Gene Expression and Localization of LAMTOR3 in the Skin Cells of Liaoning Cashmere Goats

Liaoning cashmere goats are the most precious genetic resources in China. The function of LAMTOR3 [late endosomal/lysosomal adaptor, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin activator 3/MAPK scaffold protein 1] gene is expressed in the skin of Liaoning cashmere goats. In situ hybridization (ISH) found that LAMTOR3 is expressed in the inner root sheath (IRS) of hair follicles. During the anagen or catagen phase, the expression of LAMTOR3 is higher in secondary hair follicles than in primary hair follicles. Expression of LAMTOR3 in skin cells treated with melatonin or insulin-like growth factor-1 (IGF-1) is lower than in untreated cells. In addition, the simultaneous treatment of fibroblast growth factor 5 and melatonin decrease the expression of LAMTOR3 in skin cells. The simultaneous treatment with melatonin and 10^?5?g/L IGF-1 or 10^?4?g/L IGF-1 increases the expression of LAMTOR3 gene in skin cells. If Noggin expression is decreased, then LAMTOR3 expression is increased. This hypothesis suggested that LAMTOR3 influences the character of cashmere fiber, and it may regulate the development of hair follicle and cashmere growth by inducing the MAPK signaling pathway.     

Variation and expression of KAP9.2 gene affecting cashmere trait in goats

Keratin-associated proteins 9.2 (KAP9.2) gene encodes one of the ultra high sulfur KAPs. Variation in KAP genes may affect the structure of KAPs and hence cashmere characteristics. In order to test the association between the polymorphism of KAP9.2 gene and cashmere trait, DNA sequencing was used to detect a novel C/T polymorphism of KAP9.2 gene from a genomic DNA pool. The mutation could be recognized by Pst I restriction enzyme. To Shanbei white cashmere goat, Inner Mongolia white cashmere goat and Guanzhong dairy goat, the genotypic frequencies of TT, TC and CC from total 1,236 animals were as follows: 0.047, 0.519 and 0.434; 0.180, 0.592 and 0.228; 0.431, 0.544 and 0.025. The allelic frequencies of T and C were 0.307 and 0.693; 0.476 and 0.524; 0.703 and 0.297, respectively, in breeds mentioned above. The frequency of C allele between cashmere and dairy goat was significant (P?<?0.01). To provide support for the hypothesis that SNP 586 was responsible for KAP9.2 expression, quantitative real-time PCR analysis revealed that the expression level of KAP9.2 was reduced in individuals bearing genotype CC compared with TT individuals, suggesting that C was the nucleotide causing decreased expression of KAP9.2 or was in linkage disequilibrium with the causative SNP. The 586C/T SNP found in this study might control translation or stability of KAP9.2 mRNA, which would be beneficial for marker assistant selection in cashmere goat breeding.     

Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCR method

Keratin-associated protein 9.2 (KAP9.2) and Homeobox C13 (Hoxc13) genes were chosen to study because of their biological functions involving hair formation. KAP9.2 gene belongs to the ultra high sulfur KAPs, which is important for hair formation and may have association with cashmere. Hoxc13 takes part in the formation of cashmere keratin and maintaining the normal structure of follicle. It has been reported that Hoxc13 gene exists binding site of KP and KAP genes at its promoter regions in mouse. So the expression of KAP9.2 and Hoxc13 genes was detected at anagen stage vs telogen stage by qRT-PCR. The data showed that KAP9.2 and Hoxc13 gene had similar expression trend at different stages, which indicated that there was interaction between them. KAP9.2 and Hoxc13 gene had lower expression level in anagen than that of in telogen of cashmere growth. In anagen, KAP9.2 and Hoxc13 expressed lower in high cashmere yield individuals than that of in low cashmere yield ones. In telogen, the result was reverse. The study would provide the evidence of involvement of KAP9.2 and Hoxc13 in hair periodic growth.     

