O.Wesley McBride

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN) ☆

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.

Localization of two genes encoding plasma membrane Ca2(+)-transporting ATPases to human chromosomes 1q25-32 and 12q21-23.

Human plasma membrane Ca2(+)-ATPase (PMCA) isoforms are encoded by at least four separate genes and the diversity of these enzymes is further increased by alternative splicing of transcripts. Cloned cDNAs for two of these isoforms have been used as probes to localize chromosomally the human PMCA1 (ATP2B1) gene to 12q21-q23 and PMCA4 (ATP2B2) to 1q25-q32. These results were obtained by three independent methods, including Southern analysis of human-rodent somatic cell hybrids, in situ hybridization of human metaphase spreads, and genetic linkage analysis in the CEPH pedigrees. High-frequency RFLPs detected at each locus were used in these linkage analyses. No evidence was obtained for multiple copies of the gene at either locus. A cross-hybridizing sequence was detected with PMCA4 probes on Xq13-qter at low stringency. Further studies are required to determine whether this X-chromosomal sequence represents a third member of the PMCA gene family.

Heparan sulfate proteoglycan of human colon: Partial molecular cloning, cellular expression, and mapping of the gene (HSPG2) to the short arm of human chromosome 1

We have determined the sequence of two overlapping cDNA clones encoding a portion of the human heparan sulfate proteoglycan core protein (HSPG2) from a human colon library. The cDNA clones encompassed 1.34 kb of nucleotide sequence and showed approximately 85% homology to the murine heparan sulfate proteoglycan of the EHS tumor, BPG-5. The deduced amino acid sequence revealed an identity of 87% between the human and the murine species. Of the 57 different amino acids encoded in the human clones, 20 were substituted with a similar amino acid. Among the specific features that were highly conserved was the 32 cysteine residues with a distinctive repeat pattern characteristic of epidermal growth factor. Northern blotting revealed a single, approximately 12 kb transcript in a variety of cells including human colon carcinoma, endothelial, and fibroblastic cells. The size of this transcript correlated with the estimated molecular weight (approximately 400 kDa) of the protein core precursor. Southern blot analyses of DNA from 120 human/rodent somatic cell hybrids, including subclones with specific translocations or spontaneous breaks of human chromosome 1, demonstrated the presence of HSPG2 gene on the telomeric region of the short arm of chromosome 1 (1p34-pter). Two polymorphisms with TaqI and EcoRI restriction endonucleases, respectively, were also detected. The localization of this proteoglycan locus in the human genome and the availability of new RFLPs provide the tools for future studies of human diseases where the HSPG2 proteoglycan gene is suspected to be involved.

Localization of PGI (biglycan, BGN) and PGII (decorin, DCN, PG-40) genes on human chromosomes Xq13-qter and 12q, respectively.

The genes for PGI (biglycan, BGN) and PGII (decorin, DCN) have been assigned to human chromosomes X and 12 by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these related small proteoglycans. Regional localization of BGN to Xq13-qter and DCN to 12p12.1-qter was also obtained by examining hybrids containing spontaneous breaks or well-characterized translocations involving chromosomes X and 12. Biglycan (BGN) is a single-copy gene about 6 kb in length. Hybridization with subfragment cDNA probes suggests the presence of two copies of the decorin (DCN) gene, or related sequences, at the locus on chromosome 12, although there is no evidence for function of more than one DCN gene. Efforts to detect restriction fragment length polymorphisms with these probes were unsuccessful.

The human calcitonin gene is located on the short ARM of chromosome 11

By molecular hybridization of human calcitonin cDNA probes to DNA from human-rodent hybrid cells containing identified human chromosomes, we have mapped the human calcitonin gene to the short arm of chromosome 11. This location has been confirmed by in situ hybridization, which further localized the calcitonin gene to region 11p13-15. The significance of this region regarding gene linkage and possible markers for inherited cancers is discussed.

