Keratin-associated Protein Genes Research Articles

Variation in ovineKRTAP8-1is associated with variation in wool fibre staple strength and curvature

AbstractKRTAP8-1was the initial high-glycine-tyrosine keratin-associated protein gene recognized in sheep, but little is known about the functional influence of this gene. The current study used polymerase chain reaction-single stranded conformational polymorphism analysis to genotypeKRTAP8-1in 391 Southdown × Merino-cross sheep from six sire-lines. Five previously described variants (namedAtoE) ofKRTAP8-1were identified with frequencies of 67.0, 14.2, 7.0, 10.7 and 1.0%, respectively. Of the four variants (A,B,CandD) that occurred at a frequency greater than 5%, the presence ofCwas found to be associated with a reduction in mean fibre curvature (MFC) and the presence ofDwas associated with an increase in mean staple strength (MSS), whereas the presence ofAhad a trend of association with reduced MSS. Associations were not identified with other wool traits. These results suggest that variation inKRTAP8-1affects MSS and MFC, and thatKRTAP8-1has the potential to be used as a genetic marker for improving these traits.

Identification of the Ovine Keratin-Associated Protein 21-1 Gene and Its Association with Variation in Wool Traits.

Simple SummaryKeratin-associated proteins (KAPs) are key constituents of wool and hair fibers. In this study, the ovine keratin-associated protein 21-1 gene (KRTAP21-1) was identified, and association analysis showed that its variation affected wool yield, which prompted the conclusion that the gene may have potential use as a genetic maker for improving wool yield.Keratin-associated proteins (KAPs) are key constituents of wool and hair fibers. In this study, an ovine KAP gene encoding a HGT-KAP protein was identified. The gene was different from all of the HGT-KAP genes identified in sheep, but was closely related to the human KAP21-1 gene, suggesting that it represented the unidentified ovine KRTAP21-1. Four variants (named A to D) of ovine KRTAP21-1 were found in 360 Merino × Southdown-cross lambs from four sire lines. Three sequence variations were detected among these variants. Two of the sequence variations were located upstream of the coding region and the remaining one was a synonymous variation in the coding sequence. Six genotypes were found in the Merino-cross lambs, with only two of the genotypes (AA and AC) occurring at a frequency of over 5%. Wool from sheep of genotype AA had a higher yield than that from AC sheep (p = 0.014), but tended to have a lower greasy fleece weight (GFW) than that of genotype AC (P = 0.078). This suggests that variation in KRTAP21-1 affects wool yield and the gene may have potential for use as a genetic maker for improving wool yield.

Identification of Ovine KRTAP28-1 and Its Association with Wool Fibre Diameter.

Simple SummaryKeratin-associated proteins (KAPs) are fundamental components of wool and hair fibres. They are split into three broad groups: the high sulphur (HS), the ultra-high sulphur (UHS) and the high glycine-tyrosine (HGT) KAPs. KRTAP25-1 encodes a HS-KAP protein and the gene has recently been identified in humans. Here, we report the absence of a KRTAP25-1 in sheep, and we describe a new HS-KRTAP (named KRTAP28-1) in the chromosome region where KRTAP25-1 was expected to be found. Six variants (A−F) of KRTAP28-1 containing eight single nucleotide polymorphisms (SNPs) and a TG dinucleotide repeat polymorphism were detected. One SNP was located upstream of the start codon and all the others were non-synonymous SNPs, including a nonsense SNP. The TG repeat polymorphism would lead to a reading frame shift at the carboxyl-terminal end. The association of KRTAP28-1 with wool traits was investigated with 383 Southdown × Merino-cross lambs from seven sire lines. Of the four genotypes with a frequency over 5%, lambs of genotypes AB and BD produced wool of a smaller mean fibre diameter (MFD) than lambs of genotype BC. This shows that KRTAP28-1 is associated with a key wool trait, and variation in this gene might therefore have value as a marker for improving that trait.Keratin-associated proteins (KAPs) are a diverse group of proteins and form a matrix that cross-links keratin intermediate filaments in hair and wool fibres. From over 100 KAP genes (KRTAPs) identified in mammalian species, KRTAP25-1 is a high sulphur (HS)-KAP gene, which has recently been described in humans. Here, we report the absence of KRTAP25-1 in sheep, and describe a new HS-KRTAP (named KRTAP28-1) in the chromosome region where KRTAP25-1 was expected to be found. Six variants (A−F) of KRTAP28-1 containing eight single nucleotide polymorphisms (SNPs) and a TG repeat polymorphism were detected. One was positioned 30 bp upstream of the transcription start codon and all the others were non-synonymous SNPs, including a nonsense SNP. The TG repeat polymorphism would lead to a reading frame shift at the carboxyl-terminal end. The effect of KRTAP28-1 on wool traits was investigated with 383 Southdown × Merino-cross lambs from seven sire lines. Of the four genotypes with a frequency of over 5%, lambs of genotypes AB and BD produced wool of a smaller MFD than lambs of genotype BC. This shows that KRTAP28-1 is associated with wool fibre diameter, and that variation in this gene might have potential for use as a gene marker for reducing wool fibre diameter.

