Characterization of Mouse Pmel 17 Gene and Silver Locus

Pmel 17 cDNA clones, isolated from wild-type and si/si murine melanocytes, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the si/si Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C-terminus and to extend the Pmel 17 protein by 12 residues. The mutation was confirmed by the sequence of the PCR-amplified genomic region including the mutation site. Silver Pmel 17 was not recognized by antibodies directed toward the C-terminal amino acids of wild-type Pmel 17, indicating a defect in this region. These results indicate that silver Pmel 17 protein has a major defect at the carboxyl terminus.     

Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.     

The Dominant white, Dun and Smoky color variants in chicken are associated with insertion/deletion polymorphisms in the PMEL17 gene

Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species.     

A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions

Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Mα fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Mα fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

Mutation of macrophage colony stimulating factor (Csf1) causes osteopetrosis in the tl rat

Osteopetrosis results from a heterogeneous group of congenital bone diseases that display inadequate osteoclastic bone resorption. We recently mapped tl (toothless), a mutation that causes osteopetrosis in rats, to a genetic region predicted to include the rat Csf1 gene. In this study, we sequenced the coding sequence of the rat Csf1 gene to determine if a mutation in Csf1 could be responsible for the tl phenotype. Sequencing revealed a 10-base insertion in the coding sequence of mutant animals that produces a frameshift and generates a stop codon early in the mutant Csf1 coding sequence. The 41 amino acid polypeptide predicted to be produced from the Csf1 promoter would have only the first nine amino acids of the wild-type rat protein. These data suggest that osteopetrosis develops in tl/tl rats because they cannot produce functional mCsf, a growth factor required for osteoclast differentiation and activation.     

The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos

The EMB 506 gene of Arabidopsis, required for the normal development of the embryo beyond the globular stage, has been cloned. The gene encodes a protein of predicted size 35 kDa that contains five ankyrin (ANK) repeats within the C terminal moiety. ANK repeats are conserved domains of 33 amino acids involved in specific recognition of protein partners. The EMB 506 protein was detected at different stages of silique development but accumulated preferentially in the mature cauline leaves. The rescue of homozygous emb 506 embryos by complementation with the wild-type sequence cDNA demonstrated that the emb mutation is a consequence of the T-DNA insertion and that integration and expression of the transgene occurred during gametogenesis and/or early embryo development. In addition to the drastic effect of the emb 506 mutation during embryo development, complementation experiments revealed another effect of the gene: emb 506 plants transformed with the wild-type EMB 506 sequence were able to produce viable seeds but showed a reduction of apical dominance and the presence of adventitious buds or bracts along the stem. This result supports the idea that genes essential for embryogenesis may also be required at other stages of the plant life cycle.     

A novel dominant-negative mutation of the hepatocyte nuclear factor-1alpha gene in Japanese early-onset type 2 diabetes.

We investigated the presence and the function of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations in 26 Japanese subjects with type 2 diabetes. The subjects were between 20 and 39 years of age on diagnosis and had diabetic first-degree relatives. Two different frameshift mutations were found in 2 subjects (8 %). One novel mutation, T539fsdelC (deletion of C in codon 539 for Thr), is predicted to generate a protein of normal 539 residues at the N-terminus followed by an abnormal 119 amino acid protein. The mutation, P291fsinsC (insertion of C in codon 291 for Pro) should lead to production of a truncated protein of 315 amino acids. Transfection reporter assay using MIN6 and HepG2 cells revealed both mutations to have null function in the transactivation of reporter gene expression. When transfected with wild-type gene, these mutations behaved as dominant-negative regulators in both cells. An equimolar amount of T539fsdelC reduced wild-type activity by approximately 80% in MIN6 cells, while the same concentration of P291fsinsc reduced it by 30%. The sequences responsible for the transactivation activity of HNF-1alpha are confined largely to amino acids 547-628, so that the T539fsdelC mutation, which affects this entire region, replacing amino acids 540-631 with an abnormal 119 amino acid protein, may acquire a potent dominant-negative function.     

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.     

Proprotein convertases process Pmel17 during secretion

Pmel17 is a melanocyte/melanoma-specific protein that traffics to melanosomes where it forms a fibrillar matrix on which melanin gets deposited. Before being cleaved into smaller fibrillogenic fragments the protein undergoes processing by proprotein convertases, a class of serine proteases that typically recognize the canonical motif RX(R/K)R↓. The current model of Pmel17 maturation states that this processing step occurs in melanosomes, but in light of recent reports this issue has become controversial. We therefore addressed this question by thoroughly assessing the processing kinetics of either wild-type Pmel17 or a secreted soluble Pmel17 derivative. Our results demonstrate clearly that processing of Pmel17 occurs during secretion and that it does not require entry of the protein into the endocytic system. Strikingly, processing proceeds even in the presence of the secretion inhibitor monensin, suggesting that Pmel17 is an exceptionally good substrate. In line with this, we find that newly synthesized surface Pmel17 is already quantitatively cleaved. Moreover, we demonstrate that Pmel17 function is independent of the sequence identity of its unconventional proprotein convertase-cleavage motif that lacks arginine in P4 position. The data alter the current view of Pmel17 maturation and suggest that the multistep processing of Pmel17 begins with an early cleavage during secretion that primes the protein for later functional processing.     

The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521.     

Molecular and Genetic Analysis of Unc-7, a Caenorhabditis Elegans Gene Required for Coordinated Locomotion

Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.     

Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17

A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp).     

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.     

Isolation and characterization of a novel human gene (NESH) which encodes a putative signaling molecule similar to e3B1 protein

Using a conventional cloning technique, a novel full-length cDNA was isolated and sequenced from a human placental cDNA library. This cDNA consists of 2129 bp and has a predicted open reading frame encoding 366 amino acids. It possesses a Src homology 3 (SH3) motif, proline-rich region, serine-rich region and no catalytic domain, suggesting that it seems to be a signaling protein most similar to e3B1, an eps8 SH3 binding protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 17q21.3 near the marker D17S1795.