Radiation-induced modification of the chromosome sensitivity of human somatic cells to the testing mutagenic exposure of bleomycin in vitro

Hidden chromosome instability in 53 persons who underwent radiation exposure of different intensity was evaluated with the use of the modified G₂-bleomycin sensitivity assay. A wide interindividual variability in the frequency of chromosome aberrations and absence of positive correlation between the background and bleomycin-induced cytogenetic effects in all examined individuals were found. The maximal number (57.9%) of individuals hypersensitive to the testing mutagenic activity of bleomycin was found in the group of reconvalescents of acute radiation syndrome. In the other groups, the frequency of individuals with hidden chromosome instability was practically the same and did not exceed 33.3%. The results confirmed the reality of the radiation-induced modification of genetically determined susceptibility of human somatic cell chromosomes to mutagenic stress; such susceptibility depends on the intensity and character of irradiation.     

IL9 maps to mouse chromosome 13 and human chromosome 5

Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL-9 is distal to Tcrg and Proximal to Dhfr.     

Sporadic chromosome abnormalities in human lymphocytes and previous exposure to chemicals

Sporadic abnormalities in lymphocyte cultures are often attributed to in vitro culture variations of no clinical significance. The data presented here compare the findings from 11,873 cells of 230 patients referred with histories of previous chemical exposure (usually to mixtures of solvents and/or pesticides) with 27,050 cells from 855 patients referred for other reasons. Detection of 0.38% or more, structural abnormalities (approximately 1 in 30 cells) was 27.2 times more likely in exposed persons than in controls and the finding of a single autosomal trisomic cell was 14.4 times more likely in exposed persons. These highly statistically significant findings were similar to the frequencies of abnormalities reported in other studies of persons exposed to benzene, pesticides, herbicides and irradiation. It is recommended that findings of sporadic abnormalities in lymphocytes be routinely recorded, and patients with positive findings followed up to discover whether there are past histories of significant exposures.     

Effect of a viral infection on the cellular chromosome apparatus of mice in vitro and in vivo against a background of hydrocortisone action]

A cytogenetic investigation of murine bone marrow after hydrocortison injection has been made. High doses of hormone (50 mg/kg) provoke deteriorations in bone marrow both in the structure and in the chromosome number. A dose of 5 mg/kg has no such effect. The Koksak A13 virus does not induce cytogenetic deteriorations in mice, however, it is able to produce a big mutagenic effect on the hydrocortison background. The vaccine strain of measles virus -- Leningrad-16 -- also increases its mutagenic action on the bone marrow cell chromsome apparatus of mice affected with hydrocortison. At the same time, in the cell culture of murine kidney, hydrocortison does not induce chromosome deteriorations and even lowers the frequency of cells with deteriorations in the chromosome set during the initial days after injecting the virus culture with measles virus.     

cDNA cloning of human oxysterol-binding protein and localization of the gene to human chromosome 11 and mouse chromosome 19

Cellular cholesterol metabolism is regulated primarily through sterol-mediated feedback suppression of the activity of the low-density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway. We previously described the cloning of a rabbit cDNA for the oxysterol-binding protein (OSBP), a cytosolic protein of 809 amino acids that may participate in these regulatory events. We now use the rabbit OSBP cDNA to clone the human OSBP cDNA and 5' genomic region. Comparison of the human and rabbit OSBP sequences revealed a remarkably high degree of conservation. The cDNA sequence in the coding region showed 94% identity between the two species, and the predicted amino acid sequence showed 98% identity. The human cDNA was used to determine the chromosomal localization of the OSBP gene by Southern blot hybridization to panels of somatic cell hybrid clones containing subsets of human or mouse chromosomes and by RFLP analysis of recombinant inbred mouse strains. The OSBP locus mapped to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. Along with previously mapped genes including Ly-1 and CD20, OSBP defines a new conserved syntenic group on the long arm of chromosome 11 in the human and the proximal end of chromosome 19 in the mouse.     

