N. K. Hayward, D. J. Nancarrow, P. G. Parsons, C. Kidson, and K. A. O. Ellem Queensland Institute of Medical Research, Bramston Terrace, Herston, 4006, Australia Rare alleles of the c-Ha-ras-1 locus detected by cleavage of DNA with MspI/HpaII have been reported to occur signifi- cantly more frequently in patients with various types of cancer than in healthy controls (Krontiris et al. 1985; Lidereau et al. 1986; Hayward et al. 1988). However, others have failed to find an association (Heighway et al. 1986; Thein et al. 1986; Ceccherinni-Nelli et al. 1987; Ishikawa et al. 1987; Gerhard et at. 1987; Radice et al. 1987; Wyllie et al. 1988). TaqI cleavage results in two major types of polymorphism at the human c-Ha-ras-1 locus (Thein et al. 1986; Pierotti et al. 1986; Ceccerinni-Nelli et al. 1987). One of these overlaps with the same alleles as MspI/HpaII (depending on variations in VTR length), whereas the other occurs because of the pres- ence of TaqI sites within the VTR itself, resulting in patterns designated Tp alleles (Pierotti et al. 1986). The Tp alleles appear closely linked to VTR length, occurring always within the largest (a4) common allele and in rare alleles of similar size (Thein et al. 1986; Pierotti et al. 1986). However, in one report, the Tp alleles appeared to occur in a proportion of samples with the second largest (a3) common allele, but not in the single case studied with an a4 allele (Ceccherinni-Nelli et al. 1987). Recently, melanoma patient lymphocyte DNAs have been reported to have a significantly higher frequency of Tp alleles than lymphocyte DNAs from unaffected controls (Radice et al. 1987). To investigate this association further and to clarify in which alleles the Tp fragments occur, we have undertaken a study of melanoma (MM) cell line and melanoma tumour DNA samples, together with a series of DNAs from control lymphoblastoid cell lines (LCLs) for comparison. All DNA samples were derived from Caucasian (pred- ominantly Anglo-Saxon) individuals. These comprised 66 un- affected control LCLs, 34 MM cell lines and 22 fresh MM biopsies. After overnight digestion with 20 units of TaqI, DNA fragments were electrophoresed in 1.4% agarose gels. DNA was Southern blotted onto nylon membranes and hybridised with the 6.6-kb BamHI c-Ha-ras-1 fragment, contained within the plasmid pEJ, labelled with S2p-dCTP. Hybridisation was carried out for 22h at 65°C in 6 x SSC, 5raM EDTA, 0.1% SDS, 5 × Denhardt's solution and 500gg/ml denatured sheared salmon sperm DNA. Washes were performed at 65°C Offprint requests to: N. K. Hayward for lh in 2 x SSC, 0.1% SDS followed by lh in 0.3 x SSC, 0.1% SDS at the same temperature. By studying MspI/HpaII data previously obtained from our samples (Hayward et al. 1988), we found that Tp alleles occurred without exception in all control and melanoma DNAs with MspI/HpaII VTR-containing fragments larger than or equal to 2.32kb. In no instance did a sample possessing an a3 common allele, or rare allele of similar size, give rise to Tp Fig. 1. DNAs from 7 different MM cell lines digested with TaqI and hybridised to the plasmid pEJ containing the c-Ha-ras-i genomic se- quence. All major allelic forms (Pierotti et al. 1986) are shown, e.g. lanes A, G contain bl and Tpl alleles; lanes B, F have Tml and Tpl alleles; lane C is homozygous for Tpl; lane D has bl and b2; and lane E a b3 and a Tpl allele. There is little or no microvariation of the smallest (300 bp) Tpl fragment although considerable microvariation is seen with the two Tp fragments around 690 and 830 bp (lanes A-C). The major 2200-bp Tpl allele fragment also shows a degree of micro- variation and co-migrates with a faint invariant fragment from the 5' end to the gene observed in DNAs lacking Tp alleles (lane D). Sizes of the major allelic fragments are indicated in base pairs (bp). The 300-bp fragments have run off the end of the gel in lanes E, F, G. Note: bl, b2 and b3 alleles in TaqI digested DNA are equivalent to al, a2 and a3 alleles respectively in MspIIHpaII digested DNA (Kron- tiris et al. 1985; Pierotti et al. 1986)