The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9‐kb repeating sequences. We determined the nucleotide sequence of a 15‐kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9‐kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12‐nucleotide spacer signals. Most of the somatic DH sequences found in the published VH‐DH‐JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR‐DH or DH‐DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.

In order to clarify the mechanism of pine wilt caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, nematode migration in tissues and disease symptoms in Pinus thunbergii seedlings were investigated. One-year-old seedlings were inoculated with different pathogenic isolates of PWN under two different temperatures. At an early stage of symptom development, a virulent isolate of PWN multiplied in both bark and xylem and was distributed in cortical resin canals, cortical tissue, and xylem resin canals at 30°C. Cell death and disease symptoms developed in both bark and xylem. The virulent isolate of PWN at 25°C and the avirulent isolate of PWN at 30°C were distributed mainly in cortical resin canals, but rarely in xylem resin canals and cortical tissue. Disease symptoms and cell death occurred in cortical resin canals and rarely occurred in other tissues. These results demonstrated that the virulent isolate of PWN at low temperature and avirulent nematodes could not easily migrate to xylem resin canals and cortical tissue. It was shown that cell death and early symptom development coincided with PWN migration and, therefore, PWN invasion induces cell death and early symptom development.

In mice, 12 germ-line DH genes belonging to three different families (DQ52, DSP2 and DFL16) have been identified. The DH genes other than DQ52 are clustered in the 60 kb-long region located between VH and JH genes. Since there are seven DH gene families (DHQ52, DXP, DA, DK, DN, DM and DLR) in humans, we tried to identify new DH gene families in the 60 kb-long region using human DH gene probes. Mouse and human DH genes showing the highest similarity were mouse DFL16 genes and human DA genes. Southern hybridization of the mouse clones covering the 60-kb region with human DH probes did not detect any other DH genes. Nucleotide sequence analysis of the 4.0-kb fragment containing the DFL16.1 gene confirmed this conclusion. Comparison of the 12 germ-line DH genes and more than 150 somatic DH sequences also indicated that there are not more germ-line DH genes in the mouse genome. Moreover, comparison of nucleotide sequences of DFL16.1 and DSP2.2 genes and their surrounding regions suggests that both DH gene families originate from the same primordial DH gene. Using the flanking sequences of both DH genes, the divergence date between DFL16 and DSP2 genes was estimated at around 37 million years ago.

Three Japanese patients demonstrated plasma prekallikrein (PK) deficiency (PKD) after an examination of the proband family line named 'PKD Seki'. A molecular genetic analysis of these PK genes showed homozygous amino acid substitutions Gly104Arg and Asn124Ser in exon 5, which encodes part of the apple domain 2 (A2) of the heavy chain. This is the first case involving substitutions in the heavy chain of the PK gene which affected blood coagulation. Because the apple domains of PK bind to the C-terminal domain (D6(H)) of high-molecular weight kininogen (HMWK), the two substitutions in A2 may therefore be the main cause of PKD Seki. We subsequently investigated the effects of amino acid substitutions in A2 to elucidate the binding activity of PK to HMWK using mutant A2 proteins produced in Escherichia coli. We clearly demonstrated that the Gly104Arg-substitution with the Asn124Ser-substitution in A2 reduce the binding activity of A2 to HMWK. PKD Seki is the first significant case to show the amino acid substitutions in the A2 affecting the binding capacity of PK with HMWK. Our findings therefore suggest that the binding of PK to HMWK may play a crucial role in the first step of blood coagulation.