A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Assignment of 128 genes localized on human chromosome 14q to the IMpRH map.

To provide a gene-based comparative map and to examine a porcine genome assembly using bacterial artificial chromosome-based sequence, we have attempted to assign 128 genes localized on human chromosome 14q (HSA14q) to a porcine 7000-rad radiation hybrid (IMpRH) map. This study, together with earlier studies, has demonstrated the following. (i) 126 genes were incorporated into two SSC7 RH linkage groups by CarthaGene analysis. (ii) In the remaining two genes, TOX4 linked to TCRA located in SSC7 by two-point analysis, whereas SIP1 showed no significant linkage with any gene/marker registered in the IMpRH Web Server. (iii) In the two groups, the gene clusters located from 19.9 to 36.5 Mb on HSA14q11.2-q13.3 and from 64.0 to 104.3 Mb on HSA14q23-q32.33 respectively were assigned to SSC7q21-q26. (iv) Comparison of the gene order between the present RH map and the latest porcine sequence assembly revealed some inconsistencies, and a redundant arrangement of 16 genes in the sequence assembly.

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11.

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

Assignment of 115 genes from HSA9 and HSA14 to SSC1q by RH mapping to generate a dense human-pig comparative map.

A large number of significant QTL for economically important traits including average daily gain have been located on SSC1q, which, as shown by chromosome painting, corresponds to four human chromosomes (HSA9, 14, 15 and 18). To provide a comprehensive comparative map for efficient selection of candidate genes, 81 and 34 genes localized on HSA9 and HSA14 respectively were mapped to SSC1q using a porcine 7000-rad radiation hybrid panel (IMpRH). This study, together with the cytogenetic map (http://www2.toulouse.inra.fr/lgc/pig/cyto/genmar/htm/1GM.HTM), demonstrates that SSC1q2.1-q2.13 corresponds to the region ranging from 44.6 to 63.2 Mb on HSA14q21.1-q23.1, the region from 86.5 to 86.8 Mb on HSA15q24-q25, the region from 0.9 to 27.2 Mb on HSA9p24.3-p21, the region from 35.1 to 38.0 Mb on HSA9p13, the region from 70.3 to 79.3 Mb on HSA9q13-q21 and the region from 96.4 to 140.0 Mb on HSA9q22.3-q34. The conserved synteny between HSA9 and SSC1q is interrupted by at least six sites, and the synteny between HSA14 and SSC1q is interrupted by at least one site.

Assignment of 101 genes localized in HSA10 to a swine RH (IMpRH) map to generate a dense human-swine comparative map.

Economically important traits such as growth and backfat in pigs have been shown to be influenced by genes in swine chromosome (SSC) 10q12-->qter corresponding to human chromosome (HSA) 10p. However, since gene information in the swine chromosomal region was limited, we attempted to generate a dense comparative map between SSC10 and HSA10 by mapping the 115 genes of HSA10 to a swine RH map (IMpRH map). In the mapping ten genes were assigned to SSC10, 88 to SSC14, and one to SSC3. One gene was suggested to link to SSC3, and another to SSC9. The correspondences between HSA10 and SSC10 and between HSA10 and SSC14 were essentially consistent with the observations obtained from bi/uni-directional chromosome painting or other results. This study further indicated that a large number of intrachromosomal rearrangements occurred in the synteny-conserved regions following species separation.

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse.

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine x Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.

A dense comparative gene map between human chromosome 19q13.3-->q13.4 and a homologous segment of swine chromosome 6.

The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11-->q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3-->q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3-->q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22-->q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12-->q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

Construction of a high-resolution comparative gene map between swine chromosome region 6q11-->q21 and human chromosome 19 q-arm by RH mapping of 51 genes.

