Molecular Characterization and Tissue Expression of Ovine PSAM6 Gene from Muscle Full-Length cDNA Library of Black-Boned Sheep

An ovine PSMA6 gene was obtained from muscle full-length cDNA library of black-boned sheep. The sequences for the PSAM6 gene of Romney sheep and Yunling black goat were also generated in this study. Sequence analysis revealed that nucleotide sequence of this gene was not homologous to any of the known sheep genes, and its open reading frame encodes a protein that contains the putative conserved domain of proteasome subunit alpha type 6 (PSAM6). The nucleotide sequence had higher identity with other animals. However, one mutation of A to G at the site of 383 bp, leading to an amino acid mutation of Asn to Ser, was found only in the black-boned sheep. Tissue expression analysis indicated that this gene was generally expressed in most tissues and differently expressed in tissues of black-boned sheep. This the first report of the ovine PSAM6 gene.     

Pigmentation in Black-boned sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>): association with polymorphism of the <Emphasis Type="Italic">Tyrosinase</Emphasis> gene

Measurements were made in Black-boned (n = 40) and normal (n = 23) sheep (Ovis aries) from a flock in Nanping County of Yunnan Province, China, as well as a group (n = 21) of Romney Marsh sheep (O. aries) with the view to explaining the basis of the dark pigmentation occurring in the Black-boned animals. Plasma colour was significantly darker (P < 0.01) in Black-boned sheep than in their normal flock mates, which in turn had significantly darker plasma (P < 0.01) than the Romney Marsh sheep. Similar significant (P < 0.01) differences were measured for plasma tyrosinase activity and both groups of sheep from Nanping County had similar plasma concentrations of glutathione which were significantly smaller (P < 0.01) than for the Romney Marsh sheep.A partial fragment of 750 bp of exon 1 of the gene encoding tyrosinase was constructed and found to contain two silent mutation sites (G192C and C462T) but there was no effect on amino acid sequences of tyrosinase. Using restriction fragment length polymorphism analyses two allelic variants of site G192C were identified giving rise to the genotypes GG, GC and CC; the frequencies of allele G being 0.914, 0.824 and 0.286 in the Black-boned sheep, their flock mates and the Romney Marsh sheep respectively. Plasma tyrosinase activity was similar for genotypes GG and GC and for both genotypes significantly higher (P < 0.05) than for genotype CC. The sheep from Nanping County displayed only the GG and GC genotypes and had predominantly black or black and white coat colour whereas the Romney Marsh sheep were of either genotype GC or CC and exhibited only white coat colouration. It is not appears that the dark pigmentation of the Black-boned sheep arises because of polymorphisms in the exon 1 of tyrosinase gene. However, this result could explain the differences between Black-boned and Romney Marsh sheep but not for differences between Black-boned and Nanping Normal sheep. Moreover, this result has provided evidence of genetic markers in the form of polymorphisms of the tyrosinase gene which may help to find the black traits causing mutations. There would be merit in further studies using histochemical and molecular techniques to elucidate the causes of the dark pigmentation in these Black-boned sheep.     

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the MC1R gene

Variations in vertebrate skin and hair color are due to varied amounts of eumelanin (brown/black) and phaeomelanin (red/yellow) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates. We have sequenced the entire coding region of the MC1R gene in Black-boned, Nanping indigenous and Romney Marsh sheep populations and found two silent mutation sites of A12G and G144C, respectively. PCR-RFLP of G144C showed that frequency of allele G in Black-boned, Nanping indigenous and Romney Marsh sheep was 0.818, 0.894 and 0, respectively. Sheep with GG genotype had significantly higher (P < 0.05) tyrosinase activity than sheep with CC genotype in the all investigated samples. Moreover, there was significant effect of MC1R genotype on coat color, suggesting that MC1R gene could affect coat color but not black traits. There would be merit in further studies using molecular techniques to elucidate the cause of black traits in these Black-boned sheep.     

Skin Transcriptome Profiles Associated with Skin Color in Chickens

Nutritional and medicinal benefits have been attributed to the consumption of tissues from the black-boned chickens in oriental countries. Lueyang black-boned chicken is one of the native chicken breeds. However, some birds may instead have white or lighter skin, which directly causes economic losses every year. Previous studies of pigmentation have focused on a number of genes that may play important roles in coat color regulation. Illumina2000 sequencing technology was used to catalog the global gene expression profiles in the skin of the Lueyang chicken with white versus black skin. A total of 18,608 unigenes were assembled from the reads obtained from the skin of the white and black chickens. A total of 649 known genes were differentially expressed in the black versus white chickens, with 314 genes that were up regulated and 335 genes that were down-regulated, and a total of 162 novel genes were differentially expressed in the black versus white chickens, consisting of 73 genes that were up-regulated (including 4 highly expressed genes that were expressed exclusively in the skin of the black chickens) and 89 genes that were down-regulated. There were also a total of 8 known coat-color genes expressed in previous studies (ASIP, TYR, KIT, TYRP1, OCA2, KITLG, MITF and MC1R). In this study, 4 of which showed greater expression in the black chickens, and several were up-regulated, such as KIT, ASIP, TYR and OCA2. To our surprise, KITLG, MITF and MC1R showed no significant difference in expression between the black- and white-skinned chickens, and the expression of TYRP1 was not detected in either skin color. The expression of ASIP, TYR, KIT, TYRP1, OCA2, KITLG, MITF and MC1R was validated by real-time quantitative polymerase chain reaction (qPCR), and the results of the qPCR were consistent with the RNA-seq. This study provides several candidate genes that may be associated with the development of black versus white skin. More importantly, the fact that the MC1R gene showed no significant difference in expression between the black and white chickens is of particular interest for future studies that aim to elucidate its functional role in the regulation of skin color.     