Transcriptome Analysis of Improved Wool Production in Skin-Specific Transgenic Sheep Overexpressing Ovine β-Catenin.

β-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine β-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine β-catenin gene and that the β-catenin gene could be stably inherited. To study the molecular responses to high expression of β-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that β-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes.

Associations between variation in the ovine high glycine-tyrosine keratin-associated protein gene KRTAP20-1 and wool traits.

The keratin-associated proteins (KAPs) are important constituents of wool fibers. Of the many mammalian KAP genes (KRTAPs) identified, KRTAP20-1 has been described in humans, but it has not been described in any other species. A search of the sheep genome using the human KRTAP20-1 sequence revealed a homologous open reading frame on chromosome 1, which would encode a high glycine-tyrosine KAP. PCR-single-stranded conformational polymorphism (PCR-SSCP) analysis identified 8 different banding patterns representing 8 unique DNA sequences (named A to H). The sequences had highest similarity to the human KRTAP20-1 sequence, and this suggests that they are variants of ovine KRTAP20-1. Among these variants, a 12-bp insertion/deletion and 6 single nucleotide poly- morphisms (SNPs), including one 5' untranslated region (UTR) SNP, one 3' UTR SNP, and 2 nonsynonymous SNPs, were detected. Variant A was found to be associated with a decrease in mean fiber diameter, fiber diameter standard deviation, and prickle factor, whereas variant C was associated with increased greasy fleece weight and decreased wool yield. These associations persisted after adjusting for the effect of 2 nearby KRTAPs (KRTAP6-3 and KRTAP22-1) that have also been reported to associate with these wool traits. This suggests that variation in KRTAP20-1 affects wool yield and mean fiber diameter-associated traits, and that this effect is unlikely to be the result of the clustering of these KRTAPs on chromosome 1.

A keratin-associated protein (KAP) gene that is associated with variation in cashmere goat fleece weight

The keratin-associated proteins (KAPs) are key constituents of cashmere fibres. Of the many mammalian KAP genes (KRTAPs), KRTAP20-1 has been described in humans, but it has not been described in any other species. A search for comparable sequences in the goat genome using the human KRTAP20-1 sequence as a model, revealed an open reading frame on chromosome 1 that would encode a high glycine/tyrosine KAP. Using PCR-single stranded conformational polymorphism (PCR-SSCP) analysis as a screening tool, four different banding patterns were identified in 406 cashmere goats from the Longdong District of the Gansu Province in China. These represented four unique DNA sequences that were named A - D, and six single nucleotide polymorphisms (SNPs) were detected in them. The sequences had highest similarity to the human KRTAP20-1 sequence, and this suggests they are variants of caprine KRTAP20-1. The gene was expressed in secondary fibre follicles, but not in the longissimus dorsi muscle, and tissue from goat heart, liver, lungs and kidneys. Variant A was associated with crimped (un-straightened) fibre length and increased weight of harvested cashmere fibre per goat. Variant B was associated with a reduction in the weight of harvested fibre. Genotype AA goats had a greater harvested fibre weight and crimped fibre length, than genotype BB goats.