A comprehensive and comparative map was constructed for the porcine chromosome (SSC) 6q11-->q21 region, where the gene(s) responsible for the maldevelopment of embryos are localized using swine populations of the National Institute of Animal Industry, Japan (NIAI). Since the chromosomal region corresponds to a region of human chromosome (HSA) 19q13.1-->q13.3 based on bi-directional chromosome painting, primer pairs were designed from porcine cDNA sequences identified, on a sequence comparison basis, as being transcripts from genes orthologous to those in the HSA region. Fifty-one genes were successfully assigned to a swine radiation hybrid (RH) map with LOD scores greater than 6. ERF and PSMD8 genes were assigned to SSC4 and SSC1, respectively. The remaining 49 genes were assigned to SSC6, demonstrating that the synteny between the SSC6 and HSA19 chromosomal regions is essentially conserved, therefore confirming, the results of bi-directional chromosome painting. However, when examined precisely, rearrangements have apparently occurred within the region of conserved synteny. For the ERF and PSMD8 genes assigned to SSCs other than SSC6, additional mapping using somatic cell hybrid (SCH) panels was performed to confirm the results of RH-mapping.

Elucidation of correspondence between swine chromosome 4 and human chromosome 1 by assigning 27 genes to the ImpRH map, and development of microsatellites in the proximity of 14 genes.

Loci affecting swine intramuscular fat content, backfat thickness, carcass weight, and daily weight gain were assigned to regions of swine chromosome (SSC) 4, which were shown to correspond to human chromosome (HSA) 1p22--> q25 by ZOO-FISH, bidirectional chromosome painting, as well as by the linkage map of genes. In order to select candidate genes responsible for the above traits from the human genome database, precise correspondence between SSC4 and HSA1 is a prerequisite. In the present study, 27 genes, PTGFR, GBP1, GBP2, GFI1, GCLM, ABCD3, EXTL2, KCNA3, ADORA3, KCND3, WNT2B, NRAS, SYCP1, PTGFRN, IGSF2, NOTCH2, S100A10, SHC1, SSR2, LMNA, CCT3, CD5L, PEA15, FCER1G, EAT2, DDR2, and LAMB3, located in the HSA1 region corresponding to SSC4 or possibly SSC4, were assigned to the IMpRH map. The alignment of genes from centromere to telomere in the SSC4 q arm is basically conserved in HSA1p22-->q25 with the direction from the q arm to the p arm, which is in good agreement with results from linkage mapping. In addition, the present study first demonstrated that WNT2B residing in the middle of the HSA1 region was assigned to SSC18 with a high lod score (> 5), and that at least three intrachromosomal rearrangements occurred in the region in the process of swine and human evolution. PTGFR, and LAMB3 localized at both ends of the HSA1 region were assigned to SSC6 and SSC9, respectively, which is consistent with regional correspondence reported earlier. In the course of the above analysis, microsatellite markers were developed in the proximity of eleven genes localized on SSC4, and three genes on other swine chromosomes.

Glucocorticoids activate transcription of the gene for the glucose-6-phosphate transporter, deficient in glycogen storage disease type 1b.

Deficiencies in the glucose-6-phosphate transporter (G6PT) cause glycogen storage disease type 1b (GSD-1b), a heritable metabolic disorder. The G6PT protein translocates glucose-6-phosphate from the cytoplasm to the lumen of the endoplasmic reticulum, where glucose-6-phosphatase metabolizes it to glucose and phosphate. Therefore, G6PT and glucose-6-phosphatase work in concert to maintain glucose homeostasis. To delineate the control of G6PT gene expression, we first demonstrated that transcription of the gene requires hepatocyte nuclear factor 1alpha. Consequently, hepatocyte nuclear factor 1alpha-null mice manifest a G6PT deficiency like that of GSD-1b patients. In this study, we delineated the role of glucocorticoids in the transcription of the G6PT gene. We showed that the basal G6PT promoter is contained within nucleotides -369 to -1 upstream of the translation start site, which contains three activation elements. Further, we demonstrated that glucocorticoids activate G6PT transcription and that glucocorticoid action is mediated through a glucocorticoid response element within activation element-2 of the promoter. Taken together, the results suggest that glucocorticoids play a pivotal role in regulating the G6PT gene.