绒山羊和绵羊KAP6.1基因CDS区克隆与分析

目的:克隆绒山羊、绵羊KAP6.1基因CDS全序列,分析序列特征和蛋白结构。方法:RT-PCR法克隆基因并测序,生物信息学软件分析序列特征和结构。结果:绒山羊、绵羊KAP6.1基因CDS序列全长252 bp,编码83个氨基酸;绒山羊、绵羊KAP6.1蛋白在基本理化特性、二级结构以及空间三维结构上的差异较小。结论:绒山羊与绵羊KAP6.1蛋白结构上的差异将会在一定程度上影响其功能。     

<Emphasis Type="Italic">Tβ4</Emphasis>-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics

Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tβ4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.     

Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFβ and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.     

Cashmere production,skin characteristics,and mutated genes in crimped cashmere fibre goats

A subpopulation of Yanshan cashmere goats with crimped fibre has emerged in a closed population in recent years, but little is known about differences in cashmere production performance, skin characteristics, and fibre-style-related genes between goats with different cashmere fibre styles. Therefore, the aim of this study was to investigate fibre characteristics, cashmere yield, hair follicle traits, and fibre-style-related genes in cashmere goats with the two cashmere fleece styles—non-crimped and crimped. Based on the cashmere fibre type, 80 six-month-old female Yanshan cashmere goats were used in this study: 40 goats with non-crimped fleece (NCF) and 40 with crimped fleece (CF). The growth performance and cashmere production of the goats were recorded. Skin samples were collected to determine hair follicle traits and gene sequencing. The results indicated that there were no differences in initial live weight, final live weight, average daily feed intake, and average daily gain between the two groups of goats (P > 0.05). The total yield of cashmere and the stretched length of fibre of the CF goats were higher (P < 0.01 and P < 0.05, respectively) and fibre diameter was lower (P < 0.05) than that of the NCF goats. There were no between-group differences in the density and activity of primary and secondary hair follicles, secondary-to-primary fibre ratio, depth of primary follicles, or epidermal thickness. However, the depth of secondary follicles and dermal thickness were higher (P < 0.05) in NCF goats than in CF goats. There were mutations in the KRT5, KAP8, KRT8, KRT74, KRT34, KRT1, KRT71, KRT6A, KAP6, KRT81, and KRT83 genes, four of which caused amino acid changes. The allele and genotype frequencies of base mutations in the KRT5, KAP8, KRT34, KRT1, KRT6A, KRT81, and KRT83 genes were different in the NCF and CF goats (P < 0.05). The distribution and content of the secondary structure elements and tertiary structures of proteins differed between the wide-type and mutated KRT1 and KRT6A proteins. KRT1, KRT6A, KRT71, and TGFα mRNA expression levels were significantly higher in CF goats than in NCF goats (P < 0.05). It is concluded that cashmere goats that have fleece with crimped fibres produce a greater yield of fleece with finer diameter fibres than those with conventional straight cashmere fibres. These differences in fibre properties may be associated with mutations in the genes coding for KRT1 and KRT6A.     

Seasonal hair follicle activity and fibre growth in some New Zealand Cashmere-bearing goats (Caprus hircus)

Hair follicle activity and fibre growth were studied using histological sections from the skin of five adult feral does sampled every four weeks for 18 months. The main period of guard hair growth in primary follicles was from November to April. Secondary follicles grew fine, long, nonmedullated fibres (cashmere) from December to June. Shedding of these fibres from secondary follicles had commenced by July and cashmere was absent from the fleece by November. From September to December a subsidiary hair cycle occurred in many secondary follicles which produced minute (vellus) fibres, less than 2.4 mm in length. Some secondary follicles probably shed their cashmere fibres and remain quiescent over spring. Annual pelage changes were therefore achieved with one main growth period, although many secondary follicles underwent another brief hair cycle in spring.     

