Differentiation of hairbulb pigment cell melanosomes in compound agouti and albino locus mouse mutants (Ay, a, c2J; C57BL/6J)

Our objective was to determine using electron microscopy how nonagouti (a), lethal yellow (Ay), and albino (c2J) genes affect the program of mouse hairbulb melanosome differentiation; 1,921 hairbulb melanosomes from four genotypes (a/a C/C = B,Ay/a C/C = Y, a/a c2J/c2J = BA, and Ay/a c2J/c2J = YA) were scored for developmental stage, length, and width. Qualitative and quantitative electron microscopy revealed the following. An albino locus-induced diminution of melanosome size suggests that the albino locus is involved in structural features of melanosomes not directly related to the synthesis and deployment of tyrosinase. Ratio data on melanosome length-to-width confirm that the agouti locus determines melanosome shape, either spherical or elliptical; melanization is not required for melanosomes to achieve their agouti-locus-determined shapes. YA (Ay/a c2J/c2J) melanosomes, characterized by poorly organized matrices, absence of active tyrosinase, unusually large membrane invaginations, and significantly smaller dimensions than those of BA (a/a c2J/c2J), showed additive effects of both Ay and c2J alleles. These data suggest that the albino locus plays a structural as well as functional (tyrosinase) role in the differentiation of mouse hairbulb melanosomes. The agouti locus, even in the absence of melanization, directs melanosome shape either via synthesis and deployment of agouti-locus-encoded matrix proteins or by other structural factors. The additive effects of Ay and c2J alleles in compound YA mutants document the importance of specific interactions both functional and structural between agouti and albino loci.     

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.     

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.     

Inducible system for the utilization of beta-glucosides in Escherichia coli. II. Description of mutant types and genetic analysis

Two types of mutants obtained by treating beta-gl(+) cells with nitrosoguanidine are described. One type, beta-gl(+)c, is constitutive for the biosynthesis of the aryl beta-glucoside splitting enzyme(s) and for the beta-glucoside permease; the other (beta-gl(+)sal(-)) has lost the capacity to ferment salicin, but has retained the capacity to ferment arbutin and other aryl beta-glucosides. By two successive mutational steps, beta-gl(+)sal(-)c double mutants can be obtained. Determinations of the enzymatic splitting of salicin and p-nitrophenyl beta-glucoside by beta-gl(+)sal(-) cells and extracts showed that these mutants have lost the capacity to split salicin but do split p-nitrophenyl beta-glucoside; they possess the beta-glucoside permease, and in them salicin is a gratuitous inducer for enzyme and permease biosynthesis. Studies on a beta-gl(+) strain, which splits salicin as well as p-nitrophenyl beta-glucoside, have shown that the splitting of salicin is more temperature-sensitive than that of p-nitrophenyl beta-glucoside and other beta-glucosides. Other properties of the two activities are similar. Interrupted mating experiments and cotransduction with P1kc phage showed that the genetic determinants of the beta-glucoside system map between the pyrE and ile loci. Three distinct mutational sites were found and are presumed to have the following functions: beta-glA, a structural gene for an aryl beta-glucoside splitting enzyme; beta-glB, either the structural gene for the beta-glucoside-permease or a regulatory gene; and beta-glC, a regulatory gene (or site). Escherichia coli wild-type strains are of the genotype A(+) B(-) C(+). The beta-gl(+) mutation determining the ability to ferment beta-glucosides is considered to be a permease or regulatory mutation, and the resulting genotype is A(+) B(+) C(+). The beta-gl(+)sal(-) phenotype results from a mutation in the beta-glA gene (genotype A' B(+) C(+)), and the constitutive phenotype results from a mutation in the beta-glC gene, the genotypes A(+) B(+)C(a) and A' B(+)C(a) corresponding to the phenotypes beta-gl(+)c and beta-gl(+)sal(-)c.     

A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine----serine substitution at position 85

Murine albinism is characterized by complete lack of melanin pigments in skin and retina. In order to study the molecular basis of albinism, we have cloned and characterized the tyrosinase gene of BALB/c mice (c/c). Sequence analysis of this gene reveals a point mutation at nucleotide residue 387 (G----C transversion) causing a Cys----Ser substitution at position 85 in one of the cysteine-rich domains of the tyrosinase molecule. Since this G----C transversion creates an additional DdeI site, we were able to confirm that this mutation is actually present in BALB/c genomic DNA using DNA amplification techniques. In contrast, both C57BL/6 (C/C) and DBA/2 (C/C) mouse strains carry the G residue at the same position, suggesting that this point mutation is specific for the albino mutation at the c locus. Moreover, we were able to show that the tyrosinase containing Ser-85 is not functional in transient expression of its cDNA. We therefore suggest that a G----C transversion at nucleotide residue 387 of the tyrosinase gene could lead to the albino phenotype of BALB/c mouse.     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol-1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i(1) is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol-1. Recently, mosaic black-pigmented i(1) medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol-1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

