Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.     

Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.