Sequence analysis of the human kallikrein gene locus identifies a unique polymorphic minisatellite element

Minisatellites are repetitive sequences of DNA that are present throughout the genome. Although the origin and function of these minisatellites is still unknown, they found clinical applications as markers of many diseases, including cancer. Also, they are useful tools for DNA fingerprinting and linkage analysis. Kallikreins are serine proteases that appear to be involved in many diseases including brain disorders and malignancy. We have recently characterized the human kallikrein gene locus on chromosome 19q13.4, which includes 15 kallikrein genes. In this study, we examined the kallikrein locus ( approximately 300 Kb) for all known repeat elements. About 50% of this genomic area is occupied by different repeat elements. We also identified unique minisatellite elements that are restricted to chromosome 19q13. Ten clusters of these minisatellites are distributed along the locus on either DNA strand. The clusters are located in the promoters and enhancers of genes, in introns, and in untranslated regions of the mRNA. Analysis of these elements indicates that they are polymorphic, thus they can be useful in linkage analysis and DNA fingerprinting. Our preliminary results indicate also that the distribution of the different alleles of these minisatellites might be associated with malignancy.     

Human prostate-specific antigen (APS) is a member of the glandular kallikrein gene family at 19q13

The amino acid sequence of human prostate-specific antigen (APS) suggests that it is a member of the glandular kallikrein subfamily of serine proteases. In the mouse, the kallikrein-like family is localized in a single locus on chromosome 7, while other serine proteases are distributed over a variety of different chromosomes. To investigate the physical relationship between the human kallikrein genes, we have used in situ hybridization and Southern analysis of a human x mouse somatic cell hybrid panel to map the APS gene to 19q13, concordant with the renal kallikrein KLK1 gene. This finding indicates that APS is a member of a human kallikrein-like gene family with analogous organization to that of the mouse.     

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.     

The human and mouse replication-dependent histone genes

The multigene family encoding the five classes of replication-dependent histones has been identified from the human and mouse genome sequence. The large cluster of histone genes, HIST1, on human chromosome 6 (6p21-p22) contains 55 histone genes, and Hist1 on mouse chromosome 13 contains 51 histone genes. There are two smaller clusters on human chromosome 1: HIST2 (at 1q21), which contains six genes, and HIST3 (at 1q42), which contains three histone genes. Orthologous Hist2 and Hist3 clusters are present on mouse chromosomes 3 and 11, respectively. The organization of the human and mouse histone genes in the HIST1 cluster is essentially identical. All of the histone H1 genes are in HIST1, which is spread over about 2 Mb. There are two large gaps (>250 kb each) within this cluster where there are no histone genes, but many other genes. Each of the histone genes encodes an mRNA that ends in a stemloop followed by a purine-rich region that is complementary to the 5' end of U7 snRNA. In addition to the histone genes on these clusters, only two other genes containing the stem-loop sequence were identified, a histone H4 gene on human chromosome 12 (mouse chromosome 6) and the previously described H2a.X gene located on human chromosome 11. Each of the 14 histone H4 genes encodes the same protein, and there are only three histone H3 proteins encoded by the 12 histone H3 genes in each species. In contrast, both the mouse and human H2a and H2b proteins consist of at least 10 non-allelic variants, making the complexity of the histone protein complement significantly greater than previously thought.     

The human myoglobin gene: a third dispersed globin locus in the human genome.

Human myoglobin is specified by a single gene. Unique sequence DNA probes were isolated from the cloned gene and used to test for the presence of the human myoglobin gene in a series of human rodent somatic cell hybrids containing various complements of human chromosomes. The myoglobin gene cosegregated with human chromosome 22. Somatic cell hybrids containing translocation chromosomes carrying part of chromosome 22 were used to locate the myoglobin gene to the region 22q11----22q13. The myoglobin gene is therefore not linked to the alpha-globin gene cluster on chromosome 16 or the beta-globin cluster on chromosome 11, and represents a third dispersed globin locus in the human genome.     

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.     

Regional localization of the hepatocyte growth factor (HGF) gene to human chromosome 7 band q21.1.

