DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization

We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Biochemical and genetic studies of two Heterodera avenae resistance genes transferred from <Emphasis Type="Italic">Aegilops ventricosa</Emphasis> to wheat

Two Heterodera avenae resistance genes, Cre2 from Aegilops ventricosa AP-1 and Cre5 from Ae. ventricosa #10, were shown to confer a high level of resistance to the Spanish pathotype Ha71. No susceptible plants were found in the F(2) progeny from the cross between the two accessions of Ae. ventricosa, suggesting that their respective resistance factors were allelic. However, genes Cre2 and Cre5 apparently were transferred to a different chromosomal location in the wheat line H-93-8 and in the 6M(v)(6D) substitution, respectively, as proved by F(2) segregation of their cross progeny. The induction of several defence responses during early infection by the same H. avenae pathotype in resistant lines carrying Cre2 or Cre5 genes was studied. Isoelectrofocusing (IEF) isozyme analysis revealed that peroxidase, esterase and superoxide dismutase activity increased after nematode infection, in roots of resistant lines in comparison with their susceptible parents. Differential induced isoforms were also identified when IEF patterns of resistant lines were compared. A DNA marker, absent in Cre5-carrying genotypes, was found to be linked, thought not very tightly, to the Cre2 gene in the H-93-8 line. The differences observed between the Cre2 and Cre5 genes with respect to their chromosomal location in wheat introgression lines, de-toxificant enzyme induction and behaviour against different pathotypes, suggest they are different H. avenae resistance sources for wheat breeding.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.     

High-resolution analysis of chromosomal imbalances using the Affymetrix 10K SNP genotyping chip

Array-based comparative genome hybridization is a powerful tool for detecting chromosomal imbalances at high resolution. However, the design and setup of such arrays are time consuming and expensive and thus worthwhile only when large numbers of arrays will be processed. To provide a feasible solution, we have developed an algorithm that renders the publicly available Affymetrix 10K SNP genotyping chip useful for high-resolution analysis of chromosomal imbalances. We have used our newly developed algorithm to analyze data from Affymetrix 10K chips that were hybridized with DNA probes from a variety of different sources, such as primary tumors, cell lines, and blood from patients with unbalanced translocations. In summary, we were able to (i) demonstrate the capability of our method by reproduction of published and unpublished data obtained with alternative methods and (ii) identify novel imbalances that were not shown before.     

Identification and classification of chromosomal aberrations in human induced pluripotent stem cells

Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.     

Normal inhibition of DNA synthesis following gamma-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

Inhibition of DNA synthesis was studied in gamma-irradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of gamma-rays in all of the cell lines. 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to gamma-rays. Contrary to other data in the literature these results demonstrate that radioresistant DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivity.     

The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes.

The chromosomal distribution of murine genes expressed during differentiation of skeletal muscle cells was determined by Southern blot analysis of DNA from mouse-Chinese hamster hybrid cell lines containing incomplete subsets of mouse chromosomes. All detectable myosin heavy chain genes are located on chromosome 11. The gene for the myosin light chain 2 is located on chromosome 7. The skeletal muscle alpha-actin gene and several other actin genes, or pseudogenes, are located on chromosome 3. Additional actin DNA sequences are distributed on other mouse chromosomes.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Chromosomal assignment of human genes for gastrin, thyrotropin (TSH)-beta subunit and C-erbB-2 by chromosome sorting combined with velocity sedimentation and Southern hybridization

Human genes for gastrin, thyrotropin (THS)-beta subunit and c-erbB-2 were assigned to specific chromosomes using a single-laser cell sorter. For this purpose, condensed human chromosomes prepared from a karyotypically normal lymphoblastoid cell line were preliminarily fractionated by velocity sedimentation, and then sorted using a fluorescence-activated cell sorter. DNA was then extracted from the chromosomes, cleaved by restriction enzymes, and subjected to Southern hybridization using gene-specific radioactive probes. When the assignment of specific chromosomes was not possible due to chromosomal size overlapping, sorted chromosomes from cell lines carrying chromosomal translocation or from hybrid cells carrying known human chromosomes were used in addition. The results indicate that human genes for gastrin, TSH-beta, and c-erbB-2 are located on chromosomes 17, 1 and 17, respectively.     

Maternal and paternal chromosomes 7 show differential methylation of many genes in lymphoblast DNA

Genomic imprinting, the differential expression of paternal and maternal alleles, involves many chromosomal regions and plays a role in development and growth. Differential methylation of maternal and paternal alleles is a hallmark of imprinted genes, and thus methylation assays are widely used to support the identification of novel imprinted genes. Either blood or lymphoblast DNAs are most often used in these assays, even though methylation levels may change in cell culture. We undertook a systematic survey of parent-of-origin-specific methylation of chromosome 7 genes and ESTs by comparing DNA samples from cases of maternal and paternal uniparental disomy for chromosome 7 using DNA from fresh blood and lymphoblast cell lines. Our results revealed that up to 41% of genes and ESTs show parent-of-origin-specific methylation differences in lymphoblast DNA after only a short time in culture, whereas methylation differences were not seen in blood DNA. The methylation changes occurred most commonly on paternal chromosome 7, whereas alterations on maternal chromosome 7 were more infrequent and weaker. These findings indicate that methylation patterns may change significantly during cell culture in a parent-of-origin-dependent manner and suggest that methylation is maintained differently on maternal and paternal chromosomes 7.     

A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer

The relationship between the apparently random chromosomal changes found in aneuploidy and the genetic instability driving the progression of cancer is not clear. We report a test of the hypothesis that aneuploid chromosomal abnormalities might be selected to preserve cell-survival genes during loss of heterozygosity (LOH) formations which eliminate tumor suppressor genes. The LOHs and structurally abnormal chromosomes present in the aneuploid LoVo (colon), A549 (lung), SUIT-2 (pancreas), and LN-18 (glioma) cancer cell lines were identified by single nucleotide polymorphisms (SNPs) and Spectral Karyotyping (SKY). The Mann-Whitney U and chi square tests were used to evaluate possible differences in chromosome numbers and abnormalities between the cell lines, with two-tailed P values of <0.01 being considered significant. The cell lines differed significantly in chromosome numbers and frequency of structurally abnormal chromosomes. The SNP analysis revealed that each cell line contained at least a haploid set of somatic chromosomes, consistent with our hypothesis that cell-survival genes are widely scattered throughout the genome. Further, over 90% of the chromosomal abnormalities seemed to be selected, often after LOH formation, for gene-dosage compensation or to provide heterozygosity for specific chromosomal regions. These results suggest that the chromosomal changes of aneuploidy are not random, but may be selected to provide gene-dosage compensation and/or retain functional alleles of cell-survival genes during LOH formation.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Characterisation of a bovine/murine hybrid cell panel informative for all bovine autosomes

A bovine/murine hybrid cell panel consisting of 57 cell lines was typed with 124 markers by PCR, Southern hybridisation and isozyme analysis in order to establish its utility as a resource for genome mapping. All bovine chromosomes, including the sex chromosomes were represented in the panel. Computerised analysis of syntenies indicated that there are no cell lines containing only a single bovine chromosome. The panel was used to map 10 new bovine microsatellite markers, and the MYL6 and CPE genes. This panel is informative for all bovine chromosomes other than the sex-specific region of the X chromosome and can be used in synteny mapping studies. At present, due to the relatively small number of markers typed, the resolution of the panel does not go beyond the chromosomal level.     

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose [gamma] irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.