A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Hemopexin is localized to human chromosome 11

Hemopexin, a plasma protein that migrates during electrophoresis with the -globulins, transports free heme to sites of its catabolism in the liver. A hemopexin cDNA clone has been utilized for mapping the hemopexin (HPX) gene to human chromosome 11 in the region pterp11 by somatic cell hybrid analysis.     

Mapping of the genes for human endoplasmic reticular heat shock protein gp96/grp94

The murine tumor rejection antigen gp96 (TRA1, mapped to mouse chromosome 10) is a member of the heat shock protein family. Using a fragment of the murine gp96 cDNA as a probe, three gp96-related human genes have been isolated and structurally characterized. They have been mapped to human chromosomes 1 (p22), 12 (q24.2 q24.3), and 15 (q25 q26) by Southern blot hybridization and in situ hybridization of gene-specific probes. Only one of the genes, designatedTRA1 (human chromosome 12) is a coding gene; the other genes (TRA1P1 andTRAP2) appear to be independently derived, processed pseudogenes.     

Disruption of the integrity of human peritoneal mesothelium by interleukin-1β and tumor necrosis factor-α

Inflammatory and neoplastic disease processes of the abdominal cavity are frequently associated with disruption of the integrity of the peritoneal mesothelium. In the present study, we analyzed the effects of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) on the morphology and expression of adhesion molecules of human peritoneal mesothelial cells (HPMC). Treatment of HPMC with IL-1 and TNF- resulted in a time- and dose-dependent alteration of the normal cobblestone morphology of the mesothelium with loss of polarization, cellular retraction and exposure of the submesothelial matrix. The effect was already observable after 6 h of treatment and was most pronounced at a dose of 10 ng/ml of IL-1 or TNF-. These morphological alterations were associated with a significant rearrangement of the expression of mesothelial adhesion molecules as detected by flow cytometry. IL-1 and TNF- both led to a loss of the expression of the hemidesmosomal integrin subunits 6 (P<0.01 and P<0.001) and 4 (P<0.01) and an increased expression of the integrin subunit 5 (P<0.001 and P<0.01). IL-1 furthermore upregulated the expression of the integrin subunits 1, 2 and the adhesion molecule CD44 while the latter was downregulated by TNF-. Our data indicate that IL-1 and TNF- may significantly affect disease processes of the abdominal cavity by their potential to disrupt the mesothelial basal cell-matrix adhesion and, thus, the integrity of the peritoneal mesothelial cell lining.     

Regional mapping of RBP4 to 10q23→q24 and RBP1 to 3q21→q22 in man

The human gene coding for RBP4 has been assigned to 10q2324 using a panel of somatic cell hybrids and in situ hybridization experiments. The mapping of the human RBP1, previously assigned by our group to chromosome 3 using a panel of somatic cell hybrids, was restricted to the region 3q2122 using in situ experiments and Southern blots of genomic DNA from a hybrid retaining a portion of chromosome 3.R.F. is recipient of a Research Grant from A.I.R.C.     

Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-CC-G or T-AG-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine had G-CA-T mutations at non-CpG sites; in addition, EMS induced several A-TG-C, A-TT-A, and G-CT-A substitutions. A single ICR191-induced mutant analyzed had a unique A-TG-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.     

Cognitive mediators of the social influence-exercise adherence relationship: A test of the theory of planned behavior

The purpose of this study was to examine cognitive constructs from the theory of planned behavior (i.e., attitude, perceived behavioral control, and intention) as potential mediators of the relationship between selected social influence constructs (i.e., subjective norm, social support, and cohesion) and adherence to structured exercise classes. Sixty-two participants completed self-administered questionnaires during the fourth week (social influence constructs) and eighth week (cognitive constructs) of a 12-week exercise program. Exercise adherence was monitored during weeks 9 through 12 using perceived intensity and attendance. Pearson correlations indicated that social support correlated with perceived behavioral control, whereas cohesion correlated with attitude. Path analysis supported two distinct paths from social influence to exercise adherence: (a) social support perceived behavioral control intention excersise adherence, and (b) cohesion attitude intention exercise adherence. Discussion focuses on the theoretical importance of these findings, conceptual and measurement issues regarding subjective norm, and suggestions for future research.     