A nucleotide substitution in the ovine KAP20-2 gene leads to a premature stop codon that affects wool fibre curvature.

The keratin-associated proteins (KAPs) form a matrix that cross-links the main component of the wool fibre, the keratin intermediate filaments. They are therefore believed to play an important role in defining the physical and mechanical properties of the fibre. Although over 80 functional KAP genes (KRTAPs) have been identified in humans, only 29 have been found in sheep,1-3 All of these are polymorphic, and variation in some of these ovine KRTAPs has been associated with wool traits.1-6 Variation in the gene encoding the high glycine-tyrosine KAP20-2 protein (KRTAP20-2) has been reported to affect cashmere fibre weight traits.7 A blast search in the Ovine Genome Assembly Oar_v4.0 with the caprine KRTAP20-2 coding sequence (MF973462) identified on chromosome 1 an orthologous open reading frame (NC_019458.2:12334029_123134217), which has seven nucleotide substitutions relative to the caprine sequence. The sequence was located in a region that contains 13 other known KRTAPs (Fig. S1). A comparison of the sheep KRTAP20-2 with the human and caprine homologues and with other ovine HGT-KAP genes is shown in Fig. S2. Analysis of a 273-bp PCR fragment containing this open reading frame by single-stranded conformational polymorphism (SSCP) revealed two nucleotide sequence variants (Fig. S3), corresponding to a single nucleotide polymorphism (SNP) c.160A>T. This is the only difference between these sequences and the sheep v4.0 sequence, with the A nucleotide being found in that genome assembly sequence. Both sequences have been submitted to GenBank with accession nos. MH071391 and MH071392, for the A- and T-containing sequences respectively. This introduces a premature stop codon that removes the nine C-terminal amino acid residues. In spite of a high frequency of SNPs and insertion/deletions in KRTAPs,1 a nonsense mutation has not been reported to date. The PCR-SSCP analysis of 250 Merino × Southdown-cross sheep (from three sire lines) for which wool trait data were available, revealed that the A and T variants occur at a frequency of 71.8% and 28.2% respectively. Analyses using general linear models (GLMs) in minitab version 16 indicate that the wool fibre from homozygous AA sheep have a higher mean fibre curvature (MFC) than does the wool fibre from sheep carrying the TT genotype (Table 1). Theoretically, this effect may also reflect linkage between KRTAP20-2 and other genes in the same chromosome region. However, the effect observed for KRTAP20-2 differs from the associations observed for three nearby KRTAPs (KRTAP6-1, KRTAP22-1 and KRTAP6-3), which are at a distance of approximately 73 kb, 80 kb and 87 kb respectively from KRATP20-2 (Fig. S1). Specifically, previous research on Merino-cross sheep has revealed that KRTAP22-1 appears to affect wool yield,3 whereas KRTAP6-1 and KRTAP6-3 appear to affect mean fibre diameter (MFD) associated traits, but none of these genes were associated with the curvature.4, 8 Our data also suggest an association between variation in KRTAP20-2 and MFD (P = 0.091), but this trait in these Merino-cross sheep correlated with MFC (r = 0.490; P < 0.001) (Table S1). This study was financially supported by Ningxia Agricultural Breeding Program and the Lincoln University Gene-Marker Laboratory. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Evolution of Trichocyte Keratin Associated Proteins.

The major components of hair are keratins and keratin associated proteins (KRTAPs). KRTAPs form the interfilamentous matrix between intermediate filament bundles through extensive disulfide bond cross-linking with the numerous cysteine residues in hair keratins. A variable number of approximately100-180 genes compose the KRTAP gene family in mammals. KRTAP gene family members present a typical pattern of concerted evolution, and its evolutionary features are consistent with the evolution of mammalian hair. KRATP genes might be more important in determining the structure of cashmere fibers in domestic mammals like sheep and goats. KRTAP gene variants thus should provide information for improved wool by sheep and goat breeding.

