Copy Of Human Chromosome Research Articles

PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

Cognitive Enhancement Therapy for a Model of Down Syndrome

Down syndrome is a complex condition that results from having a third copy of human chromosome 21. People with the syndrome experience problems with learning and memory that affect many aspects of their lives. In this issue of Science Translational Medicine, Salehi et al. report on successful drug treatment of learning deficits in an animal model of Down syndrome. This study highlights the function of the norepinephrine-ergic system in Down syndrome and suggests possible treatment options for people with Down syndrome.

Genetic control of telomerase and replicative senescence in human and rodent cells.

The ribonucleoprotein telomerase is detectable in most human cancer cells and immortalized cells but is absent or inactive in the vast majority of normal counterparts. Repression of telomerase activity in human somatic cells, which leads to telomere shortening and replicative senescence, may have evolved as a protective mechanism against immortalization, unfettered clonal evolution and cancer. Rodent cells in culture are far more susceptible to immortalization and malignant progression than human cells. This can be explained by our observation that normal diploid rodent (hamster) fibroblasts possess active telomerase throughout their proliferative life span, and therefore they do not require a telomerase activation step during immortalization. Monochromosome transfer techniques have enabled us to identify powerful telomerase repressive activity specifically associated with the introduction of a single copy of human chromosome 3 into human carcinoma cells. Fine-structure deletion analysis of non-repressed hybrids has permitted us to map the position of the candidate telomerase repressor gene to 3p21.1-3p21.3. A strategy for isolating the gene has been developed involving a combination of fine-structure deletion mapping and functional gene transfer approaches. The availability of cloned telomerase repressor genes will advance our understanding of telomerase regulation in the human soma and its disruption during human cancer development.

Mouse With Human Chromosome Should Boost Down Syndrome Research

GENETICSOn page [2033][1] of this issue of Science , researchers report creating a strain of mice with a nearly complete copy of human chromosome 21. The strain's unusual genome mimics the genetic makeup of people with Down syndrome, the most common inherited form of mental retardation. [1]: http://www.sciencemag.org/cgi/content/short/309/5743/2033

Recombination between homologous chromosomes does not play a dominant role in the formation of radiation-induced chromosomal aberrations

Purpose : In mammalian cells, the relevance of homologous recombination in radiation-induced double-strand break (DSB) repair is not yet well understood. In the present work, the role of recombination between homologous chromosomes and homology-directed repair of DSB were studied, using X-ray-induced chromosomal aberrations as an end-point. Materials and methods : Human-hamster hybrid cells containing one or two copies of human chromosome 8 were used. If recombination between homologous chromosomes plays a dominant role in DSB repair, it is expected that X-irradiation of cells with two copies of chromosome 8 would result in a lower frequency of aberrations involving this chromosome compared with cells with only one copy of chromosome 8. The aberrations involving human chromosome 8 were detected by fluorescence in situ hybridization (FISH). Furthermore, a comparison between the hamster cell line XR-C1 (defective in non-homologous repair), CHO-9 (the wild-type cells) and the cell line XR-C1#8 (in which the defect of XR-C1 is complemented by human chromosome 8) was made to determine, indirectly, the involvement of homology-directed recombination in DSB repair. Results : The observed frequencies of aberrations per human chromosome 8 were not significantly different between cells containing one or two copies of this chromosome. The frequency of chromatid-type aberrations was doubled in XR-C1 cells compared with CHO-9 and XR-C1#8 cells. Conclusions : In hamster cells, recombination between homologous chromosomes appears not to have a major role in the formation of radiation-induced chromosomal aberrations, while nonhomologous repair seems to be important in both the G1 and G2 phases of the cell cycle.

Construction of chicken x human microcell hybrids for human gene targeting.

Human chromosomes 1, 2, 3, and 11 were tagged with pSV2 neo and transferred via microcell fusion from mouse A9 human monochromosomal hybrids to a chicken pre-B cell line, DT40, proficient for homologous recombination. Hybrids containing two copies of human chromosome 11 were transfected with targeting vectors containing a mammalian selectable gene with either the D11S16 or HRAS genomic sequences corresponding to two different chromosome 11 loci. Analysis of stable transfectants showed a high frequency (approximately 80%) of targeted integration of these constructs into each of the homologous loci of human chromosome 11 in DT40 hybrids. The results suggest that any human genomic sequences on human chromosomes transferred into DT40 cells could be targeted at high frequency, thereby allowing for subsequent modification of human genes and chromosomes.

The use of methylthioadenosine phosphorylase activity to select for human chromosome 9 in interspecies and intraspecies hybrid cells.