Regional assignment of human amylase (AMY) to p22 → p21 of chromosome 1

A human genomic DNA segment (hal) which hybridizes with rat pancreatic amylase cDNA was used to regionally assign amylase genes in human-mouse somatic cell hybrids. The assignment of amylase genes to chromosome 1 was confirmed and regionally mapped to the short arm region p22.1p21 using a cell hybrid retaining a translocation involving chromosome 1 [46,XX,t(1;2)(p21;q37)]. Restriction length polymorphisms at the amylase loci are reported for gene linkage studies.     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

Parental origin and mechanism of formation of X chromosome structural abnormalities: Four cases determined with RFLPs

Parental origin and mechanism of formation of X chromosome structural anbormalities were studied in one each case of dup(X)(pter p11.4::p22.1qter), del(X)(qterp11:), i(X)(qtercenqter), and inv dup(X) (pterq22::q22pter) using various X-linked RFLPs as genetic markers. Segregation and densitometric analyses on polymorphic DNAs revealed that the dup(Xp) and the del(Xp) are both of paternal origin and the i(Xq) and i dic(X) are of maternal origin. The dup(Xp) had arisen by an unequal sister chromatid exchange and the del(Xp) had occurred through an intrachromosomal breakage-reunion mechanism, both in the paternal X chromosome. The i(Xq) had arisen either through centromere fission of a maternal X chromosome, followed by duplication, of its long-arm, or through a translocation between two maternal X chromosomes after meiotic crossing-over. The inv dup(X) arose through sister chromatid breakage and reunion in a maternal X chromosome. These results, together with those of previous studies, suggest that thede novo abnormalities due to events involving centromere disruption arise predominantly during oogenesis, while those due to simple breakage-reunion events occur preferentially during spermatogenesis.     

Transforming growth factor β gene maps to human chromosome 19 long arm and to mouse chromosome 7

Transforming growth factors (TGF) are defined as biologically active polypeptides which reversibly confer the transformed phenotype onto untransformed cultured cells. They have been subdivided into two classes: type and type TGFs. TGF- acts synergistically with TGF- in inducing phenotypic transformation. TGF- can also act as negative autocrine growth factor. A human 1050-bp EcoRI cDNA fragment was used to map the human locus for TGF- by Southern blotting of DNA prepared from 17 human × Chinese hamster somatic cell hybrids. The humanspecific restriction fragments segregated with human chromosome 19 in all of 14 informative hybrids. All other human chromosomes were discordant with the TGF- bands in at least four hybrids. After in situ hybridization of the tritiated TGF- probe to normal human metaphase spreads, 151 silver grains were scored in 54 cells. Of 24 grains over chromosome 19, 16 grains (11%) lay over region 19q13.1 q13.3. Of the 54 cells analyzed, 16 (30%) had label over region 19q13.1 q13.3. Thus,TGFB is assigned to chromosome 19, subbands q13.1 q13.3. TheTgf- locus in the mouse was mapped to chromosome 7 by hybridizing a murine cDNA probe to a Chinese hamster × mouse hybrid panel. Human chromosome 19 and proximal mouse chromosome 7 share another four homologous loci.     

Gene assignment of α-fucosidase and glucose dehydrogenase to the p21→pter region of chromosome 1 in man

Assignment of human genes coding for -fucosidase (FUC) and glucose dehydrogenase (GDH) to chromosome 1 has been confirmed and a location in the p21pter region demonstrated using man-mouse somatic cell hybrids. The regional location af FUC andGDH was established in cell hybrids using human cells possessing 1/2 translocation chromosomes [46,XX,t(1;2)(p21;q37)]. Hybrids which retained the 2q⁺ chromosome carrying the 1p211pter region concordantly expressed FUC, GDH, and the short-arm markers ENO₁, AK₂, and PGM₁. Hybrids which retained the 1p211qter region only expressed human PEPC and FH. Data obtained from hybrids in which spontaneous breaks in chromosome 1 had occurred indicate that the gene order in 1p21ar1pter is (ENO₁,GDH)-FUC-AK₂-PGM₁.     

Gene for murine α1 → 3-galactosyltransferase is located in the centromeric region of chromosome 2

The gene for 1 3-galactosyltransferase, termedGgta-1, was mapped to mouse chromosome 2 by Southern blot analysis of Chinese hamster × mouse somatic cell hybrids. Using an intersubspecies back-cross, this locus was positioned to the centromeric region on this chromosome, near the Hc locus.     

