Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.     

Identification of two novel clusters of ultrahigh-sulfur keratin-associated protein genes on human chromosome 11

We analyzed two novel clusters of keratin-associated protein (KAP) genes on human chromosome 11 (11p15.5 and 11q13.5) in which we identified two known human KRTAP5 genes, KerA (=KRN1) and KerB, and nine novel KRTAP5 family genes. RT-PCR analysis of these KAP genes showed preferential expression in human hair root, suggesting these gene products are required for hair formation. Based on the deduced amino acid sequences, all these KAP proteins were classified into an ultrahigh-sulfur (UHS) type KAP with high cysteine content (> 30 mol%). These KAPs also showed high glycine and serine contents (average 24.30 and 21.13 mol%, respectively), distinguishing from other UHS/HS KAP families located on human chromosomes 17 and 21. Dot-matrix analysis revealed a significant similarity between these two KAP gene clusters. We postulated a mechanism by which these two KAP gene clusters are generated via genomic duplication of a primordial gene cluster followed by genetic modification during evolution.     

角蛋白HGTP及其对羊毛发育的影响

羊毛的主要成分是角蛋白,其组分高甘氨酸-酪氨酸蛋白(HGTP)家族成员KAP6、KAP7和KAP8基因表达对羊毛细度和弯曲等特性具有重要影响。本文从羊毛的组成、角蛋白的生物学特征以及HGTP基因定位和表达对细度的影响等方面进行了综述,旨在为羊毛发育调控研究提供理论参考。     

Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.     

Polymorphisms in the human high sulfur hair keratin-associated protein 1, KAP1, gene family

Hair fiber differentiation and maturation involves the close interaction between hair keratins and their associated proteins, KAPs. Recently, a cluster of seven human KAP multigen families has been identified on chromosome 17q12-21 among which were four hKAP1 genes (hKAP1.1B, hKAP1.3, hKAP1.4, and hKAP1.5). In addition, there were previous as well as recent reports on four additional hKAP1 genes (hKAP1.1A, hKAP1.2, hKAP1.6, and hKAP1.7) with unknown chromosomal location. In this study, we have analyzed these eight hKAP1 genes in unrelated Japanese and Caucasian individuals and discovered that hKAP1.1A, hKAP1.6, and hKAP1.7 represent size polymorphisms of the hKAP1.1B gene. In addition, we show that hKAP1.2 as well as three hitherto unknown genes (hKAP1.8A, hKAP1.8B, and hKAP1.9) are size polymorphisms of the hKAP1.3 gene. In contrast, no polymorphic alleles were found for the hKAP1.4 and hKAP1.5 genes. We provide evidence that the polymorphic hKAP1.1B and hKAP1.3 alleles arose mainly by intragenic deletion and/or duplication events of distinct pentapeptide repeats typical for hKAP1 genes. We also demonstrate the occurrence of both frequent and rare population-specific hKAP1.1B and hKAP1.3 alleles, which were obviously generated after the divergence of the Caucasian and Japanese lineage. In addition, by means of a pan-hKAP1 antibody, we confirm the previous hKAP1 family mRNA localization data in the middle to upper cortex of the human anagen hair follicle.     

Genetic and biochemical aspects of keratin synthesis by hair follicles

In this review article the data about synthesis and gene regulation of keratin by hair follicles have been summarized. It has been shown that both differentiation of hair follicle matrix cells and normal growth of hair require the coordinated activities of the genes encoding structural proteins. The keratin genes are clustered in families and are usually 5-10 kb in the genome. The separate clusters of two keratin IF gene families and five KAP gene families have been discovered and some of them have been mapped. The close relation between these clusters suggests that the "global" regulatory domains might govern their expression.     

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.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

优质细毛羊羊毛细度的候选基因分析

采用PCR-SSCP的分子标记技术, 选择编码羊毛纤维组成蛋白基因中的KAP1.1、KAP1.3的部分序列、KAP6.1的外显子区作为候选基因, 通过对其多态性的研究, 探索将该基因作为候选基因来间接选择羊毛细度性状的可行性。得出其中在角蛋白辅助蛋白的多基因家族中的高硫蛋白辅助蛋白基因(KAP1.1、KAP1.3)中, 位点W08667与羊毛细度有显著的相关性(P<0.05)。在甘氨酸酪氨酸角蛋白辅助蛋白中, 外显子位点W06933的AA基因型和BB基因型与羊毛细度之间有显著的相关性(P<0.05)。     

Identification of a zeta-crystallin (quinone reductase)-like 1 gene (CRYZL1) mapped to human chromosome 21q22.1

To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA. One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified. The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids. A search of GenBank revealed that the clone has a high degree of homology to zeta-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1. Thus a new gene was named CRYZL1 (zeta-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene. The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.     

