Myung Soo Lyu

Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping.

Bone sialoprotein (BSP) is a small ( -70 ,000 Mr), highly post-translationally modified protein that is an abundant noncollagenous component of the bone matrix (Fisher et al. 1983). With a combination of immunohistochemistry and in situ hybridization, it has been shown that in the human and rat, BSP is produced by bone-forming cells and mature osteoblasts, recently entombed osteocytes, osteoclasts, and hypertrophic chondrocytes within the growth plate (Bianco et al. 1991; Chen et al. 1991). The only nonskeletal source that shows expression of BSP is the mononucleated trophoblast cells and their multinucleated syncytia in the developing placenta. Studies in the rat also showed that BSP is expressed in the odontoblasts of the developing mandibular incisor (Chen et al. 1992b). In an in vitro model of differentiating fetal bovine osteoblasts, BSP mRNA was increased 120-fold at a time point that corresponded to a dramatic formation of mineralizing nodules and trabecular-like structures (Ibaraki et al. 1992). This localization of BSP to sites of new mineral formation implicates BSP in this process. To determine the structure and describe the expression of BSP in the mouse, we have cloned and sequenced mouse BSP cDNA. In this study we report the nucleotide sequence of the murine cDNA, its deduced amino acid sequence, and we report the mapping of the mouse BSP gene (Ibsp) to Chromosome (Chr) 5. Mouse BSP cDNA was cloned from a 16-day mouse embryo cDNA library in a lambda SHlox vector (Novogen Cat #69641-1) with a 1.8-kb insert of rat cDNA (Oldberg et al. 1988a) as a probe under moderate hybridization conditions (Sambrook et al. 1989). One clone contained an insert size of 1.3 kb and was sequenced completely through-

Genetic variations at loci involved in the immune response are risk factors for hepatocellular carcinoma.

Primary liver cancer is the third most common cause of cancer‐related death worldwide, with a rising incidence in Western countries. Little is known about the genetic etiology of this disease. To identify genetic factors associated with hepatocellular carcinoma (HCC) and liver cirrhosis (LC), we conducted a comprehensive, genome‐wide variation analysis in a population of unrelated Asian individuals. Copy number variation (CNV) and single nucleotide polymorphisms (SNPs) were assayed in peripheral blood with the high‐density Affymetrix SNP6.0 microarray platform. We used a two‐stage discovery and replication design to control for overfitting and to validate observed results. We identified a strong association with CNV at the T‐cell receptor gamma and alpha loci (P < 1 × 10−15) in HCC cases when contrasted with controls. This variation appears to be somatic in origin, reflecting differences between T‐cell receptor processing in lymphocytes from individuals with liver disease and healthy individuals that is not attributable to chronic hepatitis virus infection. Analysis of constitutional variation identified three susceptibility loci including the class II MHC complex, whose protein products present antigen to T‐cell receptors and mediate immune surveillance. Statistical analysis of biologic networks identified variation in the “antigen presentation and processing” pathway as being highly significantly associated with HCC (P = 1 × 10−11). SNP analysis identified two variants whose allele frequencies differ significantly between HCC and LC. One of these (P = 1.74 × 10−12) lies in the PTEN homolog TPTE2. Conclusion: Combined analysis of CNV, individual SNPs, and pathways suggest that HCC susceptibility is mediated by germline factors affecting the immune response and differences in T‐cell receptor processing. (HEPATOLOGY 2010)

Genetic mapping of six mouse peroxiredoxin genes and fourteen peroxiredoxin related sequences

