Chromosomes In Somatic Cell Hybrids Research Articles

Conversion Technology for the Separation of Maternal and Paternal Copies of Any Autosomal Chromosome in Somatic Cell Hybrids

Conversion Technology (CT) is a streamlined version of somatic cell hybrid preparation that was developed to be amenable for use as a mutation detection method and as a diagnostic tool for individual patients. It is also a powerful research tool. There are two broad categories of potential applications for CT: research applications in gene mapping and identification, and clinical applications in the detection of disease-causing mutations in individual patients. CT may emerge as the gold standard for both mutation detection and haplotyping. It will likely prove valuable in mutation detection due to its ability to detect mutations that are not amenable to detection by sequencing (e.g., deep intron or promoter mutations). It will likely prove valuable in gene identification projects because genotyping of haploid chromosomes yields unequivocal haplotypes.

Epigenetic predisposition to expression of TIMP1 from the human inactive X chromosome

BackgroundX inactivation in mammals results in the transcriptional silencing of an X chromosome in females, and this inactive X acquires many of the epigenetic features of silent chromatin. However, not all genes on the inactive X are silenced, and we have examined the TIMP1 gene, which has variable inactivation amongst females. This has allowed us to examine the features permitting expression from the otherwise silent X by comparing inactive X chromosomes with and without TIMP1 expression.ResultsExpression was generally correlated with euchromatic chromatin features, including DNA hypomethylation, nuclease sensitivity, acetylation of histone H3 and H4 and hypomethylation of H3 at lysines 9 and 27. Demethylation of the TIMP1 gene by 5-azacytidine was able to induce expression from the inactive X chromosome in somatic cell hybrids, and this expression was also accompanied by features of active chromatin. Acetylated histone H3 continued to be observed even when expression was lost in cells that naturally expressed TIMP1; while acetylation was lost upon TIMP1 silencing in cells where expression from the inactive X had been induced by demethylation. Thus ongoing acetylation of inactive X chromosomes does not seem to be simply a 'memory' of expression.ConclusionWe propose that acetylation of H3 is an epigenetic mark that predisposes to TIMP1 expression from the inactive X chromosome in some females.

Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines

X inactivation requires XIST, a functional RNA that is expressed exclusively from, and localizes to, the inactive X in female somatic cells. In mouse, low-level unstable transcription of Xist is observed prior to the time of inactivation, and an antisense transcript, Tsix, is a critical regulator of early Xist expression. To examine the presence and impact of an antisense transcript in humans we have characterized the extent of sense and antisense transcription in human somatic, transgenic, and embryonal carcinoma (EC) cell lines. Downstream antisense expression at the human XIST locus was not detected in somatic cells, but was detected in the EC line N-Tera2D1 and in somatic cells with an ectopic XIST locus. Presence of the antisense did not disrupt the stability or localization of the sense transcript. We have also identified additional sense transcripts in EC and female somatic cells and demonstrate that the 5′ flanking JPX/ENOX gene is expressed from both the active and the inactive X chromosome in somatic cell hybrids, delimiting the extent of inactive X-specific transcriptional control in somatic cells. These analyses reveal similarities to and differences from the murine Xist and Tsix transcripts and generate a complex picture of developmentally regulated transcription through the region.

Detection of human chromosomes in somatic cell hybrids by PCR analysis.

The detection of human chromosomes in somatic cell hybrids is usually made by chromosomal analysis, Southern blot analysis with human probes, and starch-gel electrophoresis of isoenzymes. We describe here a new, quick, and very efficient method to detect human chromosomes in somatic cell hybrids between human and rodent (rat and mouse) cells. The method is based on the polymerase chain reaction to promote amplification of human DNA, using primers derived from localized genes or DNA fragments from each human chromosome.

An evaluation of the inactive mouse X chromosome in somatic cell hybrids.

The expression of mouse Zfx, Rps4, Ube1x, and Xist was evaluated in hamster-mouse somatic cell hybrids containing either an active or an inactive mouse X chromosome using polymerase chain reaction of reverse transcribed RNA (RT-PCR). The results showed that Zfx, Rps4, and Ube1x are expressed exclusively from the active mouse X, while Xist is expressed exclusively from the inactive X. These findings confirm the pattern of X inactivation for these mouse genes reported previously based on expression in somatic tissues of F1 females from interspecific crosses. These results demonstrate the existence of differences between human and mouse X inactivation, as the corresponding human genes, ZFX, RPS4X, and UBE1 escape X inactivation.

