The gene content of the MHC class I telomerically adjacent region, in linkage disequilibrium with hereditary hemochromatosis, has not been well characterized yet. In the present work, we established three bacterial clone contigs, including mainly P1-derived artificial chromosomes. These contigs cover 89% of the 1.2-Mb 6p-subtelomeric region encompassing locus D6S105. Terminal exon trapping was applied to selected clones from these contigs. Forty-six independent terminal exons were identified and mapped within the region, 2 of which matched perfectly to expressed sequence tags. These 3' exons are all expressed in human fetal brain but differentially expressed in four tissues and two cell lines. The high number of exons identified indicates that the high gene density observed in the MHC class I region extends to this telomerically adjacent region.

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase IA12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.

We report the sequencing and identification on chromosome X of Saccharomyces cerevisiae of an open reading frame whose product, designated yClC-1, displays significant structural similarity to a voltage-gated Cl- channel family. This putative protein contains 13 hydrophobic domains very similar to transmembrane domains exhibited by known members of this family. Some amino acids in the domains and at the loops between them are well conserved among all members. This is the first voltage-gated Cl- channel described in the yeast S. cerevisiae. The identification of yClC-1 will facilitate the functional analysis of Cl- channels in general, and should also assist in the identification of other ClC genes in higher eukaryotes.

A new anti-B19 IgM ELISA was developed taking advantage of antibody-capture with biotinylated fusion protein as antigen. Specificity was examined using serum IgM antibody positive for rubella, hepatitis B core antigen, cytomegalovirus and Epstein-Barr virus as well as with sera positive for rheumatoid factors or antinuclear antibodies. The specificity was found to be 96%. Of one hundred serum samples compared using the new ELISA or the standard MACRIA tests for the presence of B19 IgM, 88 gave the same results. Fifty-three were negative and 35 were positive. Six sera were ELISA-negative MACRIA-positive, and six MACRIA-negative ELISA-positive. Thus, the ELISA gave 90% agreement with MACRIA. In a clinical study with 725 sera from suspected B19 infections, 161 (22%) were found positive by ELISA. The positive sera were from patients suffering from arthritis (35%), rash (35%), acute or chronic erythroblastopenia (21%), pancytopenia (5%), vascular purpura (2%) and lymphadenopathy (2%). A series of serum specimens obtained from two-B19 infected individuals were also studied. The IgM antibody became undetectable after four months.

A 1.4 kb fragment (nucleotides 2430 to 3901) encoding portions of the human parvovirus B19 structural proteins was inserted into the pRIT2 plasmid expression vector containing the gene encoding staphylococcal Protein A under the control of the phage lambda promoter PR. The fusion protein was used to raise antibodies in rabbits. The sera were shown by immune electron microscopy to agglutinate B19 particles and were also shown to recognize the VP2 B19 capsid protein, by Western blot analysis. The B19 antigenicity of the fusion protein was confirmed by immunoblot and enzyme immunoassay with IgG and IgM anti-B19-positive reference human sera.

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton & Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.