Mapping of the thrombospondin gene to human chromosome 15 and mouse chromosome 2 by in situ hybridization

We have mapped the thrombospondin gene (THBS1) to a single locus on human chromosome 15 (band q15) and on mouse chromosome 2 (region F). Thrombospondin has been implicated in a variety of cell-matrix and cell-cell interactions. The finding of a single locus suggests that the different functions of thrombospondin are not due to a closely related family of genes. These results also confirm a region of homology between the proximal part of human chromosome 15 and region F of mouse chromosome 2.     

Localization of the human gene allowing infection by gibbon ape leukemia virus to human chromosome region 2q11-q14 and to the homologous region on mouse chromosome 2.

Retrovirus receptors remain a largely unexplored group of proteins. Of the receptors which allow infection of human and murine cells by various retroviruses, only three have been identified at the molecular level. These receptors include CD4 for human immunodeficiency virus, Rec-1 for murine ecotropic virus, and GLVR1 for gibbon ape leukemia virus. These three proteins show no homology to one another at the DNA or protein level. Therefore, work to date has not shown any general relationship or structural theme shared by retroviral receptors. Genes for two of these receptors (CD4 and Rec-1) and several others which have not yet been cloned have been localized to specific chromosomes. In order to assess the relationship between GLVR1 and other retroviral receptors, we mapped the chromosome location of GLVR1 in human and mouse. GLVR1 was found to map to human chromosome 2q11-q14 by in situ hybridization and somatic-cell hybrid analysis. This location is distinct from those known for receptors for retroviruses infecting human cells. Glvr-1 was then mapped in the mouse by interspecies backcrosses and found to map to chromosome 2 in a region of linkage conservation with human chromosome 2. This mouse chromosome carries Rec-2, the likely receptor for M813, a retrovirus derived from a feral Asian mouse. These data raise the interesting possibility that Rec-2 and Glvr-1 are structurally related.     

Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsolin and dopamine beta-hydroxylase map to mouse chromosome 2.

The mapping of human chromosome 9 (HSA9) and mouse chromosome 2 (MMU2) has revealed a conserved syntenic region between the distal end of the long arm of chromosome 9 and proximal mouse chromosome 2. Two genes that map to human chromosome 9q34, gelsolin (GSN) and dopamine beta-hydroxylase (DBH), have not previously been located in the mouse. We have used an interspecific backcross to map each of these genes, by Southern blot analysis, to mouse chromosome 2. Gelsolin (Gsn) is tightly linked to the gene for complement component C5 (Hc), and dopamine beta-hydroxylase (Dbh) is just proximal to the Abelson leukemia virus oncogene (Abl) and alpha-spectrin 2 (Spna-2). The loci for gelsolin and dopamine beta-hydroxylase therefore form part of the conserved synteny between HSA9q and MMU2.     

Early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice

Background

Early-life respiratory viral infections, notably with respiratory syncytial virus (RSV), increase the risk of subsequent development of childhood asthma. The purpose of this study was to assess whether early-life infection with a species-specific model of RSV and subsequent allergen exposure predisposed to the development of features of asthma.

Methods

We employed a unique combination of animal models in which BALB/c mice were neonatally infected with pneumonia virus of mice (PVM, which replicates severe RSV disease in human infants) and following recovery, were intranasally sensitised with ovalbumin. Animals received low-level challenge with aerosolised antigen for 4 weeks to elicit changes of chronic asthma, followed by a single moderate-level challenge to induce an exacerbation of inflammation. We then assessed airway inflammation, epithelial changes characteristic of remodelling, airway hyperresponsiveness (AHR) and host immunological responses.