Organisms living in aerobic environments require mechanisms that prevent or limit cellular damage caused by reactive oxygen species (O2 , H2O2, and HO) that arise from the incomplete reduction of oxygen during respiration. Alternatively, damage can result from exposure to external agents such as light, radiation, redox-cycling drugs, or stimulated host phagocytes (Sies 1993; Halliwell and Gutteridge, 1989). The reactive oxygen species cause damage to all major classes of biological macromolecules leading to protein oxidation, lipid peroxidation, and DNA base modifications and strand breaks. To guard against these destructive processes, organisms have developed a battery of antioxidant defenses (Halliwell and Gutteridge 1989; Amstad et al. 1991). The preventive antioxidant systems include enzymes that decompose peroxides and superoxide anion and compounds that sequester metal ions. These types of antioxidants reduce or eliminate the generation of free radicals. Chain-breaking antioxidants, such as ascorbate and a-tocopherol, scavenge transient free radicals and inhibit the attack of these reactive species on biological targets. We have previously purified a 25-kDa enzyme from yeast that prevents damage induced by the thiol oxidation system but not by the ascorbate oxidation system, despite the fact that the degree of oxidative stress is similar for the two systems as judged by the comparable extent of induced inactivation of glutamine synthetase (Kim et al. 1988). Thus, we originally named this protein thiolspecific antioxidant (TSA). Although the exact nature of the oxidant eliminated by TSA was not known at that time, the importance of TSA as an antioxidant was readily apparent as the application of oxidative pressure to yeast resulted in an increase in the synthesis of TSA, and TSA protein constituted 0.7% of total soluble protein from yeast grown aerobically (Kim et al. 1989). Yeast TSA gene was cloned and sequenced (Chae et al. 1993). It shows no significant homology to any known catalase, superoxide dismutase, or peroxidase enzymes. This lack of homology is consistent with the observation that TSA does not possess catalytic activity characteristic of conventional antioxidant enzymes. A yeast mutant that cannot produce TSA was constructed by homologous recombination (Chae et al. 1993). The mutant and wild-type trains grew at equal rates under anaerobic conditions. However, under aerobic conditions, especially under oxidative stress, the growth rate of mutant yeast was significantly lower than that of wild-type yeast. A database search revealed a number of proteins from a variety organisms that show similarity to TSA (Chae et al. 1994b). These homologous proteins have now been named the peroxiredoxin (PRDX) family. We recently demonstrated that the antioxidant activity of TSA is attributable to its ability to reduce H 2O2. The apparent specific requirement for a thiol for antioxidant function was due to the fact that an intermolecular disulfide linkage of oxidized TSA can be reduced by a thiol but not by ascorbate. We have shown that thioredoxin (TRX) is the physiological electron donor for the reduction of TSA (Chae et al. 1994a). TSA was thus the first peroxidase to be identified for which TRX is the immediate electron donor, and it was therefore renamed TRX peroxidase (TPX). Despite this finding, the TSA homologs (the PRDX gene family) were not termed the TPX family because not all members use TRX as the hydrogen donor. For example, enteric bacteria homolog AhpC and trypanosomatid homolog C22 receive electron from AhpF and C30 proteins, respectively, for the reduction of H2O2 (Jacobson et al. 1989; Montemartini et al. 1998). Furthermore, mammalian PRDX-V is capable of reducing H 2O2 in the presence of dithiothreitol but not in the presence of TPX (Kang et al. 1998). The complete amino acid sequences of 15 mammalian members of the PRDX family have been determined: six (PAG, NKEFA, NKEFB, TSA, MER5, and AOE372) from human, six (MSP23, OSF3, TSA, MER5, AOP1, and AOP2) from mouse, two (TSA and HBP23) from rat, and one (SP22) from cow. With the exception of TSA, all mammalian PRDX proteins were initially characterized without reference to antioxidant function (Chae et al., 1994c; Prosperi et al. 1993; Shau et al. 1994; Yamamoto et al. 1989; Jin et al. 1997). Among the six reported human PRDX sequences, there are four distinct human PRDX proteins: PAG/ NKEFA, TSA/NKEFB, MER5, and AOE372. Similarly, the six reported mouse sequences actually correspond to four distinct proteins: MSP23/OSF3, TSA, MER5/AOP1, and AOP2. The four human PRDX proteins show only 60–80% sequence identity to each other, but all except AOE372 share >90% identity with a corresponding mouse homolog. Each of the two rat proteins (HBP23 and TSA) and bovine SP22 show >92% sequence identity to one of the human or mouse proteins. Therefore, the mammalian PRDX proteins can be grouped into one of five types: PRDX-I, represented by PAGA; PRDX-II, represented by TSA; PRDX-III, represented by MER5; PRDX-IV, represented by AOE372; and * Present address:Laboratory of Population Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Mapping of the human and mouse bone sialoprotein and osteopontin loci.

~School of Biological Sciences and Departments of Dental Medicine and Surgery, 3.239, Stopford Building, University of Manchester, Manchester, M13 9PT, UK 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA 3Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 4Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA

Characterization of a Polytropic Murine Leukemia Virus Proviral Sequence Associated with the Virus Resistance Gene Rmcf of DBA/2 Mice

ABSTRACT The DBA/2 mouse Rmcf gene is responsible for in vivo and in vitro resistance to infection by the polytropic mink cell focus-forming (MCF) virus subgroup of murine leukemia viruses (MLVs). Previous studies suggested that Rmcf resistance is mediated by expression of an interfering MCF MLV envelope (Env) gene. To characterize this env gene, we examined resistance in crosses between Rmcfr DBA/2 mice and Mus castaneus, a species that lacks endogenous MCF env sequences. In backcross progeny, inheritance of Rmcf resistance correlated with inheritance of a specific endogenous MCF virus env-containing 4.6-kb EcoRI fragment. This fragment was present in the DBA/2N substrain with Rmcf-mediated resistance but not in virus-susceptible DBA/2J substrain mice. This fragment contains a provirus with a 5′ long terminal repeat and the 5′ half of env; the gag and pol genes have been partially deleted. The Env sequence is identical to that of a highly immunogenic viral glycoprotein expressed in the DBA/2 cell line L5178Y and closely resembles the env genes of modified polytropic proviruses. The coding sequence for the full-length Rmcf Env surface subunit was amplified from DNAs from virus-resistant backcross mice and was cloned into an expression vector. NIH 3T3 and BALB 3T3 cells stably transfected with this construct showed significant resistance to infection by MCF MLV but not by amphotropic MLV. This study identifies an Rmcf-linked MCF provirus and indicates that, like the ecotropic virus resistance gene Fv4, Rmcf may mediate resistance through an interference mechanism.