Expression of the murine wild-type tyrosinase gene in transgenic rabbits

The tyrosinase gene is known to be essential for melanization and has been shown to rescue pigmentation in albino mice. Previously we have described the strict copy-number-dependent expression of a murine wild-type tyrosinase gene construct over several generations in transgenic mice. In this study, we analysed the same gene construct as a marker gene for the transmission and expression of transgenes in rabbits. Using an albino hybrid strain, we produced transgenic rabbits expressing the murine tyrosinase gene. Strict correlation between integration and expression of the transgene and stable germline transmission of the integrated gene construct according to the Mendelian pattern of inheritance was observed. Thus, breeding control was facilitated by simple phenotypic examination of the transgenic animals. In contrast to mice transgenic for the same gene construct, tyrosinase-transgenic rabbits showed a greater variety in hue, intensity and extent of coat pigmentation, which is caused by the diversity in the loci affecting the melanization. Benefits and limitations of tyrosinase as a marker gene for the detection of homozygous individuals in the albino hybrid strain used are discussed.     

Proper folding and endoplasmic reticulum to golgi transport of tyrosinase are induced by its substrates, DOPA and tyrosine

Tyrosinase is essential for pigmentation and is a source of tumor-derived antigenic peptides and cellular immune response. Wild type tyrosinase in melanoma cells and certain albino mutants in untransformed melanocytes are targeted to proteolytic degradation by the 26 S proteasome due to retention of the misfolded protein in the endoplasmic reticulum and its subsequent retranslocation to the cytosol. Here, we demonstrate that the substrates DOPA and tyrosine induced in melanoma cells a transition of misfolded wild type tyrosinase to the native form that is resistant to proteolysis, competent to exit the endoplasmic reticulum, and able to produce melanin. Because the enzymatic activity of tyrosinase is induced by DOPA, we propose that proper folding of the wild type protein, just like mutant forms, is tightly linked to its catalytic state. Loss of pigmentation, therefore, in tyrosinase-positive melanoma cells is a consequence of tumor-induced metabolic changes that suppress tyrosinase activity and DOPA production within these cells.     

Expression of Tyrosinase Gene in Transgenic Albino Mice: The Heritable Patterned Coat Colors

To elucidate the regulatory mechanism for tyrosinase gene expression in vivo, we microinjected a mouse tyrosinase minigene, mg-Qrs-J, into the fertilized eggs of BALB/c albino mice. As a result, we obtained six pigmented founder mice that exhibited non-standard coat color variations as well as the wild-type phenotype. These founder mice were subsequently crossed with BALB/c albino mice to establish the transgenic lines. As a consequence, two primary lines and five sublines have been obtained from four of the six founder mice. We found that not only uniformly pigmented phenotypes but also patterned phenotypes were inherited by their descendants. The possible underlying mechanism of the patterned phenotypes is discussed.     

The Molecular Basis of Brown, an Old Mouse Mutation, and of an Induced Revertant to Wild Type

The murine b locus encodes the tyrosinase related protein, TRP-1, a putative membrane-bound, copper-containing enzyme having about 40% amino acid identity with tyrosinase. The protein is essential for production of black rather than brown hair pigment. We show that skin of mutant brown mice contains the same amount of TRP-1 mRNA as wild type. On sequencing the coding region of the mutant mRNA we find four nucleotide differences from the wild-type (Black) sequence. Two of these differences result in different amino acid residues encoded by the brown allele. By sequencing the TRP-1 gene from a mouse in which a reversion from brown to Black has been induced by ethylnitrosourea we are able to show that only one of these amino acid changes, which substitutes a tyrosine for a conserved cysteine, is the cause of the brown phenotype. This mutation is adjacent to another cysteine at which, in the analogous position in tyrosinase a mutation results in the albino phenotype. The sequence of the revertant is the first report of DNA sequence of an ethylnitrosourea-induced genetic change in mouse.     

Extrinsic modulation of retinal ganglion cell projections: analysis of the albino mutation in pigmentation mosaic mice

Tyrosinase is a key enzyme involved in the synthesis of melanin in the retinal pigment epithelium (RPE). Mice that are homozygous for the albino allele at the tyrosinase locus have fewer retinal ganglion cells with uncrossed projections at the optic chiasm. To determine the site of the albino gene action we studied the projections of retinal ganglion cells in two types of pigmentation mosaic mice. First, we generated mosaic mice that contain a translocated allele of the wild-type tyrosinase on one X chromosome but that also have the lacZ reporter transgene on the opposite X chromosome. In these lacZ/tyrosinase mice, which are homozygous for the albino allele on chromosome 7, X-inactivation ensures that tyrosinase cannot be functional within 50% of the retinal ganglion cells and that these individual cells can be identified by their expression of the lacZ reporter gene product, beta-galactosidase. The proportion of uncrossed retinal ganglion cells expressing beta-galactosidase was found to be identical to the proportion that did not express it, indicating that the albino mutation associated with axonal behavior at the optic chiasm must affect ganglion cells in a cell-extrinsic manner. Second, to determine whether the RPE is the source of the extrinsic signal, we generated aggregation chimeras between pigmented and albino mice. In these mosaic mice, the extent of the uncrossed projection corresponded with the amount of pigmented cells within the RPE, but did not correspond with the genotypes of neural retinal cells. These studies demonstrate that the albino mutation acts indirectly upon retinal ganglion cells, which in turn respond by making axonal guidance errors at the optic chiasm.     