Serological evidence of Toxoplasma gondii and Neospora caninum infection in black-boned sheep and goats in southwest China

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49–41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02–11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ² = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ² = 8.558, df = 2, P<0.05), age (χ² = 16.631, df = 3, P < 0.01), gender (χ² = 11.219, df = 1, P < 0.01) and species (χ² = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94–5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats.     

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.     

Mammalian melanogenesis: tyrosinase versus peroxidase involvement, and activation mechanisms

A study of the enzymes functioning in murine melanogenesis was carried out on tissue homogenates of the black mouse. Several major points were resolved: (a) while the enzyme peroxidase is capable of converting tyrosine to melanin in vitro, it is not responsible for observed melanogenesis in the mouse, (b) a proteolytic activation system for tyrosinase, such as that described for amphibian skin, does not seem to function in mammalian tyrosinase activation, and (c) tyrosinase activity in normal murine tissues can be stimulated with a variety of treatments.     

GPR143在绵羊皮肤组织中的表达及定位分析

G蛋白偶联受体143(G-protein coupled receptor143, GPR143)在黑素体的生物合成中起重要作用,本文旨在研究GPR143基因在不同毛色绵羊皮肤组织中的差异表达及定位,探索GPR143基因与毛色形成的相关性。通过qRT-PCR方法和免疫印迹方法分别检测不同毛色绵羊皮肤组织中GPR143基因mRNA水平和蛋白水平的表达差异;运用免疫荧光法对不同毛色绵羊皮肤组织中的GPR143基因进行定位并对结果进行光密度值分析。qRT-PCR结果显示,GPR143基因在黑色绵羊皮肤组织中mRNA相对表达量为白色绵羊的7.84倍,二者差异极显著(P<0.01);免疫印迹结果显示,黑色绵羊皮肤组织中GPR143蛋白表达量是白色绵羊的1.3倍,二者差异显著(P<0.05)。免疫荧光结果显示,GPR143蛋白的主要表达部位为绵羊皮肤组织毛囊外根鞘和表皮层,经光密度值分析后发现,GPR143在黑色绵羊皮肤毛囊外根鞘和表皮层的表达量显著高于白色绵羊。本研究结果表明不同毛色绵羊皮肤组织均能表达GPR143基因,但黑色绵羊皮肤组织中该基因的mRNA和蛋白水平都显著高于白色绵羊,说明GPR143的mRNA和蛋白在黑色绵羊皮肤组织中表达上调,在白色绵羊皮肤组织中表达下调。GPR143基因可能通过调控MITF水平和黑素体的数量、大小、运动和成熟进而参与绵羊毛色的形成过程。     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.     

Genetic controls over melanocyte differentiation: interaction of agouti-locus and albino-locus genetic defects

Tyrosinase activities and dopachrome conversion activity were evaluated in extracts made from skins of 6-day-old mice that were mutant at the agouti and albino loci. Dopa oxidase (DO) activity of tyrosinase in fully pigmented (C/C) mice is reduced in extracts made from skins of yellow 6-day-old mice as compared to those of black mice. Dopachrome conversion (DC) activity is absent from skin extracts of normal yellow mice and is present in normal black mice. DC activity is a characteristic of a separate enzyme which has been called dopachrome conversion factor or dopachrome oxidoreductase. We measured the dopa oxidase activity and dopachrome conversion activity in skin extracts of yellow mice and black mice that were mutant at the albino (C) locus. Extracts made from extreme-dilution (ce/ce) mice do not have DO activity. Those from yellow extreme-dilution mice do not have DC activity, while those from black, extreme-dilution mice do. The DO and DC activities that characterize skin extracts made from platinum (cp/cp) yellow mice are similar to those of platinum black mice. These observations suggest possible mechanisms by which the functions controlled by the agouti and albino loci interact to control melanogenesis.     