Complementary physical and genetic techniques map the vinculin (VCL) gene on chromosome 10q.

Vinculin is a cytoskeletal protein component of adherens type cell junctions. The gene had been mapped to 10q11.2-qter. We have used a combination of physical and genetic mapping techniques to refine this localization. Hybridization of the vinculin cDNA probe, HV1, to a human-rodent somatic hybrid panel initially suggested a position of either 10q11.2 or 10q22.1-10q23. Genetic recombination mapping in three-generation families with multiple endocrine neoplasia type 2 (MEN2) indicated a position distal to D10S22 (10q21.1) in 10q22.1-10q23. This was confirmed by hybridization of the vinculin cDNA to flow-sorted translocation derivative chromosomes containing the q21-qter portion of chromosome 10. We conclude that the vinculin locus maps in 10q22.1-q23, distal to D10S22.     

An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains

Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1-linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C-->T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called "puratrophin-1" (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the spectrin repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1--normally expressed in a wide range of cells, including epithelial hair cells in the cochlea--was aggregated in Purkinje cells of the chromosome 16q22.1-linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5' untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5' UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans.     

Restriction fragment length polymorphism analysis and assignment of the metalloproteinases stromelysin and collagenase to the long arm of chromosome 11

Collagenase and stromelysin are two metalloproteinases produced mainly by connective tissue cells and involved in the breakdown of the extracellular matrix. cDNA clones for both of these genes have been isolated and sequencing has shown them to be closely related. The collagenase and stromeylsin cDNA clones have been used to assign these genes to the long arm of chromosome 11 in the regions 11q21-22.1 and 11q22.2-22.3, respectively. This has been achieved using somatic cell hybrids and in situ hybridization. In addition a Taq1 restriction fragment length polymorphism has been demonstrated using the stromelysin cDNA.     

Genome-wide scan and fine-mapping linkage study of androgenetic alopecia reveals a locus on chromosome 3q26

Androgenetic alopecia (AGA, male pattern baldness) is the most common form of hair loss. The origin of AGA is genetic, with the X chromosome located androgen receptor gene (AR) being the only risk gene identified to date. We present the results of a genome-wide linkage study of 95 families and linkage fine mapping of the 3q21-q29, 11q14-q25, 18p11-q23, and 19p13-q13 regions in an extended sample of 125 families of German descent. The locus with strongest evidence for linkage was mapped to 3q26 with a nonparametric linkage (NPL) score of 3.97 (empirical p value = 0.00055). This is the first step toward the identification of new susceptibility genes in AGA, a process which will provide important insights into the molecular and cellular basis of scalp hair loss.     

Characterization and expression analysis of KAP7.1, KAP8.2 gene in Liaoning new-breeding cashmere goat hair follicle

Keratin-associated protein is one of the major structural proteins of the hair, whose content in hair has important effect on the quality of cashmere. In order to study the relationship between HGTKAP gene expression and cashmere fineness, the quantitative real-time RT–PCR (qRT–PCR) was firstly used to detect the levels of KAP7.1, KAP8.2 gene expression in the primary and secondary hair follicles; semi-quantitative RT–PCR was used to detect whether KAP7.1, KAP8.2 gene are expressed in heart, liver, spleen, lung, kidney tissues; and in situ hybridization(ISH) to detect KAP7.1 gene expression location. qRT–PCR result showed that the expression of both KAP7.1 and KAP8.2 gene in the secondary hair follicles are significantly higher than that in the primary follicles, relative quantitative analysis obtained that KAP7.1 was 2.28 times, while KAP8.2 was 2.71 times. Semi-quantitative RT–PCR results revealed that KAP 7.1 and KAP8.2 mRNA were not detected in the heart, liver, spleen, lung and kidney tissues, demonstrating that KAP7.1 and KAP8.2 were specially expressed in hair follicles, participating in hair formation. Moreover, KAP7.1 gene has a strong expression in the cortical layer, inner root sheath of the primary follicles and the cortical layer, inner root sheath and hair matrix of the secondary hair follicles by ISH analysis. Taken together, the evidence presented here indicated that in the formation of cashmere and wool, differential expression of these two genes in the primary and secondary hair follicles may have an important role in regulating the fiber diameter.