Association of polymorphisms of paraoxonase 1 and 2 genes,alone or in combination,with bone mineral density in community-dwelling Japanese

Oxidative stress may affect cellular functions in various pathological conditions, including osteoporosis. Paraoxonase 1 confers antioxidant properties on high-density lipoprotein, with which it is associated, by reducing the accumulation of lipid peroxidation products. We have now examined whether the 584AG (Gln192Arg) and 172TA (Leu55Met) polymorphisms of the paraoxonase 1 gene and the 959GC (Cys311Ser) polymorphism of the paraoxonase 2 gene are associated with bone mineral density (BMD) in community-dwelling Japanese (1,087–1,094 women and 1,112–1,125 men). The subjects were aged 40 –79 years and were randomly recruited to a population-based prospective cohort study of aging and age-related diseases. BMD for the lumbar spine and right femoral neck was measured by dual-energy X-ray absorptiometry. Genotypes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. The 584AG and 172TA polymorphisms of the paraoxonase 1 gene and the 959GC polymorphism of the paraoxonase 2 gene were associated with BMD for the lumbar spine or femoral neck in postmenopausal women, with the 584GG, 172TT, and 959CC genotypes representing risk factors for reduced bone mass. None of these three polymorphisms was associated with BMD in premenopausal women or in men. Our results suggest that the paraoxonase 1 and 2 genes are candidate loci for reduced bone mass in postmenopausal Japanese women.     

Distribution of glycoconjugates in the optic vesicle and optic cup

Summary Lectin histochemical methods and immunohistochemical techniques have been utilized to investigate and partially characterize glycoconjugates in the developing eye. Peanut-lectin-binding sites associated with radial glial cells were found in the diencephalon. In the optic primordia, binding sites associated with radial glia were masked by terminal sialic acid, and only reacted with peanut lectin when pretreated with sialidase. This finding indicates that glycoconjugates associated with diencephalic radial glia contain terminal galactose--(13)N-acetyl galactosamine, but glycoconjugates associated with radial glia in the optic primordia contain sialic acidgalactose-(13)N-acetyl galactosamine. The selective distribution of galactose, N-acetyl galactosamine and fucose associated with radial glial cells has also been demonstrated. We postulate that these distributions mediate the shaping of the developing eye.This work was supported by NIH Grants # NS 11066 and # DE 05832     

The role of acidic residues in the “fusion segment” of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Summary To clarify the role of acidic amino acid residues in the fusion segment of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA₂19D G), –K11 (HA₂11E K) and –K19(HA₂19D K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA₂11E G) induced limited polykaryon formation and SVHA-G11,19 (HA₂11E G, 19D G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the fusion segment at low pH is not important for interaction of the fusion segment with the target lipid bilayer or for triggering the membrane fusion.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Human interstitial retinol-binding protein (IRBP): cloning,partial sequence,and chromosomal localization

A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86–88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity,suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2q11.2), although a secondary site of hybridization at q2425 was also observed.     

Spatial properties of the visual detectability of moving spatial white noise

Summary We determined the spatial parameters that describe the visual detection of spatio-temporal correlation in moving two-dimensional noise patterns. The target field (5.21×5.31 deg of visual angle) was divided into horizontal stripes of equal width D. Adjacent bars alternately contained noise patterns moving with velocity v₁ and v₂. We varied D, v₁ and v₂.Roughly three different percepts occurred. If the stripes were very broad the different movements in alternate stripes were perceived together with the division of the field into stripes. If the stripes were very narrow the division into stripes was not seen, but the moving noise patterns with velocities v₁ and v₂ were perceived as transparent sheets moving through each other. For intermediate stripe widths the target field looked incoherent and the subject was not clear about the percept. In this region the subject found it difficult and sometimes impossible to discriminate these patterns from a completely uncorrelated spatiotemporal white noise pattern (snow).To quantify the detectability, the patterns were masked with snow (spatio-temporal white noise). The r.m.s. contrast of the total stimulus was kept at a constant value, whereas the subject set the signal-to-noise ratio (SNR) to a threshold value. At certain barwidths the thresholds reached a maximum value. These critical barwidths depended on the velocities v₁ and v₂.These critical barwidths were interpreted in terms of a simple general model for the detection of spatiotemporal correlation. In these terms the span of the elementary correlators rose monotonically with the velocity to which the correlator is most sensitive.Supported in part by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Regional assignment of human protooncogenec-myb to 6q21→qter

Using human-mouse somatic cell hybrids containing different parts of chromosome 6 and a DNA probe of the oncogene (V-myb) of avian myeloblastosis virus (AMV), we regionally mapped by Southern blot techniques the human cellular myb (cmyb) protooncogene to 6q21qter.