Identification of the ovine keratin-associated protein 15-1 gene (KRTAP15-1) and genetic variation in its coding sequence

Keratin-associated proteins (KAPs) are the major component of wool fibres. The gene encoding the high sulphur-protein KAP15-1 has been described in humans and mice, but it has not been identified in sheep. A BLAST search of the Ovine Genome Assembly v4.0 using the coding sequence of caprine KRTAP15-1, identified a putative ovine KAP15-1 gene clustered within eleven other KAP genes on chromosome 1. PCR-Single Stranded Conformational Polymorphism (PCR-SSCP) analysis was used to screen amplicons derived from the gene in 732 sheep from six Chinese sheep breeds. Four different PCR-SSCP patterns, representing four unique DNA sequences (designated A-D) were detected. These sequences shared lower identity with other ovine KRTAP sequences, but the highest similarity was found with goat and human KRTAP15-1 sequences. This suggests that the sequences are variants of ovine KRTAP15-1. Among these four sequences, six nucleotide substitutions were identified in the coding region and four of the substitutions were non-synonymous. Breed differences in variant frequencies were observed. A was the most common variant in all the breeds. The next common variant differed in frequency between breeds. These results indicate that ovine KRTAP15-1 is variable and the differences between the Chinese breeds investigated suggest the variation might underpin variation in wool traits.

Identification of novel variants for KAP 1.1, KAP 8.1 and KAP 13.3 in South African goats

The yield and quality of animal fibres such as mohair, cashmere and cashgora are primarily influenced by the expression of various keratin associated protein genes, such as KAP 1.1, KAP 8.1, and KAP 13.3. Recent developments in molecular genetics provide the opportunity to characterize KAP genes at a base-pair level, which can lead to improved selection and genetic progress in mohair fibre production. PCR and sequencing technology was used to identify and characterize KAP 1.1, KAP 8.1, and KAP 13.3 in 108 goats, representing the South African Angora, Boer and Angora x Boer goat populations. The three genes showed varying degrees of polymorphism with between 4 and 18 alleles identified per locus. Some discrepancies in the current gene sequence of KAP 1.1 were discovered. Nineteen novel variants were identified in total, seven for KAP 1.1, one for KAP 8.1 and eleven for KAP 13.3. Observed heterozygosity values closely matched expected heterozygosity values, with Boer goats consistently having the lowest levels of expected and observed heterozygosity (approximately 0.5) for all three of the genes. The greatest variation for each gene existed between the Angora and Boer goat breeds, with FST values of 0.28, 0.13, and 0.24 for KAP 1.1, 8.1, and 13.3 respectively. Predominant alleles differed between the various populations, indicating the need for further research into possible allelic and fibre quality associations.

Wool Keratin-Associated Protein Genes in Sheep-A Review.

The importance of sheep’s wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses.

Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression

The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3–3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5′ flanking/5′ untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5′ upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays.

Effects of Food-Derived Collagen Peptides on the Expression of Keratin and Keratin-Associated Protein Genes in the Mouse Skin

Oral ingestion of collagen peptides (CP) has long been suggested to exert beneficial effects on the skin, but the molecular events induced by CP on the skin remain unclear. Here, we investigated the effects of oral CP administration on gene expression in hairless mouse skin and of prolyl-hydroxyproline (Pro-Hyp), a collagen-derived dipeptide, on gene expression in a coculture of mouse skin keratinocytes and fibroblasts. Using microarray analysis, we found that oral administration of CP to hairless mice for 6 weeks induced increased expression of Krtap and Krt genes in the skin. Annotation analysis using DAVID revealed that a group of the up-regulated genes, Gprc5d, Sprr2a1, Krt27 and Krtap16-7, is associated with the development of the epidermis and the hair cycle. In addition, the presence of Pro-Hyp (200 μ<smlcap>M</smlcap>) induced an increase in the expression of Krtap16-7, Krtap15, Krtap14 and Krtap8-2 in keratinocytes in coculture, partially resembling the in vivo result. The Pro-Hyp-induced up-regulation of these genes was not observed when keratinocytes were cultured without fibroblasts, suggesting that the presence of fibroblasts is essential for the effects of Pro-Hyp. Our study presents new insights into the effects of CP on the skin, which might link to the hair cycle.

