No abstract

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.

PKD1, the gene that is mutated in approximately 85% of autosomal dominant polycystic kidney disease (ADPKD) cases in humans, has recently been identified (Eur. PKD Consortium. Cell 77: 881-894, 1994; also, erratum in Cell 78: 1994). The longest open-reading frame of PKD1 encodes polycystin, a novel approximately 460-kDa protein that contains a series of NH2-terminal adhesive domains (J. Hughes, C. J. Ward, B. Peral, R. Aspinwall, K. Clark, J. San Millan, V. Gamble, and P. C. Harris. Nat. Genet. 10: 151-160, 1995; and Int. PKD Consortium. Cell 81: 289-298, 1995) and several putative transmembrane segments. To extend studies of polycystin to an experimentally accessible animal, we have isolated a cDNA clone encoding the 3' end of Pkd1, the mouse homologue of PKD1, and raised a specific antibody to recombinant murine polycystin. This antibody was used to determine the subcellular localization and tissue distribution of the protein by Western analysis and immunocytochemistry. In the mouse, polycystin is an approximately 400-kDa molecule that is predominantly found in membrane fractions of tissue and cell extracts. It is expressed in many tissues including kidney, liver, pancreas, heart, intestine, lung, and brain. Renal expression, which is confined to tubular epithelia, is highest in late fetal and early neonatal life and drops 20-fold by the third postnatal week, maintaining this level into adulthood. Thus the temporal profile of polycystin expression coincides with kidney tubule differentiation and maturation.

The amino acid sequence of the capsid (C) protein was deduced from the nucleotide sequence of the C gene. This part of the viral 42S RNA genome was transcribed into double-stranded cDNA. The cDNA was cloned in the Escherichia coil X1776-pBR322 host-vector system and then the base sequence was determined with the technique described by Maxam and Gilbert. The amino acid sequence of the C protein shows a clustering of basic amino acids and prolines within the first 110 amino acids.from the virus. This specificity can probably be explained by the formation of bonds between the C protein in the nucleocapsid and the spanning segments of the spike glycoproteins (9).A more detailed understanding of SFV structure and assembly at the molecular level is difficult without the knowledge of the amino acid sequences of the structural proteins. We report here the primary structure of the C protein.Semliki Forest virus (SFV) is a simple membrane virus of the alphavirus group. It has been used extensively as a model system to study the structure and assembly of cellular membranes (1). The virus particle consists of an icosahedral nucleocapsid surrounded by a membrane. The nucleocapsid is a complex of about 240 capsid proteins (C protein, Mr = 30,000) (2, 3) and a RNA molecule (42S), the viral genome (4). The membrane consists of a lipid bilayer with about 240 external glycoprotein spikes (5). Each spike contains three different glycopolypeptides: El (Mr = 49,000), E2 (Mr = 52,000), and E3 (Mr = 10,000) (6, 7). The E2 polypeptide spans the membrane; there are about 30 amino acid residues present on the internal side of the viral membrane (8, 9).The virus enters the host cell by absorptive endocytosis (10). Inside the lysosomes of the cell, the low pH probably triggers a fusion of the viral membrane with the lysosomal membrane (10,11). This allows the nucleocapsid to enter the cell cytoplasm, where the viral genome is uncoated so that it can act as a mRNA for synthesis of polymerase molecules. The viral RNA polymerase synthesizes new 42S RNA molecules and smaller 26S RNA molecules. The latter molecule is homologous to the 3' end of the viral genome (12) and functions as a mRNA for the SFV structural proteins, which are translated from a single initiation site (13). The C protein is made first. As soon as it is completed it is cleaved from the growing polypeptide chain, and the ribosomes continue to read off the membrane proteins in the order E3, E2, and El (8, 14). The membrane proteins are cotranslationally translocated across the membrane of the endoplasmic reticulum and transported to the plasma membrane (15, 16).The assembly of the nucleocapsid in the cell cytoplasm is not understood. Newly synthesized capsid proteins are known to be associated with the large subunit of the ribosome before they complex with the 42S RNA into nucleocapsids (17). The final step in SFV assembly, budding, takes place at the cell surface (18). The nucleocapsid binds to the cytoplasmic aspect of the plasma membrane which folds around the nu...