Molecular detection of altered X-inactivation patterns in the diagnosis of genetic disease.

It is widely assumed that when a female carrier of a genetic disorder exhibits clinical signs of the disorder it is due to chance non-random X-inactivation in particular tissues. Recently molecular methods have become available for the analysis of X-chromosome inactivation status. These are based either on the methylation patterns of DNA from the active and inactive chromosomes or on the rescue of active X chromosomes in somatic cell hybrids. As a consequence of the molecular studies, it has become obvious that there are some special cases of non-random X-inactivation patterns. These include females carrying X-linked immunodeficiencies and, sometimes, one of a pair of identical female twins.

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flowsorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.

Differential staining of human and murine chromatin in situ by hybridization with species-specific satellite DNA probes

Human and murine chromatin was differentially labeled by hybridization with DNA probes that bind to species-specific satellite DNA. The targets for in situ hybridization were the mouse-specific major or gamma satellite DNA and the human alpha satellite DNA. These sequences typically are localized at or near the chromosome centromeres, and remain their tight localization throughout the cell cycle. DNA probes were synthesized in vitro by primer directed DNA amplification using the polymerase chain reaction. In typical applications like the differentiation of cells derived from chimeric animals or the characterization of chromosomes in somatic cell hybrids, the two DNA probes are differently labeled and detected using label-specific reagents that fluoresce at different wavelengths. The rapid technique for chromatin discrimination described here combines high specificity with unprecedented signal intensity.

Identification of equine chromosomes in horse x mouse somatic cell hybrids.

Giemsa-11 (G-11) staining and in situ hybridization were used to identify the equine chromosome complement of horse x mouse somatic cell hybrids. The presence of horse chromosomes in somatic cell hybrids was determined by differential G-11 staining. The slides were then destained and hybridized with biotinylated total horse (Equus caballus) genomic DNA without suppression. Fluorescence detection permitted rapid confirmation of horse chromosomal DNA in the hybrid cells.

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequence as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

“PCR-Karyotype” of human chromosomes in somatic cell hybrids

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes was performed by the polymerase chain reaction (PCR) using primers directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a “PCR-karyotype.” This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosomes produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.

Heterogeneity of clinical severity and molecular lesions in Aircardi syndrome

All patients with Aicardi syndrome are female or have a 47,XXY karyotype. This finding, along with a report of an Aicardi syndrome patient with an Xp22/autosome translocation, led to the hypothesis that Aicardi syndrome might be caused by an X-linked dominant, male-lethal mutation on the short arm of the X chromosome. To study this hypothesis, we investigated X chromosome inactivation patterns in peripheral lymphocytes from seven patients. We used two methods: methylation-sensitive restriction enzyme analysis and segregation of the active X chromosome in somatic cell hybrids. We found that three of seven cytogenetically normal girls with Aicardi syndrome had profoundly skewed X-inactivation in their lymphocytes, supporting the concept that Aicardi syndrome is X linked. Three of the five girls with the greatest degree of psychomotor retardation and the poorest seizure control had skewed X-inactivation. In contrast, the two highest-functioning children had random X-inactivation. We screened DNA using eight polymorphic probes from the Xp22 region but were unable to identify a deletion in any of the seven patients. Nonrandom X-inactivation in lymphocytes and possibly other tissues in some, but not all, patients with Aicardi syndrome may reflect heterogeneity of their molecular lesions.