Results

Allergic airway inflammation, including recruitment of eosinophils, was prominent only in animals that had recovered from neonatal infection with PVM and then been sensitised and chronically challenged with antigen. Furthermore, only these mice exhibited an augmented Th2-biased immune response, including elevated serum levels of anti-ovalbumin IgE and IgG₁ as well as increased relative expression of Th2-associated cytokines IL-4, IL-5 and IL-13. By comparison, development of AHR and mucous cell change were associated with recovery from PVM infection, regardless of subsequent allergen challenge. Increased expression of IL-25, which could contribute to induction of a Th2 response, was demonstrable in the lung following PVM infection. Signalling via the IL-4 receptor α chain was crucial to the development of allergic inflammation, mucous cell change and AHR, because all of these were absent in receptor-deficient mice. In contrast, changes of remodelling were evident in mice that received chronic allergen challenge, regardless of neonatal PVM infection, and were not dependent on signalling via the IL-4 receptor.

Conclusion

In this mouse model, interaction between early-life viral infection and allergen sensitisation/challenge is essential for development of the characteristic features of childhood asthma, including allergic inflammation and a Th2-biased immune response.     

Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.     

Assignment of the erythropoietin receptor (EPOR) gene to mouse chromosome 9 and human chromosome 19

Erythropoietin (EPO), the primary regulator of mammalian erythropoiesis, binds and activates a specific receptor on erythroid progenitors. The human and mouse cDNAs for this receptor (EPOR) have recently been isolated. These cDNAs were used to establish the genomic location of the EPOR gene. By somatic cell hybrid analysis, the locus for the EPOR maps to human chromosome (Chr) 19pter-q12. By interspecific backcross mapping the locus is tightly linked to the murine Ldlr locus near the centromere of mouse Chr9. This region of mouse Chr9 is homologous to a region of human Chr 19p13 carrying the human LDLR and MEL loci, strongly suggesting that the human EPOR gene is at 19p13 near the human LDLR locus.     

Use of comparative physical and sequence mapping to annotate mouse chromosome 16 and human chromosome 21

Distal mouse chromosome 16 (MMU16) shares conserved linkage with human chromosome 21 (HSA21), trisomy for which causes Down syndrome (DS). A 4.5-Mb physical map extending from Cbr1 to Tmprss2 on MMU16 provides a minimal tiling path of P1 artificial chromosomes (PACs) for comparative mapping and genomic sequencing. Thirty-four expressed sequences were positioned on the mouse map, including 19 that were not physically mapped previously. This region of the mouse:human comparative map shows a high degree of evolutionary conservation of gene order and content, which differs only by insertion of one gene (in mouse) and a small inversion involving two adjacent genes. "Low-pass" (2.2x) mouse sequence from a portion of the contig was ordered and oriented along 510 kb of finished HSA21 sequence. In combination with 68 kb of unique PAC end sequence, the comparison provided confirmation of genes predicted by comparative mapping, indicated gene predictions that are likely to be incorrect, and identified three candidate genes in mouse and human that were not observed in the initial HSA21 sequence annotation. This comparative map and sequence derived from it are powerful tools for identifying genes and regulatory regions, information that will in turn provide insights into the genetic mechanisms by which trisomy 21 results in DS.     

High susceptibility of chromosome 16 to radiation-induced chromosome rearrangements in human lymphocytes under in vivo and in vitro exposure

The aim of the present study was to investigate whether chromosome 16p presents breakpoint regions susceptible to radiation-induced rearrangements. The frequencies of translocations were determined by fluorescence in situ hybridization (FISH) using cosmid probes C40 and C55 mapping on chromosome 16p, and a chromosome 16 centromere-specific probe (pHUR195). Peripheral lymphocytes were collected from normal individuals and from seven victims of 137Cs in the Goiania (Brasil) accident (absorbed doses: 0.8-4.6 Gy) 10 years after exposure. In vitro irradiated lymphocytes (3 Gy) were also analyzed. The mean translocation frequency/cell obtained for the 137Cs exposed individuals was 2.4-fold higher than the control value (3.6 x 10(-3) +/- 0.001), and the in vitro irradiated lymphocytes showed a seven-fold increase. The genomic translocation frequencies (FGs) were calculated by the formula Fp = 2.05 fp(1-fp)FG (Lucas et al., 1992). For the irradiated lymphocytes and victims of 137Cs, the FGs calculated on the basis of chromosome 16 were 2- to 8-fold higher than those for chromosomes 1, 4 and 12. Our results indicate that chromosome 16 is more prone to radiation-induced chromosome breaks, and demonstrate a non-random distribution of induced aberrations. This information is valuable for retrospective biological dosimetry in case of human exposure to radiation, since the estimates of absorbed doses are calculated by determining the translocation frequency for a sub-set of chromosomes, and the results are extrapolated to the whole genome, assuming a random distribution of induced aberrations. Furthermore, the demonstration of breakpoints on 16p is compatible with the reports about their involvement in neoplasias.     