Deficient melanosome formation in some coat-color mutant mice revealed by a monoclonal antibody against melanosome

Some coat-color loci in mice are considered to control melanosome formation. In order to investigate genetic control of melanosome-associated proteins, we prepared monoclonal antibodies against mouse melanosomes. Melanosomes were isolated from B16 mouse melanoma through differential fractionation. BALB/c mice were immunized with an SDS-solubilized melanosome fraction. The spleen cells were subsequently fused with mouse myeloma cells, the resulting hybridomas cloned. Their secreted IgG was screened for reactivity to the SDS-solubilized melanosome fraction. One monoclonal antibody, M10, was shown to react to melanosomes by immunoelectronmicroscopy. It recognized a single protein band of 61,000 dalton on immunoblots of gel-fractionated melanosomes. The reactivities of M10 to skin homogenates from various coat-color mutants were examined by the ELISA method. Five congenic genotypes, non-agouti (a/a), brown (b/b), albino (c/c), dilute (d/d), and pink-eyed dilution (p/p) were examined. Among these, b/b and p/p showed significantly lower reactivities than a/a. Our results seem to suggest that the pigment abnormalities in these mutants result from abnormalities of the melanosomal proteins. In the case of albino mice, the reactivity of M10 to skin homogenate was almost the same as the wild-type mouse. It seems that the albino mice are capable of producing the melanosomal protein.     

Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in regenerating hairbulb melanocytes of lethal yellow (Ay/a), black (a/a), agouti (AwJ/AwJ/) and albino (a/a/c2J/c2J) mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in particulate and soluble fractions of hairbulb melanocytes of lethal yellow (Ay/a C/C), nonagouti black (a/a C/C), and albino (a/a c2J/c2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, Ay/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (AwJ/AwJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and Ay/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and Ay/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in Ay/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol‐1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i¹ is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol‐1. Recently, mosaic black‐pigmented i¹ medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol‐1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.     

Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson's disease

We have used quantitative 2D gel electrophoresis to analyze serum proteins from 422 patients with neurodegenerative diseases and normal individuals in an unbiased approach to identify biomarkers. Differences in abnormal serum levels were found between amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and related disorders for 34 protein biomarker spots, nine of which were related to the complement system. Of these nine, four spots originated from the Complement C3b-alpha-chain (C3c(1), C3c(2a), C3c(2b), and C3dg). The C3c spots (C3c(1), C3c(2a), and C3c(2b)) had the same amino acid sequence and glycosylation, though only C3c(1) was phosphorylated. In addition, Complement Factors H, Bb, and Pre-Serum amyloid protein displayed different serum concentrations in ALS, PD, and normal sera, whereas Complement C4b gamma-chain and Complement Factor I did not. The differential expression of the complement proteins provides potentially useful biomarkers as well as evidence for the involvement of inflammatory processes in the pathogenesis of ALS and PD.     

Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice.

The c-locus of the mouse is thought to encode tyrosinase, the key enzyme for melanin synthesis in melanocytes of the skin and the eye. Recently, a mouse cDNA was isolated and shown to confer tyrosine activity on a cell line which expressed no specialized functions for melanin synthesis. To verify that the isolated tyrosinase gene is encoded at the genetically well characterized c-locus, a minigene was assembled from tyrosinase cDNA and tyrosinase genomic DNA and used for generation of transgenic mice. Following microinjection of this construct into fertilized eggs of an albino mouse strain, transgenic mice were obtained which showed pigmentation in skin and eyes. By in situ hybridization, we show expression of the transgene in melanocytes of the hairbulb and in the pigmented cell layers of the eye. We conclude that we have rescued the albino mutation (c/c) by introduction and expression of a functional tyrosinase gene.     

黑线仓鼠及其白化突变系酪氨酸酶基因的克隆与序列分析

目的 对黑线仓鼠及其白化突变系酪氨酸酶基因进行比较研究,揭示黑线仓鼠白化突变系白化性状产生的分子机理.方法 根据小鼠与大鼠的酪氨酸酶基因保守区设计3对引物,利用RT-PCR方法,从黑线仓鼠及其白化系皮肤总RNA中扩增得到酪氨酸酶的cDNA基因,并对其二者进行克隆测序.结果 成功获得了黑线仓鼠及其白化突变系的酪氨酸酶基因,对二者的序列比较分析结果表明,二者的编码区没有差异.结论 黑线仓鼠白化突变系白化性状产生的原因与已知小鼠的白化性状产生原因不同,并不是由酪氨酸酶基因编码区突变造成的,其白化性状产生的机理有待进一步的研究.