Human clathrin heavy chain (CLTC): Partial molecular cloning, expression, and mapping of the gene to human chromosome 17q11-qter

The nucleotide sequence of a 916-bp human cDNA clone isolated from a human colon lambda gt11 cDNA library was determined. Sequence analysis showed this cDNA to have 88% homology to the nucleotide sequence of the heavy chain of rat clathrin. The deduced amino acid sequence was 98.7% identical to the rat sequence, a change of only four amino acids. The mRNA identified in both human and rat cells with the human clathrin clone revealed transcripts of approximately 6.5 kb, which is consistent with the predicted 180 kDa molecular weight of the clathrin heavy chain. Southern analysis of human/rodent somatic cell hybrids localized the human clathrin heavy chain gene (CLTC) to chromosome 17. Additional analyses using panels of human/rodent somatic cell hybrids with specific chromosomal translocations and deletions mapped the human clathrin heavy chain gene locus to 17q11-qter.

Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1)

Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences (Tarbp2-rs1, Tarbp2-rs2). The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells.

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

SummaryA collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.

Cloning of the cDNA encoding human histidine decarboxylase from an erythroleukemia cell line and mapping of the gene locus to chromosome 15

The biogenic amine histamine is an important modulator of numerous physiological processes, including neurotransmittance, gastric acid secretion and smooth muscle tone. The biosynthesis of histamine is catalyzed by the enzyme, L-histidine decarboxylase (HDC). We have previously reported the cloning and sequence of the cDNA encoding rat HDC. Utilizing the rat HDC cDNA as probe the full-length cDNA encoding human HDC was identified and characterized. The encoded protein of 662 amino acid residues has a molecular weight of 74,148. Homology comparisons of the deduced amino acid sequence with rat HDC and dopa decarboxylases from three species have revealed highly related regions. These comparisons have identified domains of amino acid decarboxylases that are highly conserved and are likely important for enzyme-substrate interaction. A dissimilar region in human and rat HDC primary translated protein near the C-terminus would appear not to be important for catalysis and may be removed by proteolysis. This processing phenomenon could be in part responsible for regulation of HDC activity. The human HDC cDNA was also utilized to map the chromosomal location of the human HDC gene locus (HDC). Analysis of human-rodent cell hybrids revealed that the HDC gene segregates with Chromosome 15. No restriction length polymorphisms in the human population were detected after cleavage of the DNAs with 12 restriction endonucleases.

The Cornified Cell Envelope: Loricrin and Transglutaminases

The cornified cell envelope (CE) of terminally differentiated human epidermis is a complex structure consisting of several defined protein constituents. The CE is the most insoluble component of the epidermis due to crosslinking by disulfide bonds as well as isodipeptide bonds that are formed by the action of transglutaminases (TGases). We have recently determined that loricrin is the major component of CE. We now have isolated and characterized its gene and showed that it has a simple structure with a single intron. We also show that the loricrin gene maps to position 1q21, which, coincidentally, is similar to the location of the profilaggrin and involucrin genes. Human loricrin in 26 kDa and consists of three long glycine‐serine‐cysteine rich sequence domains that contain quasi‐repeating peptides and which form the novel glycine loop motif. These are interspersed by lysine + glutamine rich domains involved in isodipeptide crosslinks. The glycine loops are thought to be involved in organization of epidermal proteins and maintenance of the flexibility of the epidermis. By use of PCR analyses, we have found that human loricrin consists of two allelic size variants, due to sequence variations in the second glycine loop domain only, and these variants segregate in the human population by normal Mendelian mechanisms. Futhermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. In order to study the expression and role of TGases in the formation of CE, we have isolated and sequenced cDNA and genomic clones encoding the TGasel enzyme. The deduced complete sequence has 816 amino acid residues of 90 kDa. It shares 49–53% sequence homology with the other TGases of known sequence but has unique sequences in its amino‐ and carboxyltermini. The TGM1 gene has been isolated and characterized. It is 14.1 kbp long, contains 15 exons and is similar in structure to other TGase genes. It was located to chromosome position 14q11. It will now be possible to further study the regulation of the expression of the loricrin and TGase1 gene systems in normal and abnormally keratinizing epidermis.