Methylthioadenosine phosphorylase (MTAP) is an enzyme that functions in a salvage pathway for adenine synthesis. The locus that encodes MTAP activity has been mapped to human chromosome 9 (9q12-9pter) by analysis of mouse x human somatic cell hybrids. Cells that have MTAP activity will stop proliferating, and eventually die in the presence of azaserine, an inhibitor of de novo purine synthesis, but can be rescued by the addition of methylthioadenosine (MTA) to the culture medium. Some mouse and human tumor cells lack MTAP activity and can not grow in the presence of azaserine and MTA. We fused MTAP competent human fibroblast cells to MTAP deficient mouse L-cells and selected for somatic cell hybrids, containing MTAP activity, in medium containing azaserine and MTA. In a separate experiment, a CHO cell x human fibroblast somatic cell hybrid, containing a normal copy of human chromosome 9, was used to prepare microcells, which were fused to an MTAP-deficient human leukemic cell line, CCRF-CEM. Somatic cell and microcell hybrids were shown to retain human chromosome 9 by fluorescence in situ hybridization using probes that hybridize to the interferon-alpha and -beta 1 genes on human chromosome 9 (9p21), and the centromere of human chromosome 9. This is the first report of complementation for MTAP activity being used to select for somatic cell hybrids and microcell hybrids that retain a human chromosome 9.

Assignment of proteins to human chromosome 21 using two-dimensional gel electrophoresis and somatic cell genetics: An approach to the study of Down syndrome

A mouse hybrid cell line (WA17d) was derived which contained multiple copies of human chromosome 21 and no other human chromosome. Cell extracts of this line were prepared and subjected to two-dimensional gel electrophoresis. Several proteins were identified whose synthesis was altered by the presence of chromosome 21 and 6 proteins were identified as being specific to this human chromosome. These gene products might be involved in the pathogenesis of Down syndrome and be related to the neurologic defects and premature aging seen in this common chromosome abnormality.

Tandem integration of complete and defective SV40 genomes in mouse-human somatic cell hybrids

We have analyzed the arrangement of SV40 DNA sequences integrated in human chromosome 7 in two lines of mouse-human somatic cell hybrids: one containing only one human chromosome 7 per cell and the other containing an average of about three. We found that the integration site differs in both the viral and host sequences in the two clones. However, the sites of integration into the several copies of human chromosome 7 of one clone are identical. Each chromosome 7 in both clones carries approximately six viral genomes tandemly linked. Some of these genomes lack about 20% of the DNA from the late region, including the Eco RI site.

Cell-mediated cytotoxicity to SV40-specific tumour-associated antigens

CYTOTOXIC thymus-derived lymphoctyes (T cells) from mice infected with lymphocytic choriomeningitis virus, ectromelia virus, vaccinia virus, or parainfluenza virus interact only with H–2 compatible virus-infected target cells1–6. When congenic H–2 recombinant mice7 and mice containing mutations within genes mapped at H–2K end specificities6,8 are used, recognition of virus-infected target cells by the effector lymphocytes requires compatibility at either the K or D locus of the H–2 gene complex. These observations led to the hypothesis that cytotoxic T cells recognise either a complex of viral and histocompatibility antigens or virus-induced alterations of the histocompatibility antigens. The same restriction has been described for the cytotoxic T cell response to lymphocytes modified by trinitrophenyl (TNP)9, to minor histocompatibility antigens10,11 and to the male Y antigen12. To investigate the possibility of a similarly restricted specificity of the effector cells of cell-mediated immunity to tumours, cytotoxic cells specific for SV40 tumour-associated specific antigens (SV40-TASA) were generated in two inbred strains of mice, C57BL/6J and BALB/cAn. The SV40-transformed lines C57SV (SV40-transformed C57BL/6 mouse embryo fibroblasts), MKS/Bu100 (SV40-transformed BALB/c kidney cells13) and LN-SV (SV40-transformed human skin fibroblasts14) were used as immunising and target cells (Table 1). In addition, clones of human–mouse somatic cell hybrids, obtained by fusion of LN-SV and mouse peritoneal macrophages from inbred strains15 were used. These hybrid clones contain the complete mouse genome and, in addition, one to several copies of human chromosome 7, in which the SV40 genome is presumably integrated14,15. C121, N8 and N9 cells are derived from LN-SV fused with C57BL/6 macrophages16 and C136 cells from LN-SV fused with BALB/c macrophages17. These clones express the SV40 tumour (T) antigen (a nuclear antigen)16,17, and the histocompatibility antigens of the murine parental cells (our unpublished results). Neither LN-SV nor any hybrid clones derived from LN-SV make infectious SV4018. Only after fusion with permissive monkey kidney cells can some SV40 particles be rescued. This defective virus exhibits no biological activity. Both LN-SV and human–mouse somatic cell hybrids containing the human chromosome 7 derived from LN-SV exhibit tumour-specific transplantation antigens (TSTA), as demonstrated by their ability to protect SV40-inoculated newborn hamsters from developing SV40 tumours19. Both the SV40-transformed mouse cell lines (our unpublished results) and the hybrid clones16,17 are tumorigenic in immunodeficient “nude” mice, but these cells do not grow routinely in immunocompetent syngeneic mice, probably because of strong TSTA20.