Interleukin-12 gene expression after viral infection in the mouse.

Interleukin-12 is a lymphokine that triggers gamma interferon secretion by various cells and differentiation of T-helper lymphocytes towards the Th1 subtype. Since viruses are potent inducers of gamma interferon production and elicit immune responses most probably mediated by Th1 cells, like B-cell immunoglobulin G2a secretion, we analyzed interleukin-12 message expression after infection of mice with lactate dehydrogenase-elevating virus, mouse hepatitis virus, and mouse adenovirus. Our results indicated that the message for the p40 component of interleukin-12 was transiently increased shortly after infection. Interleukin-12 was expressed mainly by macrophages. Therefore, production of interleukin-12 might constitute the initial event that would determine the subsequent characteristics of the immune response elicited by viral infections.     

Assignment of d-amino-acid oxidase gene to a human and a mouse chromosome

Summary. A part of d-amino-acid oxidase gene was amplified in the human and mouse by polymerase chain reaction. The amplified fragments were ligated to plasmids and then cloned. The plasmids containing the parts of d-amino-acid oxidase gene were biotinylated and hybridized to human and mouse metaphase chromosomes. The chromosomal slides were treated with fluorescein isothiocyanate (FITC)-conjugated avidin. The hybridized signals were amplified with biotinylated anti-avidin antibody and FITC-avidin. The chromosomes were counter-stained with diamidino-phenylindole for assignment of the signal to a specific band. Using this fluorescence in situ hybridization (FISH), d-amino-acid oxidase gene was assigned to human chromosome 12q23–24.1 and mouse chromosome 5E3-F. Since these regions are syntenic between human and mouse, the present results indicate that the locus for this enzyme has been conserved through evolution. Received July 11, 2000 Accepted November 10, 2000     

CD19 maps to a region of conservation between human chromosome 16 and mouse chromosome 7

CD19 is a B lymphocyte cell surface protein expressed from the earliest stages of B lymphocyte development unitl their terminal differentiation into plasma cells. In this report the human CD19 gene (hCD19) was localized to band p11.2 on the proximal short arm of chromosome 16 by in situ hybridization to metaphase chromosomes, using hCD19 cDNA as probe. hCD19 gene localization was confirmed by polymerase chain reaction based analysis with hCD19-specific primers, using a panel of human/hamster somatic cell hybrid DNA as templates. The mouse CD19 gene (MCd19) was mapped to bands F3-F4 of chromosome 7 by in situ hybridization to metaphase chromosomes, using a mCD19 cDNA probe. Segregation analysis of nucleotide sequence polymorphisms in inter-specific backcross progeny revealed linkage of mCd19 with hemoglobin (Hbb), Int-2, and H19, other loci previously mapped to the same region of mouse chromosome 7, confirming the localization of mCd19 to this region. The order of these loci was determined to be centromere — Hbb — mCd19 — H19 — Int-2 —telomere. The genetic distance between the loci examined, calculated from the recombination frequencies, suggested that mCd19 was located centrally between Hbb and H19. This region of mouse chromosome 7 is homologous to the region of human chromosome 16 to which the hCD19 gene maps. Multiple genes with a lymphocyte-related function also map to this conserved region including genes encoding the IL-4 receptor, CD11a, CD11b, CD11c, CD43 (leukosialin), and protein kinase C polypeptide.