Complex Changes in the Apoptotic and Cell Differentiation Programs during Initiation of the Hair Follicle Response to Chemotherapy

Chemotherapy has severe side-effects for normal rapidly proliferating organs, such as hair follicle, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in doxorubicin-treated hair follicles versus the controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL receptors 1/2), as well as of a large number of the keratin-associated protein genes were seen after doxorubicin treatment. Hair follicle apoptosis induced by doxorubicin was significantly inhibited by either TRAIL neutralizing antibody or caspase 8 inhibitor, thus suggesting a novel role for TRAIL receptor signaling in mediating doxorubicin-induced hair loss. These data demonstrate that the early phase of the hair follicle response to doxorubicin includes upregulation of apoptosis-associated markers, as well as substantial re-organization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies towards the design of novel approaches for management of chemotherapy-induced hair loss.

The sheep KAP8-2 gene, a new KAP8 family member that is absent in humans.

The keratin-associated proteins (KAPs) are fundamental components of hair and wool fibres, and are believed to in part be responsible for some of the properties of these fibres. KAPs can be divided into three groups: the high sulphur (HS) KAPs, the ultra-high sulphur (UHS) KAPs and the high glycine-tyrosine (HGT) KAPs. KAP8 is a HGT-KAP family and was believed to be coded for by a single gene in both humans and sheep. However, the recent identification of a KAP8-2 gene in goats led us to investigate whether a KAP8-2 gene exists in sheep.A BLAST search of the Ovine Genome Assembly v2.0 using the coding sequence of caprine KRTAP8-2 identified a homologous region on sheep chromosome 1 (OAR1:123005473_123005664; E = e-101). This region was clustered with a number of previously identified KAP genes including (in order from the centromere) KRTAP11-1, KRTAP7-1, KRTAP8-1, KRTAP6-2, KRTAP6-1, KRTAP13-3 and KRTAP24-1. PCR-SSCP analysis of the notional gene revealed two dissimilar PCR-SSCP banding patterns, representing two DNA sequences. A single nucleotide difference 21 bp upstream of the TATA box was identified. The two sequences did not have great homology with known ovine KRTAP sequences, but high sequence identity was found with KRTAP8-2 from goats and reindeer. These results suggest that sheep possess a KAP8-2 gene and that this gene is polymorphic.The notional KAP8-2 protein is comprised of 63 amino acid residues and is rich in glycine and tyrosine, but has a low cysteine content. In contrast to other HGT-KAPs, ovine KAP8-2 contains more acidic amino acid residues, and this would likely result in a lower isoelectric point (pI) of 6.3.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-528) contains supplementary material, which is available to authorized users.

Mammalian keratin associated proteins (KRTAPs) subgenomes: disentangling hair diversity and adaptation to terrestrial and aquatic environments.

BackgroundAdaptation of mammals to terrestrial life was facilitated by the unique vertebrate trait of body hair, which occurs in a range of morphological patterns. Keratin associated proteins (KRTAPs), the major structural hair shaft proteins, are largely responsible for hair variation.ResultsWe exhaustively characterized the KRTAP gene family in 22 mammalian genomes, confirming the existence of 30 KRTAP subfamilies evolving at different rates with varying degrees of diversification and homogenization. Within the two major classes of KRTAPs, the high cysteine (HS) subfamily experienced strong concerted evolution, high rates of gene conversion/recombination and high GC content. In contrast, high glycine-tyrosine (HGT) KRTAPs showed evidence of positive selection and low rates of gene conversion/recombination. Species with more hair and of higher complexity tended to have more KRATP genes (gene expansion). The sloth, with long and coarse hair, had the most KRTAP genes (175 with 141 being intact). By contrast, the “hairless” dolphin had 35 KRTAPs and the highest pseudogenization rate (74% relative to the 19% mammalian average). Unique hair-related phenotypes, such as scales (armadillo) and spines (hedgehog), were correlated with changes in KRTAPs. Gene expression variation probably also influences hair diversification patterns, for example human have an identical KRTAP repertoire as apes, but much less hair.ConclusionsWe hypothesize that differences in KRTAP gene repertoire and gene expression, together with distinct rates of gene conversion/recombination, pseudogenization and positive selection, are likely responsible for micro and macro-phenotypic hair diversification among mammals in response to adaptations to ecological pressures.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-779) contains supplementary material, which is available to authorized users.

