Hermansky-Pudlak syndrome (HPS) is an often-fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single-gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.

Mutations in the P gene of humans and the homologous p-locus of mice, respectively, result in the homologous disorders oculocutaneous albinism type 2 (OCA2) and pink-eyed dilution. Although clearly required for melanin biosynthesis, the specific function of the P gene product, a melanosomal transmembrane protein expressed in melanocytes of the skin, hair, and eyes, is not yet known. Here we describe lines of immortal melanocytes and melanoblasts from mice of the null genotype p(cp)/p(25H). These p-null melanocytes were severely hypopigmented, although they and the melanoblasts expressed mRNAs for a number of melanosomal proteins. Proliferation of the p-null melanoblasts was normal. Both diploid and immortal p-null melanocytes grew more slowly than wild-type melanocytes, however, and were unusually susceptible to the antibiotic G418; these abnormalities were corrected by culture in high concentrations of L-tyrosine. Transfection of the p-null melanocytes with full-length normal human P cDNA resulted in complementation of deficient melanin biosynthesis and hypopigmentation. In contrast, transfection with mutant human P cDNAs containing amino acid substitutions (A481T, V443I) found in patients with OCA2 resulted in minimal or partial correction, consistent with the corresponding pigmentation phenotypes in patients with these mutations. These results demonstrate the utility of this model system for distinguishing true OCA2 mutations from nonpathologic polymorphisms and for quantitating the effect of these mutations on P function.

During V(D)J recombination two proteins, RAG1 and RAG2, assemble as a protein-DNA complex with the appropriate DNA targets containing recombination signal sequences (RSSs). The properties of this complex require a fairly elaborate set of protein-protein and protein-DNA contacts. Here we show that a purified derivative of RAG1, without DNA, exists predominantly as a homodimer. A RAG2 derivative alone has monomer, dimer, and larger forms. The coexpressed RAG1 and RAG2 proteins form a mixed tetramer in solution which contains two molecules of each protein. The same tetramer of RAG1 and RAG2 plus one DNA molecule is the form active in cleavage. Additionally, we show that both DNA products following cleavage can still be held together in a stable protein-DNA complex.A site-specific DNA recombination mechanism, termed V(D)J recombination, is used in the developing adaptive immune system to assemble active rearranged genes for the antigen receptors from arrays of inherited inactive segments (reviewed in references 6 and 11). Targets for this recombination reaction are identified by recombination signal sequences (RSSs) which specify the recombination site in the DNA immediately adjacent to the coding sequences. The RSS is composed of a conserved heptamer (CACAGTG) and nonamer (ACAAAAACC) motif, separated by a spacer of either 12 or 23 bp in length (called 12RSS or 23RSS, respectively). A productive rearrangement in cells always occurs between pairs of DNA segments bordered by RSS elements of the two different spacer lengths (the 12/23 rule [26]). Within a chromosomal locus, similar segments generally carry RSSs of the same length. Owing to the 12/23 rule, this organization permits a V segment (for example) to join to a D segment but not a second V segment. Several recent investigations have shown that binding to individual DNA molecules containing single RSS as well as simultaneous binding that obeys the 12/23 rule can be mediated by RAG1 and RAG2 (5,7,8,30,31) and is aided by the sequence-nonspecific DNA bending protein HMG1 (9,21,27). Invariably, these studies have analyzed the behavior of the RAG proteins as part of a DNA-protein complex. Since we and others have shown that the RAG proteins can bind DNA in specific and nonspecific manners (1, 15, 25), it was not initially clear to us whether protein-protein interactions independent of protein-DNA interactions play a role in the complex formation. For example, in the most extreme case, each protein could be present in the complex purely through its contact with DNA. We undertook this study from the perspective that a description of the simpler protein-protein interactions and stoichiometries with and without single RSS-containing DNA molecules would give us a better understanding of the components available for future studies of the more complicated structure containing two RSSs.The DNA recombination occurs through a cutting and pasting mechanism in which specific double-strand breaks are generated adjacent to each RSS (13). This cleavage reaction occurs in two steps...

Hermansky-Pudlak syndrome (HPS) is a rare, often fatal, autosomal recessive disorder in which albinism, bleeding and lysosomal storage are associated with defects of diverse cytoplasmic organelles, including melanosomes, platelet dense granules and lysosomes. Similar multi-organellar defects occur in the Chediak-Higashi syndrome (CHS), as well as in a large number of different mouse mutants. The HPS gene is located in 10q23, and two genetically distinct mouse loci, pale ear (ep) and ruby-eye (ru), both with mutant phenotypes similar to human HPS, map close together in the homologous region of murine chromosome 19, suggesting that one of these loci might be homologous to human HPS. We recently identified the human HPS gene, which encodes a novel ubiquitously-expressed transmembrane protein of unknown function. Here, we describe characterization of the mouse Hps cDNA and genomic locus, and identification of pathologic Hps gene mutations in ep but not in ru mice, establishing mouse pale ear as an animal model for human HPS. The phenotype of homozygous ep mutant mice encompasses those of both HPS and CHS, suggesting that these disorders may be closely related. In addition, the mouse and human HPS genes both contain a rare 'AT-AC' intron, and comparison of the sequences of this intron in the mouse and human genes identified conserved sequences that suggest a possible role for pre-mRNA secondary structure in excision of this rare class of introns.

The Prader-Willi syndrome (PWS) usually results from a paternal deletion of 15q11-q13 or maternal disomy for chromosome 15. Reduced pigmentation of skin, hair, and eyes is common in PWS and was suggested previously to be associated with the 15q11-q13 deletion. The P gene, located in this same region, is associated with OCA2, an autosomal recessive disorder that is the most frequent form of tyrosinase-positive oculocutaneous albinism. We studied 28 individuals with PWS and found that hemizygosity for the P gene was significantly correlated with the occurrence of hypopigmentation among PWS patients. However, we found little or no relationship between the occurrence of hypopigmentation and the polymorphism haplotype of the intact P allele. Thus, our results indicate that hypopigmentation is likely the result of deletion of the P gene in the context of PWS but do not support the linked hypothesis that hypopigmentation results from hemizygosity for variant P alleles with reduced function.