B67 reveals the B*6701 allele to be a subtype of B39 which differs from B*39011 by substitution at residues 67-71 of the alpha 1 helix. In the region of difference B*6701 is identical in sequence to B7, B22, B27 and related molecules that express the epitope recognized by the ME1 monoclonal antibody. That the HLA-B67 molecule binds strongly to the ME1 antibody was demonstrated by immunoprecipitation and cell surface binding assays. Identical B*6701 nucleotide sequences were obtained for the B67 alleles isolated from 2 unrelated Japanese and 1 North American caucasoid.

Structural heterogeneity in HLA-B70, a high-frequency antigen of black populations.

Although the B70 antigen exhibits allele frequencies of 8-23% in African and American black populations, it remains poorly defined. Cloning and sequencing of cDNA encoding B70 antigens from six cell lines has identified a group of three closely related alleles: B*1503, B*1509 and B*1510, that form a subgroup of the B15 family. The sequences of these alleles and, in particular, B*1503, are close to that of the HLA-B consensus consistent with the difficulty in their serological definition. The products of the three alleles correspond to three electrophoretically detected variants of the B70 antigen and some correlation with the B71 and B72 subspecificities of the B70 antigen can be made. A fourth allele, B*7901, previously described by Choo et al. (J. Immunol. 147: 174-180, 1991) that was not serologically typed as B70, differs by a single nucleotide substitution from B*1510. The sensitivity of alloantibodies to single differences in peptide binding residues suggest a role for bound peptides in the HLA-B70 alloantigenic specificities. The heavy chains encoded by the four alleles differ at four peptide binding residues of the antigen recognition site, the evolutionary modification of which can be explained in terms of interallelic recombination events.

The molecular cloning, sequence and expression of the hdcB gene from Lactobacillus 30A.

We previously cloned the structural gene hdcA, which encodes the enzyme histidine decarboxylase (HDC; EC 4.1.1.22), from Lactobacillus 30a and found what appeared to be the start of a second gene 59 nucleotide (nt) downstream from the hdcA stop codon [Vanderslice et al., J. Biol. Chem. 32 (1986) 15186-15191]. Here we report the complete nt sequence of this second gene, which we have named hdcB, and show that it encodes a 20-kDa protein, HDCB, which was purified from Escherichia coli. The hdcA and hdcB genes together comprise an operon, the transcription from which is shown to be increased threefold by the presence of histidine in the growth medium. Western blots were used to quantitate the rise in concentrations of both gene products during histidine induction of the hdc operon. This increase was found to be proportional to the observed threefold increase in the concentration of the respective mRNAs. Transcription of the hdc operon in the mutant-3 strain of Lactobacillus 30a [Recsei and Snell, Biochemistry 12 (1973) 365-371] was shown to be constitutively 15-fold greater than in uninduced wild type cells and was unaffected by histidine. The transcription start point was defined as a guanine 73 nt 5' to the start codon of the hdcA gene. Of the transcripts initiated at this promoter, 15% include both hdcA and hdcB sequences, the remainder terminate in the intergenic region and thus encode only hdcA.

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver.

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.

Purification of nuclear and mitochondrial uracil-DNA glycosylase from rat liver. Identification of two distinct subcellular forms.

Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular weight of approximately 35 000. It has a native molecular weight of 33 000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil from both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containing a 3'-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20 000 as determined by molecular sieving, (iii) a distinct NaCl inhibition profile, and (iv) a longer half-life during thermal denaturation.