Characterization and polymorphism of Keratin Associated Protein 1.4 gene in goats

Keratin-associated proteins (KAPs) are among the main structural components of the animal fibers and form semi-rigid matrix wherein the keratin intermediate filaments (KIFs) are embedded. Variation in the KAP genes has been reported to affect the structure of KAPs and hence fiber characteristics. As no information is available on this gene in Capra hircus therefore, present work was undertaken to characterize and explore the different polymorphic variants of KAP1.4 gene at DNA level in different breeds/genetic groups of goats of Kashmir. Cashmere (Changthangi, 30 animals) and non-Cashmere (Bakerwal and Kargil goats, 20 animals each) goats formed the experimental animals for the study. Single strand conformation polymorphism technique was employed for exploring variability at gene level. On exploring the size variability in KAP1.4 gene between Ovine and Caprine, it was concluded that sheep KAP1.4 gene has a deletion of 30 nucleotides. In comparison to published nucleotide sequences of sheep, goat sequences explored are differing at positions 174, 462 and 568 and at these positions “G”, “T” and “T” nucleotides are present in sheep, but are replaced by “A”, “C” and “C” respectively, in goats. By SSC studies, two genotypes were observed in each genetic group and in Bakerwal goats the genotypes were designated as A1A1 (0.40) and A1A2 (0.60) and were formed by two alleles A1 (0.70) andA2 (0.30). The different SSC patterns observed in Kargil goats were designated as B1B1 (0.35) and B1B2 (0.65) genotypes with frequencies of B1 and B2 alleles as 0.675 and 0.325, respectively. Similarly, two genotypes C1C1 (0.60) and C1C2 (0.40) were observed in Changthangi goats and the frequencies of C1 and C2 alleles were 0.80 and 0.20, respectively. These alleles were later confirmed by sequencing. The sequences of these alleles are available in NCBI under Acc. No's. JN012101.1, JN012102.1, JN000317.1, JN000318.1, JQ436929 and JQ627657. It was concluded that all the alleles observed in a breed were unique to the breed. The designated A1 and A2 alleles of Bakerwal goats differ from each other at positions 245 and the nucleotides observed were “C” or “A” and at position 605 of the nucleotide sequence “T” or “C”, were observed. The designated B1 and B2 alleles of Kargil goats differed from each other at positions 224, 374, 375 and 521. The nucleotides observed in two SSC pattern were C→G, A→G, G→A and T→C, respectively. The designated C1 and C2 alleles of Changthangi goats differed from each other at one position 440 with the change of “A”→“C”.Only two mutations C224G and G375A in Kargil goats resulted in change of the Cysteine (C)→Serine (S) and Alanine (A)→Threonine (T), respectively. The nucleotide sequences of KAP 1.4 gene in Bakerwal, Kargil and Changthangi goats showed 99.7% similarity with each other and 96.7% with sheep and 74.4% with mice. Average guard fiber length and diameter were 81.02±0.16mm and 67.53±0.97μm, respectively, and average down fiber length and diameter was 48.38±0.70mm and 13.32±0.29μm, respectively for Changthangi goats. Average guard fiber length and diameter were 63.51±4.52mm and 105.31±4.48μm, respectively for Bakerwal goats and 62.60±5.03mm and 107.18±2.30μm, respectively for Kargil goats. The effects of the observed genotypes on Cashmere fiber diameter, Cashmere fiber length in Changthangi goats and guard fiber length and guard fiber diameters in Changthangi, Kargil and Bakerwal goats were found to be non-significant (P>0.05).The nonsignificant association between the polymorphism and fiber attributes reported herein may be due to small sample size.