The PCR-SSCP and DNA sequencing methods detecting a large deletion mutation at KAP6.2 locus in the cashmere goat

It is known that keratin-associated proteins 6.2 gene (KAP6.2) is an important structural gene responsible for the cashmere. So in this study, the polymorphism of KAP6.2 gene was firstly detected by PCR-SSCP and DNA sequencing methods in 1052 cashmere goat samples. The results showed that two alleles were detected at the locus, which were named as allele O and X. Frequencies of KAP6.2-O allele in Inner Mongolia White cashmere (n = 847) and Shaanbei White cashmere goat breeds (n = 205) were 1.000 and 0.856, respectively. Inner Mongolia White cashmere goat was monomorphic at this locus, while Shaanbei White cashmere goat was at low polymorphic level. Therefore, the allele KAP6.2-X was considered as the breed characterization of Shaanbei White cashmere goat at KAP6.2 locus. Further sequencing analysis showed that a 24-bp deletion mutation was described for the first time in Shaanbei, while Inner Mongolia White cashmere goat did not have the deletion. The study indicated that deletion mutation was predicted as a possible cause for the multiple pattern cashmere in Shaanbei White cashmere goat.     

aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat

Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.     

Analysis of the expression pattern of the carrier protein transthyretin and its receptor megalin in the human scalp skin and hair follicles: hair cycle-associated changes

Transthyretin is a serum and cerebrospinal fluid protein synthesized early in development by the liver, choroid plexus and several other tissues. It is a carrier protein for the antioxidant vitamins, retinol, and thyroid hormones. Transthyretin helps internalize thyroxine and retinol-binding protein into cells by binding to megalin, which is a multi-ligand receptor expressed on the luminal surface of various epithelia. We investigated the expression of transthyretin and its receptor megalin in the human skin; however, their expression pattern in the hair follicle is still to be elucidated. This study addresses this issue and tests the hypothesis that “the expression of transthyretin and megalin undergoes hair follicle cycle-dependent changes.” A total of 50 normal human scalp skin biopsies were examined (healthy females, 53–62 years) using immunofluorescence staining methods and real-time PCR. In each case, 50 hair follicles were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). Transthyretin and megalin were prominently expressed in the human scalp skin and hair follicles, on both gene and protein levels. The concentrations of transthyretin and megalin were 0.12 and 0.03 Ul/ml, respectively, as indicated by PCR. The expression showed hair follicle cycle-associated changes i.e., strong expression during early and mature anagen, very weak expression during catagen and moderate expression during telogen. The expression values of these proteins in the anagen were statistically significantly higher than those of either catagen or telogen hair follicles (P ≤ 0.001). This study provides the first morphologic indication that transthyretin and megalin are variably expressed in the human scalp skin and hair follicles. It also reports variations in the expression of these proteins during hair follicle cycling. The clinical ramifications of these findings are open for further investigations.     

Identification of the ovine KAP11-1 gene (<Emphasis Type="Italic">KRTAP11</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) and genetic variation in its coding sequence

Keratin-associated proteins (KAPs) are a structural component of the wool fibre and form the matrix between the keratin intermediate filaments (KIFs). The gene encoding high sulphur-protein KAP11-1 has been identified in human, cattle and mouse, but not yet in sheep, despite the economic importance of wool. In this study, PCR using primers based on the cattle KAP11-1 gene sequence produced an amplicon of the expected size with sheep DNA. Upon using PCR–Single Stranded Conformational Polymorphism (PCR–SSCP) analysis in 260 sheep, six different PCR–SSCP patterns were detected. Either one or a combination of two banding patterns was observed for each sheep, suggesting they were either homozygous or heterozygous for this gene. Sequencing of the amplicons confirmed the occurrence of six DNA sequences. All of these were unique, and the greatest homology was with KRTAP11-1 sequences from cattle, human and mouse, suggesting that they were derived from the ovine KAP11-1 gene and were allelic variants. The ovine KAP11-1 gene had an open reading frame of 477 nucleotides encoding 159 amino acids. The putative protein was rich in serine, cysteine, and threonine which account for 18.2–18.9, 12.6 and 12.0 mol%, respectively. Of these, approximately 20 of the serine and threonine residues might be phosphorylated. Five nucleotide substitutions were identified, and one was non-synonymous and would result in an amino acid change at a potential phosphorylation site. The genetic variation found in KRTAP11-1 may influence its expression, protein structure, and/or post-translational modifications, and consequently affect wool fibre structure and wool traits.     