Novel use of a chimpanzee pseudogene for chromosomal mapping of human cytochrome [formula omitted] oxidase subunitIV

We have isolated a chimpanzee processed pseudogene for subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1) by screening a chimpanzee genomic library in λCharon 32 with a bovine liver cDNA encoding COX subunit IV (COX IV), and localized it to a 1.9-kb HindIII fragment. Southern-blot analysis of genomic DNA from five primates showed that DNAs from human, gorilla, and chimpanzee each contained the 1.9-kb pseudogene fragment, whereas orangutan and pigtail macaque monkey DNA did not. This result clearly indicates that the pseudogene arose before the divergence of the chimpanzee and gorilla from the primate lineage. By screening Chinese hamster × human hybrid panels with the human COX4 cDNA, we have mapped COX4 genes to two human chromosomes, 14 and 16. The 1.9-kb HindIII fragment containing the pseudogene, COX4P1, can be assigned to chromosome 14, and by means of rearranged chromosomes in somatic cell hybrids, to 14q21-qter. Similarly, the functional gene, COX4, has been mapped to 16q22-qter.

Mapping human chromosomes in somatic cell hybrids using a low-copy-number repetitive sequence.

We have isolated a human-specific repetitive sequence with a copy number of several hundred (p11L26). Using Southern blots of EcoR1-digested DNA from somatic cell hybrids containing one or a few human chromosomes, band patterns specific for those chromosomes can be generated by hybridization with p11L26. Genomic copies of the sequence have also been mapped to subchromosomal regions using translocations. The probe offers a useful addition to the standard techniques for mapping human chromosomes in hybrid cell lines.

Detection of species specific chromosomes in somatic cell hybrids.

We describe an in situ hybridization technique which allows rapid identification of species-specific chromosomes in somatic cell hybrid lines. Chromosome preparations from rodent-human hybrid lines are hybridized to biotinylated total human DNA which is subsequently detected by a series of immunocytochemical reactions which culminate in a peroxidase reaction visible by light microscopy. This technique not only allows identification of intact human chromosomes but also fragmented and rearranged human chromosomal segments. We have detected as little as 1 X 10(7) bp of human DNA inserted into a mouse chromosome using this procedure and estimate that the sensitivity of the technique would allow detection of sequences 5- to 10-fold smaller. The usefulness of the technique for screening hybrid cell gene mapping panels is discussed.

Expression of fragile site on the human X chromosome in somatic cell hybrids between human fragile X cells and thymidylate synthase-negative mouse mutant cells.

Fragile sites appear to be associated with a higher rate of breakage and specific chromosome rearrangement in cancer. A fragile site located on the human X chromosome at band Xq27 is known to be expressed under conditions of thymidylate stress. In order to obtain a model cell system suitable for studying the mechanism of expression of the fragile X site, interspecific somatic cell hybrids were constructed by cell fusion between human skin fibroblasts derived from a male patient with fragile X-linked mental retardation and thymidylate synthase-negative mouse mutant cells. The primary isolated hybrid clones were thymidine-prototrophic and expressed the fragile X site under conditions of thymidylate stress caused by 5-fluoro-2'-deoxyuridine treatment. In a thymidine-auxotrophic hybrid clone segregated from a thymidine-prototrophic hybrid clone, the expression of the fragile X site was induced in thymidylate stress conditions achieved by thymidine deprivation alone. This result provides direct evidence that expression of the fragile X site is dependent upon a lowered supply of thymidylate.

Wilms' tumor-aniridia association: segregation of affected chromosome in somatic cell hybrids, identification of cell surface antigen associated with deleted area, and regional mapping of c-Ha-ras-1 oncogene, insulin gene, and beta-globin gene.

Fusion of an auxotrophic mutant hamster cell with the skin fibroblasts of a child with the Wilms' tumor-aniridia association produced clones which, on the one hand, contained the child's normal chromosome 11 and, on the other, the chromosome 11 with the 11p13 deletion associated with the syndrome. Both hybrids were positive for human LDH-A by enzymatic assay. Clones containing the normal human chromosome 11 were killed by a cytotoxic monoclonal antibody to a cell surface antigen previously mapped to the 11p13----11pter region of chromosome 11. Clones with the abnormal 11 were not killed. Thus, we have produced hybrids from the same patient distinct from each other on the basis of their chromosome 11. These hybrids have been used to map the locus for a cell surface antigen to the deleted region on chromosome 11 of a patient with the Wilms tumor-aniridia association. The linkage between this antigen and the syndrome should be helpful in further study of the genetics of this disease. In addition, we have found that the c-Ha-ras-1 oncogene is distal to the p13 region of chromosome 11 and the position of insulin and beta-globin on the chromosome. Finally, by producing segregants of the hybrids containing the abnormal chromosome 11, we have provided evidence that chromosome 11-associated c-Ha-ras-1 is syntenic with chromosome 11 and not moved to a different portion of the genome.

Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids

We have analysed metaphase chromosomes of man-mouse somatic cell hybrids using a sequential staining procedure involving the fluorescent DNA-binding stains, Hoechst 33258 and quinacrine mustard. This was found to be a simple and reliable means of differentiating the chromosomes of the two species and of identifying specific human chromosomes. In addition, this method will permit the study of the segregation of human chromosome homologues that are discordant for quinacrine mustard fluorescent polymorphisms.

The persistence of chromosomes in somatic cell hybrids correlates with the enzymatic hypermethylation of their dna

1. 1. Somatic cell hybrids have been constructed between A9 mouse L fibroblasts and Ehrlich ascites tumor cells. These hybrids carry all or nearly all of the parental chromsomes. In mouse-swine hybrid lines constructed between mouse A9 L fibroblasts and porcine pancreatic cells the swine chromosomes tend to be eliminated after several cell divisions. 2. 2. Factor(s) responsible for the stability or elimination of chromosomes is (are) generally unknown. It is possible, however, that a modification and restriction like system may be involved. 3. 3. In a search for such a process, the enzymatic methylation of DNA in somatic cell hybrids was compared with that in parental cells. The extent of enzymatic methylation of DNA was analysed by the restriction enzymes HpaII and MspI which cleave at the sequence 5'-CCGG-3'. 4. 4. The endonucleolytic activity of Hpall is inhibited if the internal cytosine has been enzymatically methylated prior to restriction. MspI is not inhibited by this modification. 5. 5. Total extent of 5-methylcytosines in the nuclear DNA was estimated by incorporation of [ 14C]deoxycytidine derived radioactivity in 5-methylcytosines and cytosines of DNA. 6. 6. Although no information about the extent of methylation of DNA in eliminated chromosomes could be obtained, the finding that chromosomes persisting in somatic cell hybrids contain hypermethylated DNA is compatible with the existence of a modification and restriction like system in eukaryotic cells.

Isozyme studies on the association of the herpes simplex virus type 1 thymidine kinase gene with human chromosomes in somatic cell hybrids

To investigate chromosomal site(s) of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in biochemically transformed [HeLa(BU25)/KOS 8-1] cells, these human cells which had been transformed by ultraviolet light-irradiated HSV-1 were fused with TK-negative mouse LM(TK-) cells, human-mouse somatic cell hybrid clones (LH81 clones 1-20) were isolated by HATG-ouabain selection and their chromosomes and isozymes were analyzed. Electrophoretic and serological analyses showed that all 20 clones expressed type-specific HSV-1 TK. Isozyme analyses on 29 gene-enzyme systems representing 22 human chromosomes revealed that all of the HSV-1 TK-positive clones expressed human aminoacylase-1 (ACY-1) and esterase D (ESD), which have been mapped to human chromosomes 3 and 13, respectively. Other human isozymes were detected in only one to four clones or in none of the clones. Chromosome analyses showed that: (1) the hybrid clones retained only a few human chromosomes; (2) a marker chromosome, designated M7, consisting of a chromosome 17 translocated to the short arm of chromosome 3, occurred in 36 out of the 41 metaphases examined of LH81-4 clones 1 to 4 and in 31 out of the 33 metaphases examined of LH81-12 clone 10; (3) a modified M7 chromosome, (M7/m), in which the distal 2/3 of the long arm of M7 was translocated to a small acrocentric mouse chromosome, was the only human chromosome found in metaphases of LH81-13 clone 17; and (4) an intact human chromosome 13 was not present in LH81-12 clone 10 or LH81-13 clone 17 cells. Counterselection with BrdUrd resulted in the isolation of subclones lacking HSV-1 TK, human ACY-1 and ESD, and the human marker M7 chromosomes. The experiments indicate that the HSV-1 TK gene is probably associated in HeLa (BU25)/KOS 8-1 cells with marker chromosome M7, but the possibility is not excluded that the segment of human chromosome 13 which codes for ESD is involved.