Development and application of an enzyme immunoassay for tenascin.

A sandwich enzyme immunoassay system for detecting tenascin in human serum was established using purified antibodies to tenascin. The assay system comprises polystyrene balls with immobilized polyclonal antibody F(ab')2 fragments and monoclonal antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli. The assay system has a minimum detectable sensitivity of 10 ng/ml of tenascin in human serum, with an assay range of 3 micrograms/ml. The assay system was found not to cross-react with laminin, vitronectin, human epidermal growth factor, fibrinogen, or fibronectin. Coefficients of variation within-run and between-run for the assay of human serum tenascin were less than 10%. Serum samples from healthy adults (n = 86) contained about 800 ng/ml and serum tenascin concentrations of patients with carcinoma (n = 47) were increased. These results suggest that tenascin in serum might be a marker substance for carcinoma.

Cloning and chromosomal assignment of the porcine interleukin-2 receptor alpha (IL-2Ralpha) gene.

Porcine genomic DNA encoding a 55 kDa subunit of interleukin-2 receptor (IL-2R), which is termed alpha chain (IL-2Ralpha), was cloned by repeated plaque hybridization using IL-2Ralpha cDNA as a probe. Two different lambda phage clones, one of which encoded exon 1 and the 5'-upstream flanking region of IL-2Ralpha gene and another encoded the sequence from exon 2 to exon 8, were isolated. By analysis of the 5'-upstream region of the gene, putative binding motifs for transcription factors such as GATA family proteins, Ikaros, NF-kappaB, NF-IL2Ralpha and SRF, were found as described in human, murine and bovine genes. Two additional motifs for STAT4 binding were also found in this region. Moreover, using the FISH technique, we assigned the porcine IL-2Ralpha locus to the distal end of the long arm of chromosome 10 (10q6-qter) where the vimentin gene had been assigned nearby.

Assignment of 117 genes from HSA5 to the porcine IMpRH map and generation of a dense human-pig comparative map.

Twenty-two and eight significant quantitative trait loci for economically important traits have been located on porcine chromosomes (SSC) 2q and SSC16 respectively, both of which have been shown to correspond to human chromosome 5 (HSA5) by chromosome painting. To provide a comprehensive comparative map for efficient selection of candidate genes, we assigned 117 genes from HSA5 using a porcine radiation hybrid (IMpRH) panel. Sixty-six genes were assigned to SSC2 and 48 to SSC16. One gene was suggested to link to SSC2 markers and another to SSC6. One gene did not link to any gene, expressed sequence tag or marker in the map, including those in the present investigation. This study demonstrated the following: (1) SSC2q21-q28 corresponds to the region ranging from 74.0 to 148.2 Mb on HSA5q13-q32 and the region from 176.0 to 179.3 Mb on HSA5q35; (2) SSC16 corresponds to the region from 1.4 to 68.7 Mb on HSA5p-q13 and to the region from 150.4 to 169.1 Mb on HSA5q32-q35 and (3) the conserved synteny between HSA5 and SSC2q21-q28 is interrupted by at least two sites and the synteny between HSA5 and SSC16 is also interrupted by at least two sites.

Alignment of VIM, MRC1, GAD2, and IL2RA genes on swine chromosome 10q by in situ hybridization and RH mapping.

Since the distal half of swine chromosome (SSC) 10q was shown to contain a quantitative trait locus (QTL) influencing swine growth, the precise correspondence between this chromosome region and the orthologous human chromosome region (HSA10p) was investigated using chromosomal fluorescence in situ hybridization and RH mapping of type I loci spanning the growth QTL. The goal was to align this critical region of swine with the corresponding region in human for the purpose of identifying candidate genes. The HSA10p type I loci mapped in swine were VIM, MRC1, GAD2, and IL2RA. Locus order on SSC10q was shown to be centromere-VIM-MRC1-GAD2-IL2RA, while in human the order is centromere-GAD2-MRC1-VIM--IL2RA, indicating that the chromosome segment marked by VIM, MRC1 and GAD2 has been inverted relative to the centromere and IL2RA.