Detection of Mouse Tyrosinase With a Monoclonal Antibody MAT-1 Against Human Tyrosinase

In this study we explored the possible application of MAT-1, which has been established as a monoclonal antibody against human tyrosinase, for detection of mouse tyrosinase. The MAT-1 reacted with B16 mouse melanoma cells, but not with tyrosinase-negative NIH-3T3 mouse fibroblasts. In western blot analysis of the large granule fraction (LGF) of B16 cells, MAT-1 detected a single protein of 80 kDa, whose size was close to that of human tyrosinase detected with MAT-1 in extracts of human melanocytes. Furthermore, the 80 kDa band that was detected with MAT-1 in the LGF of B16 cells was also detected by DOPA reaction. In order to confirm that the protein detected with MAT-1 is tyrosinase, a transient expression assay was carried out. When mouse tyrosinase or mouse tyrosinase-related protein 1, which shares high homology with human tyrosinase, was transiently expressed in tyrosinase-negative K1735 mouse melanoma cells by cDNA transfection, MAT-1 reacted only with the cells expressing mouse tyrosinase. These results indicate that MAT-1 specifically reacts with mouse tyrosinase.     

Homozygous tyrosinase gene mutation in an American black with tyrosinase-negative (type IA) oculocutaneous albinism

We have identified a tyrosinase gene mutation in an American black with classic, tyrosinase-negative oculocutaneous albinism. This mutation results in an amino acid substitution (Cys----Arg) at codon 89 of the tyrosinase polypeptide. The proband is homozygous for the substitution, suggesting that this mutation may be frequently associated with tyrosinase-negative oculocutaneous albinism in blacks.     

Mapping and characterization of the dominant black colour locus in sheep

Dominant black pigment synthesis in sheep is caused by alterations of the melanocortin-1 receptor (MC1-R) coding sequence. Using five bovine microsatellite markers we have mapped the sheep MC1-R gene to chromosome 14, corresponding to the location in other mammalian species. The existence of two independent mutations, both causing an amino acid substitution, in distantly related breeds of sheep, support the hypothesis that the observed black pigment synthesis is caused by a mutual effect of the two mutations. As similar mutations are found separately at both locations in dominant black variants of other animal species, it is also possible that any of the two mutations could be sufficient for a partial pigment switch.     

应用兔抗大熊猫免疫球蛋白G血清探讨大熊猫的分类地位

从大熊猫血清中纯化出免疫球蛋白(IgG),以此作为抗原免疫家兔,获得兔抗大熊猫IgG血清。以黑熊、小熊猫、狗、猫等动物血清为抗原,兔抗大熊猫IgG 血清为抗体．进行了免疫扩散和微量免疫电泳实验。 实验结果表明,收集的食肉目动物：黑熊、小熊猫、狗、猫的血清都可与兔抗大熊猫GIg血清进行沉淀反应,其中尤以黑熊的反应最强且与大熊猫的反应沉淀线完全融合;小熊猫、狗、猫反应较弱且融入大熊猫反应沉淀线后形成树板状。从此看出大熊猫lgG 与黑熊的IgG最相似,从亲缘关系上讲,二者更为接近,大熊猫反应属熊科。     

Mutations in ASIP and MC1R: dominant black and recessive black alleles segregate in native Swedish sheep populations

By studying genes associated with coat colour, we can understand the role of these genes in pigmentation but also gain insight into selection history. North European short‐tailed sheep, including Swedish breeds, have variation in their coat colour, making them good models to expand current knowledge of mutations associated with coat colour in sheep. We studied ASIP and MC1R, two genes with known roles in pigmentation, and their association with black coat colour. We did this by sequencing the coding regions of ASIP in 149 animals and MC1R in 129 animals from seven native Swedish sheep breeds in individuals with black, white or grey fleece. Previously known mutations in ASIP [recessive black allele: g.100_105del (D₅) and/or g.5172T>A] were associated with black coat colour in Klövsjö and Roslag sheep breeds and mutations in both ASIP and MC1R (dominant black allele: c.218T>A and/or c.361G>A) were associated with black coat colour in Swedish Finewool. In Gotland, Gute, Värmland and Helsinge sheep breeds, coat colour inheritance was more complex: only 11 of 16 individuals with black fleece had genotypes that could explain their black colour. These breeds have grey individuals in their populations, and grey is believed to be a result of mutations and allelic copy number variation within the ASIP duplication, which could be a possible explanation for the lack of a clear inheritance pattern in these breeds. Finally, we found a novel missense mutation in MC1R (c.452G>A) in Gotland, Gute and Värmland sheep and evidence of a duplication of MC1R in Gotland sheep.     

Molecular Cloning and Biochemical Analysis of Tyrosinase from the Crested Ibis in China

The crested ibis, one of the most endangered birds in the world, could benefit from research into its genetic diversity as a tool for conservation in the future. Tyrosinase is thought to play a major role in the production of common yellow to black melanins in birds. We have cloned and sequenced four exons of the crested ibis tyrosinase gene and discovered that the amino acid sequence has high similarity to zebra finch tyrosinase (93?%), followed by chicken (91?%) and quail (91?%). Some functional and structural domains in the crested ibis tyrosinase coding area were found to be conserved during evolution. Nine sequence variants were found in the partial coding sequence, one in exon 1 and eight in exon 4. Sequence variant 1 (SV1) shows intermediate polymorphism (0.25?<?PIC?<?0.5), and further study is needed to determine whether it can be used as a potential molecular marker in crested ibis artificial breeding programs.