Genetic mapping of the human and mouse phospholipase C genes

To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human x rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8.

Characterization of the mouse gene, human promoter and human cDNA of TSCOT reveals strong interspecies homology.

The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon alpha inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.

Secretion of a murine retroviral Env associated with resistance to infection.

Fv4 is an endogenous defective murine leukaemia virus (MuLV) which expresses high levels of an envelope protein (Env) closely related to that of the ecotropic class of MuLVs. Mice bearing the natural Fv4 gene or a transgenic version are resistant to infection by ecotropic MuLVs. Fv4 mice secrete the surface peptide (SU) of the Fv4 Env in their serum and this secreted Env can block infection of NIH3T3 cells. To study the secretion of Fv4, we metabolically labelled cells expressing Fv4 Env or Env from infectious MuLVs and followed synthesis, glycosylation, proteolytic processing and secretion of Env species. We found no difference in the kinetics of synthesis or processing of Fv4 Env compared to the envelopes of infectious MuLVs, but Fv4 Env associated more weakly with its transmembrane anchor and was shed from the surface of cells.

Genetic mapping of the gene encoding cysteine string protein

Species: Pig (Sus scrofa domestica) Locus name: Nuclear Factor I/CTF Locus symbol: NFI/CTF Map position: 2q12-13 Method of mapping: Fluorescence in situ hybridization (FISH) Molecular reagents: An EMBL3A phage containing 40 kb of pig NF1/CTF gene [1] was biotinylated and detected by avidin-FITC. Previously identified homologs: NFIC maps to human Chromosome (Chr) 19p13 [2]. Discussion: Nuclear factor NFI/CTF is a member of a family of dimeric DNA-binding proteins. Its recognition sequence [TGG(C/ A)Ns] is present in a number of DNA viruses and in mammalian and avian genomes [see 3]. The proteins are involved in cellular transcription regulation and adenovirus transcription initiation. In the human genome, NFI-A,B,C and NFI-X genes have been identified and mapped [2,4]. NFI-C and NFI-X are located in close proximity on human Chr 19p 13, whereas NFI-A and -B genes map to lp31.2-31.3 and 9p24.1, respectively [2]. In the mouse, Nfix has been assigned to 8C1-2 [7]. Of all mammalian nuclear factor I genes isolated to date, the genomic structure of the porcine gene has been characterized in most detail [5]. Gross regional homologies between human and porcine genomes have recently been demarcated by Zoo-FISH [6,q]. These studies revealed the presence of two conserved syntenic segments between HSA19 and SSC2cen-q21 and SSC6cen-q21 [7]. Comparative gene mapping data suggest that the p and q arms of HSA 19 are each conserved as a single block in artiodactyl genomes [8]. The mapping of the porcine NFI/CTF gene to SSC2q 12-13 corroborates the conserved synteny between HSA19p and the homologous segment at SSC2cen-q21, which has also been demonstrated by a recent forward and reverse Zoo-FISH approach [9].

Genetic mapping of gene encoding the large subunit of replication factor C (A1-p145) to mouse Chromosome 5

Species: mouse Locus names: collagen 4cO and collagen 4c~2 Locus symbols: Col4al, Col4a2 Map position: Chr 8. Fcer2a-(0.9 + 0.9)-Pcp2-(3.8 + 1.9)Col4al, Col4a2-3.2 + 1.8-Plat, Polb, Ram1 (Fig. 1). Method of mapping: Determined from analysis of the progeny of the cross (NFS/N x Mus spretus) x M. spretus or C58/J [1]. Molecular reagents: Col4al was typed by Southern blotting using as probe a 1.1-kb SmaI-XbaI fragment of the enhancer region of this gene [2]. All additional markers were typed as described previously [3]. Allele detection: BamHI digestion produced fragments of 19.1 kb in M. spretus and 17.0 kb in NFS/N and C58/J. Previously identified homologs: Human COL4A1 and COL4A2 have been mapped to 13q34 [4,5]. Discussion: Collagen IV is a heterotrimer composed of two c~1 chains and one ct2 chain. The mouse genes for these two alpha chains are separated by 130 bp and arranged in a head-to-head formation. These genes use a bidirectional promoter located between the two genes. The enhancer for this promoter (and the probe used in this study) is located in the first intron of the cd gene