Prevalence of appendectomy in Japanese families.

We investigated the prevalence of appendectomy in Japan to determine the tendency of familial aggregation on appendicitis using a large scale study. A total of 517 Japanese women (aged 20 to 45 years) served as the subjects and were questioned regarding their family history of appendectomy due to appendicitis. We compared the prevalence of appendectomy in children (subjects+their siblings) of three groups of families: (i) both parents affected; (ii) one parent affected; (iii) no parents affected. The differences between groups were statistically significant using contingency table and Chi-squared test. Our results suggest that about 40% of children with both parents affected, and about 20% of children with one parent affected may develop the disorder during childhood. This study may provide useful information in the nature of the development of appendicitis.

A neuroblastoma cell line derived from a case detected through a mass screening system in Japan: a case report including the biologic and phenotypic characteristics of the cell line.

BACKGROUND: A mass screening system in Japan, which involves measuring urinary catecholamine metabolites, has resulted in an increasing number of cases of neuroblastoma, most of which have favorable biologic properties and some of which are associated with tumor regression or involution. At the time this study was begun, the characteristics and biologic nature of the neuroblastomas had not been fully defined, because a cell line had not yet been established with tumor tissue taken from a neuroblastoma detected in the mass screening. METHODS: The authors established a cell line by tissue culture for over 50 generations from a neuroblastoma found during mass screening, which was characterized by favorable histology, with a triploid DNA stemline and without N-myc gene amplification. The morphologic and biologic characteristics of the new cell line were investigated in vitro. RESULTS: The cell line, designated MASS-NB-SCH-1, has neuronal properties, such as neurite-like processes and neurofilaments, as well as the expression of vimentin and fibronectin in studies of the cell morphology and immunohistochemistry. Karyotype analysis detected the presence of 42-46 chromosomes, with a deletion of the short arm of 1 of the 3 copies of chromosome 1. DNA ploidy was near-diploid in association with 20-fold amplification of N-myc genes. CONCLUSIONS: The cell line has a nature distinct from the original tumor tissue. It may be characterized by phenotypic change caused by clonal selection or evolution of aggressive, proliferative properties in vitro. This cell line will be a useful model to investigate the properties of the neuroblastoma in relation to the N-myc amplification mechanism.

Epstein-Barr virus infection, Hodgkin's disease, non-Hodgkin's lymphoma, and reactive follicular hyperplasia in Japanese children: evaluation of paraffin-embedded specimens using polymerase chain reaction and immunohistochemistry.

Epstein-Barr virus (EBV) infections are common in Japanese children, with infectious by EBV type 1. The relationships between EBV infection and lymphadenopathies in Hodgkin's disease (HD), non-Hodgkin's lymphomas (NHL), reactive follicular hyperplasia (RFH), and infectious mononucleosis (IM) in 37 Japanese children were evaluated. Formalin-fixed, paraffin-embedded lymph node specimens that were obtained at surgical resection or biopsy were evaluated for the presence of EBV DNA and the latent membrane protein-1 (LMP-1) using polymerase chain reaction (PCR) and immunohistochemical staining. The PCR detected EBV DNA in nine of 13 (69.2%) patients with RFH, including a case of IM, all three (100%) patients with HD, and one of 21 (4.8%) patients with NHL. All EBV-positive samples contained EBV type 1. Reed-Sternberg's cells in HD were immunohistochemically positive for LMP-1, whereas all cases of RFH and NHL were negative for LMP-1. Results suggest that EBV infection may be related to HD. Although no proof exists that EBV infection contributes to the transformation of cells, thus causing RFH or NHL, the present authors suggest that the EBV-positive cases in Japanese children demonstrate a relationship between the clinical and histopathological features of the lymphadenopathy and EBV-type 1 infection.