Hepatocyte growth factor (HGF) is a polypeptide involved in liver regeneration. Its amino acid sequence and gene structure are similar to those of coagulation-related serine proteases. We have used a cDNA clone of HGF and flow-sorted human chromosomes to assign this gene to chromosome 7. Fluorescence in situ hybridization of the HGF genomic clones to human metaphase chromosome spreads showed the localization of this gene to 7q21. Estimation of fluorescent signals relative to arbitrary reference points (ARPs) allowed further localization to 7q21.1.     

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.      

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.     

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.     

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.     

A genomic region encompassing a cluster of olfactory receptor genes and a myosin light chain kinase (MYLK) gene is duplicated on human chromosome regions 3q13-q21 and 3p13

The olfactory receptor (OR) multigene family is widely distributed in the human genome. We characterize here a new cluster of four OR genes (HGMW-approved symbols OR7E20P, OR7E6P, OR7E21P, and OR7E22P) on human chromosome 3p13 that is contained in an approximately 250-kb region. This region has been physically mapped, and a 106-kb portion containing the OR genes has been sequenced. All the OR sequences are disrupted by frameshifts and stop codons and appear to have arisen through local duplications. A myosin light chain kinase pseudogene (HGMW-approved symbol MYLKP) lies at one end of the OR gene cluster. Sequences spanning the entire region are also present at 3q13-q21, the site of the functional MYLK gene. This region duplicated locally before the divergence of primates, and the two paralogous copies were later separated to sites on either side of the centromere. This study increases our understanding of the evolution of the human genome. The 3p13 cluster is the first example of a tandem array of OR pseudogenes, and duplications of such clusters may account for the accumulation of a large number of pseudogenes in the human genome.     

Chromosomal distribution of the RTVL-H family of human endogenous retrovirus-like sequences

We have analyzed the chromosomal distribution of a large family of human endogenous retrovirus-like sequences termed RTVL-H. In situ hybridizations suggest that these sequences are found on all human chromosomes. These results also indicate that clusters or concentrations of RTVL-H elements may exist on chromosomes 1p and 7q. Southern blotting experiments using somatic cell hybrids containing either the human chromosome 3 or the X chromosome confirm the presence of multiple dispersed RTVL-H sequences on these two chromosomes. These experiments also demonstrate that distinct RTVL-H banding patterns can be detected for each chromosome. Thus, RTVL-H probes may be useful in genome mapping studies.     

A review and comparison of the murine alpha1-antitrypsin and alpha1-antichymotrypsin multigene clusters with the human clade A serpins

The major human plasma protease inhibitors, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, are each encoded by a single gene, whereas in the mouse they are represented by clusters of 5 and 14 genes, respectively. Although there is a high degree of overall sequence similarity within these groupings, the reactive-center loop (RCL) domain, which determines target protease specificity, is markedly divergent. The literature dealing with members of these mouse serine protease inhibitor (serpin) clusters has been complicated by inconsistent nomenclature. Furthermore, some investigators, unaware of the complexity of the family, have failed to distinguish between closely related genes when measuring expression levels or functional activity. We have reviewed the literature dealing with the mouse equivalents of human alpha(1)-antitrypsin and alpha(1)-antichymotrypsin and made use of the recently completed mouse genome sequence to propose a systematic nomenclature. We have also examined the extended mouse clade "a" serpin cluster at chromosome 12F1 and compared it with the syntenic region at human chromosome 14q32. In summarizing the literature and suggesting a standardized nomenclature, we aim to provide a logical structure on which future research may be based.     

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.     

Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells

The identification of transforming genes in human tumor cells has been made possible by DNA mediated gene transfer techniques. To date, it has been possible to show that most of these transforming genes are activated cellular analogues of the ras oncogene family. To better understand the relationship between these oncogenes and other human genes, we have determined their chromosomal localization by analyzing human rodent somatic cell hybrids with molecularly cloned human proto-oncogene probes. It was possible to assign N-ras to chromosome 1 and regionally localize c-K-ras-1 and c-K-ras-2 to human chromosomes 6pter-q13 and 12q, respectively. These results along with previous studies demonstrate the highly dispersed nature of ras genes in the human genome. Previous reports indicated that the c-myb gene also resides on chromosome 6. It has been possible to sublocalize c-myb to the long arm of chromosome 6 (q15-q21). The non-random aberrations in chromosomes 1, 6 and 12 that occur in certain human tumors suggest possible etiologic involvement of ras and/or myb oncogenes in such tumors.