Cosmid clones containing the gene for human adenosine deaminase (ADA) were isolated. The gene is 32 kb long and split into 12 exons. The exact sizes and boundaries of the exon blocks including the transcription start sites were determined. The sequence upstream from this cap site lacks the TATA and CAAT boxes characteristic for eukaryotic promoters. Nevertheless, we have shown in a functional assay that a stretch of 135 bp immediately preceding the cap site has promoter activity. This 135‐bp DNA fragment is extremely rich in G/C residues (82%). It contains three inverted repeats that allow the formation of cruciform structures, a 10‐bp and a 16‐bp direct repeat and five G/C‐rich motifs (GGGCGGG) disposed in a strikingly symmetrical fashion. Some of these structural features were also found in the promoter region of other genes and we discuss their possible function. Knowledge of the exact positions of the intron‐exon boundaries allowed us to propose models for abnormal RNA processing that occurs in previously investigated ADA‐deficient cell lines.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA‐SCID patient. In addition, wild‐type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.

BACKGROUND Alloimmunization against the high‐frequency Vel blood group antigen may result in transfusion reactions or hemolytic disease of fetus and newborn. Patients with anti‐Vel alloantibodies require Vel‐negative blood but Vel‐negative individuals are rare (1:4000). Identification of Vel‐negative donors ensures availability of Vel‐negative blood; however, accurate Vel blood group typing is difficult due to variable Vel antigen expression and limited availability of anti‐Vel typing sera. We report the production of a recombinant anti‐Vel that also identifies weak Vel expression. STUDY DESIGN AND METHODS A recombinant anti‐Vel monoclonal antibody was produced by cloning the variable regions from an anti‐Vel–specific B cell isolated from an alloimmunized patient into a vector harboring the constant regions of immunoglobulin (Ig)G1‐kappa or IgM‐kappa. Antibody Vel specificity was tested by reactivity to SMIM1‐transfected HEK293T cells and by testing various red blood cells (RBCs) of donors with normal, weak, or no Vel expression. High‐throughput donor screening applicability was tested using an automated blood group analyzer. RESULTS A Vel‐specific IgM class antibody was produced. The antibody was able to distinguish between Vel‐negative and very weak Vel antigen–expressing RBCs by direct agglutination and in high‐throughput settings using a fully automated blood group analyzer and performed better than currently used human anti‐Vel sera. High‐throughput screening of 13,288 blood donations identified three new Vel‐negative donors. CONCLUSION We generated a directly agglutinating recombinant anti‐Vel IgM, M3F5S‐IgM, functional in manual, automated agglutination assays and flow cytometry settings. This IgM anti‐Vel will improve diagnostics by facilitating the identification of Vel‐negative blood donors.

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.

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