Overexpression of kinase-associated phosphatase (KAP) in breast and prostate cancer and inhibition of the transformed phenotype by antisense KAP expression

Accumulating evidence suggests that phosphatases play an important role in regulating a variety of signal transduction pathways that have a bearing on cancer. The kinase-associated phosphatase (KAP) is a human dual-specificity protein phosphatase that was identified as a Cdc2- or Cdk2-interacting protein by a yeast two-hybrid screening, yet the biological significance of these interactions remains elusive. We have identified the KAP gene as an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy. Here we report that breast and prostate malignancies are associated with high levels of KAP expression. The sublocalization of KAP is variable. In normal cells, KAP is primarily found in the perinuclear region, but in tumor cells, a significant portion of KAP is found in the cytoplasm. Blocking KAP expression by antisense KAP in a tetracycline-regulatable system results in a reduced population of S-phase cells and reduced Cdk2 kinase activity. Furthermore, lowering KAP expression led to inhibition of the transformed phenotype, with reduced anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings suggest that therapeutic intervention might be aimed at repression of KAP gene overexpression in human breast and prostate cancer.     

Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1

Analysis of the EBI/GeneBank(TM) data base using non-human hair keratin-associated protein (KAP) cDNA sequences as a query resulted in the identification of a first domain of high glycine-tyrosine and high sulfur KAP genes located on human chromosome 21q22.1. This domain, present on the DNA accession numbers and, was approximately 535 kb in size and contained 17 high glycine-tyrosine and 7 high sulfur KAP genes, as well as 9 KAP pseudogenes. Based on amino acid sequence comparisons of the encoded proteins, the KAP genes could be divided into seven high glycine-tyrosine gene families (KAP6-KAP8, and KAP19-KAP22) and four high sulfur gene families (KAP11, KAP13, KAP15, and KAP23). The high glycine-tyrosine genes described here appear to represent the complete set of this type of KAP genes present in the human genome. Both systematic cDNA isolation studies from an arrayed scalp cDNA library and in situ hybridization expression studies of all of the KAP genes identified in the 21q22.1 region revealed varying degrees and regions of expression of 11 members of the high tyrosine-glycine genes and 6 members of the high sulfur KAP genes in the hair forming compartment.     

Molecular Cloning and Expression Analysis of Prostaglandin E Receptor 2 Gene in Cashmere Goat (Capra hircus) Skin during Hair Follicle Development

As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin.     

Cloning,chromosomal localization,expression profile and association analysis of the porcine <Emphasis Type="Italic">WNT10B</Emphasis> gene with backfat thickness

The Wingless-type MMTV integration site (Wnt) family encodes secreted glycoproteins that are ligands for the frizzled family of seven-transmembrane receptors and the low density lipoprotein receptor-related protein family of co-receptors. The WNT10B gene inhibits differentiation of preadipocytes in vitro and impairs adipose development in vivo. In the present study, a 1,615-bp cDNA sequence of the porcine WNT10B gene was obtained by RT–PCR. The porcine WNT10B gene was assigned to 5p11-p15 by using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel. One SNP in the 3′-untranslated region (3′-UTR) was found and association analysis suggested that the SNP was associated with backfat thickness. Semi-quantitative RT–PCR showed that the porcine WNT10B gene was expressed in all tissues examined in 35d and adult pigs and the mRNA expression of WNT10B in fat tissue of Tongcheng pigs was dramatically higher than that in Large White pigs.