Identification and sequence analysis of the keratin-associated protein 24‐1 (KAP24-1) gene homologue in sheep

Keratin-associated proteins (KAPs) are major structural components of hair and wool fibres, and play a critical role in determining the properties of the fibre. While over 100 KAP genes that have been grouped into 27 KAP families have been identified in mammals, most homologues remain unidentified in sheep.A BLAST search of the Ovine Genome Assembly v2.0 using a human KRTAP24-1 coding sequence (NM_001085455), identified a putative ovine KAP24-1 gene clustered with six other known KAP genes on chromosome 1. The KAP24-1 gene was amplified from the genomic DNA of 260 New Zealand Romney-cross sheep and stem–loop conformational polymorphism (SLCP) analysis of the amplicons revealed four unique banding-patterns, representing four different DNA sequences. These sequences were not closely homologous with any known ovine KRTAP and the highest similarity was with KRTAP24-1 sequences from humans, cattle, dog, pig, Sumatran orangutan and northern white-cheeked gibbon. This suggests that the sequences were allelic variants of ovine KRTAP24-1. Among these four sequences, seven nucleotide substitutions in the coding region were identified and four of the substitutions were non-synonymous.The putative ovine KAP24-1 polypeptide consisted of 252 amino acids. While probably belonging to the high-sulphur KAP group, the polypeptide had a moderate level of cysteine, but a high content of serine and tyrosine. The polypeptide possesses two putative N-glycosylation sites and a number of residues that may be O-glycosylated and/or phosphorylated.

Characterization of the Human Hair Keratin–Associated Protein 2 (KRTAP2) Gene Family

Hair keratin-associated proteins (KRTAPs) are one of the major structural components of the hair shaft. Approximately 100 KRTAP genes have been identified in humans to date, with each of the genes classified into a number of families based on their sequence homology and the nature of the repeat structures. The biophysical features of KRTAPs, however, have remained largely unknown. In this study, we investigated the characteristics of the human KRTAP2 family members at the DNA, RNA, and protein levels. We initially found that these genes had various size polymorphisms that were mainly due to differences in the length of the 3'-noncoding sequences. Reverse transcriptase-PCR experiments further detected the presence of KRTAP2 transcripts in plucked human hairs. Using indirect immunofluorescence with an anti-KRTAP2 antibody, we found that there was a predominant expression of the KRTAP2 proteins in the keratinizing zone of the human hair shaft cortex. In addition, we showed that the KRTAP2 proteins interacted with each other and preferentially bound to hair keratins, but not to epithelial keratins. Finally, we determined that the head domain of the hair keratins was essential for the affinity to KRTAP2 proteins. Our results further enhance the crucial roles of KRTAPs in hair shaft keratinization in humans.

In vivo skin leptin modulation after 14 MeV neutron irradiation: a molecular and FT-IR spectroscopic study

This paper discusses gene expression changes in the skin of mice treated by monoenergetic 14 MeV neutron irradiation and the possibility of monitoring the resultant lipid depletion (cross-validated by functional genomic analysis) as a marker of radiation exposure by high-resolution FT-IR (Fourier transform infrared) imaging spectroscopy. The irradiation was performed at the ENEA Frascati Neutron Generator (FNG), which is specifically dedicated to biological samples. FNG is a linear electrostatic accelerator that produces up to 1.0 × 10(11) 14-MeV neutrons per second via the D-T nuclear reaction. The functional genomic approach was applied to four animals for each experimental condition (unirradiated, 0.2 Gy irradiation, or 1 Gy irradiation) 6 hours or 24 hours after exposure. Coregulation of a subclass of keratin and keratin-associated protein genes that are physically clustered in the mouse genome and functionally related to skin and hair follicle proliferation and differentiation was observed. Most of these genes are transiently upregulated at 6 h after the delivery of the lower dose delivered, and drastically downregulated at 24 h after the delivery of the dose of 1 Gy. In contrast, the gene coding for the leptin protein was consistently upregulated upon irradiation with both doses. Leptin is a key protein that regulates lipid accumulation in tissues, and its absence provokes obesity. The tissue analysis was performed by monitoring the accumulation and the distribution of skin lipids using FT-IR imaging spectroscopy. The overall picture indicates the differential modulation of key genes during epidermis homeostasis that leads to the activation of a self-renewal process at low doses of irradiation.