Use Of Somatic Cell Hybrids Research Articles

Francis H. Ruddle (1929–2013): A Pioneer in Human Gene Mapping

Frank was born August 19, 1929, in West New York, New Jersey, to immigrant parents. It was said that his Welsh-English heritage defined his personality and his unique sense of humor. He joined the Air Force in 1946 to serve our country before finishing high school. After excellent service, he left the Air Force in 1949. The GI Bill helped him to obtain higher education, and he obtained his BA and MS degrees from Wayne State University. He went on to the University of California at Berkeley, where he obtained his PhD degree in zoology in 1960. Frank began his research while in Detroit, and he was involved in isolating epithelial cells from different sources, including tumors, and analyzing them. He pursued his interest in mammalian cell culture at Berkeley, where he trained with Morgan Harris and worked on cell culture and cytogenetics of pig kidney cells. Frank went on to do postdoctoral work with John Paul at the University of Glasgow in Scotland (1960–1961). He was recruited to a faculty position in the Biology Department at Yale University, where he remained throughout his career. Frank began to work on mouse genetics and began studying variant forms of mammalian enzymes (isozymes). Some of the early work was conducted in collaboration with a fellow Welshman, Tom Roderick of the Jackson laboratory. The two of them became lifelong friends. Frank’s first graduate student at Yale was Charlotte Boone, who started work on the use of somatic cell hybrids that sustained his laboratory for many years to come. Frank brought his skills with cell culture, analysis of isozymes, and cytogenetic skills to begin a program on mapping human genes. In a landmark paper that was published in Nature in 1970, Frank was able to use somatic cell hybrids between mouse and human cells to establish linkage between genes coding for lactate dehydrogenase A and B and Peptidase B. The work on the use of somatic cell hybrids to establish linkage between genes coding for enzymes and assign these genes to individual chromosomes went through an explosive phase in his laboratory for many years afterward. I had the privilege of joining his laboratory in 1972, where I stayed for 3 years. From my perch there, I was able to watch the rapid expansion of human gene mapping, which sustained my interest for many years to come, including my participation in the human genome mapping and sequencing effort in the 1990s and during the early phase of the 21st century.

Targets of extinction: identification of genes whose expression is repressed as a consequence of somatic fusion between cells representing basal and luminal mammary epithelial phenotypes.

The use of somatic cell hybrids has led to an increased understanding of the 'negative' regulation of cellular phenotype. Using somatic cell hybrids constructed between human breast cells that represent differing stages of malignancy but also display differing phenotypes from the same tissue, we present experimental results suggesting that luminal epithelial characteristics are controlled by repressive mechanisms. Fusion of HBL 100 cells, non-tumorigenic and characteristic of the basal cell lineage, with MCF-7 or MDA-MB-468 malignant breast cancer cells, characteristic of the luminal lineage, resulted in hybrid cells that displayed the phenotype of the HBL 100 cells. Using representational difference analysis, a panel of genes whose expression was repressed in the hybrid between HBL 100 and MDA-MB-468 was identified. This analysis revealed markers of luminal epithelial cells to be repressed, including Ep-CAM, cytokeratin 19 and E-cadherin. These markers were found to be coordinately re-expressed in variant hybrid cells indicating that the observed repression is reversible. Integrin (alpha)(v)(beta)(3) expression was found to be in mutual exclusivity to the luminal epithelial markers, thereby revealing a bidirectional 'switch' in the pattern of gene expression in this system. Finally, the expression of Ep-CAM was found to be lost in heterokaryons produced by fusion of HBL 100 and MCF-7 or MDA-MB-468 cells suggesting that the extinction of this gene in hybrid cells is the consequence of a trans-acting factor(s) synthesized by the HBL 100 cells. These data suggest that a number of markers of luminal cell differentiation in the mammary gland can be controlled through negative mechanisms and that such control of phenotype is highly coordinated.

A model system to study genomic imprinting of human genes.

Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study known imprinted genes and to identify as-yet-unknown imprinted genes. Testing gene expression by using reverse transcription followed by PCR, we show that functional imprints are maintained at four previously characterized 15q11-q13 loci in hybrids containing a single human chromosome 15 and at two chromosome 11p15 loci in hybrids containing a single chromosome 11. In contrast, three gamma-aminobutyric acid type A receptor subunit genes in 15q12-q13 are nonimprinted. Furthermore, we have found that differential DNA methylation imprints at the SNRPN promoter and at a CpG island in 11p15 are also maintained in somatic-cell hybrids. Somatic-cell hybrids therefore are a valid and powerful system for studying known imprinted genes as well as for rapidly identifying new imprinted genes.

Localization of PiUS, a stimulator of cellular phosphate uptake to human chromosome 3p21.3.

A novel gene, PiUS, was recently cloned and shown to increase phosphate uptake when expressed in oocytes, indicating that it may be an important regulator of cellular phosphate homeostasis. The phosphate wasting disease autosomal dominant hypophosphatemic rickets (ADHR) was previously mapped to chromosome 12p13 by linkage analysis. PiUS' role as a modulator of phosphate transport, as well as its intestinal and renal expression made the gene an appropriate candidate for ADHR. The purpose of our study was to determine the chromosomal localization of the human PiUS gene through the use of somatic cell hybrids and radiation hybrid mapping. In the present work, PiUS was localized to human chromosome 3p21.3 and is therefore not the ADHR gene.

Mapping of the Gene for the p60 Subunit of the Human Chromatin Assembly Factor (CAF1A) to the Down Syndrome Region of Chromosome 21

Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescencein situhybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakovet al.(1992,Nature359: 380) YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome.

CpG island clones for Chromosome 11p?a resource for mapping and gene identification

A NotI end fragment library has been constructed for human Chromosome (Chr) 11p. Seventy-two clones were mapped to chromosomal subregions by use of somatic cell hybrids. The clones detect 44 different CpG islands, and we have isolated cosmid contigs for 36 of them. Extrapolation from the known 11p13 NotI restriction map suggests that every second CpG island from 11p containing a Not site is already represented in the clone collection. By sequence analysis all of the 11p13 clones exhibit typical features of CpG islands, and cross-species hybridization has been detected with at least one fragment in most cases. The cosmids serve as valuable linking clones for long-range restriction mapping. They also provide excellent starting material for transcript isolation procedures to identify genes on chromosome 11p associated with developmental anomalies and various tumor types. Several transcribed sequences have already been isolated with some of these clones.

The structural genes, MEP1A and MEP1B, for the α and β subunits of the metalloendopeptidase meprin map to human chromosomes 6p and 18q, respectively

Meprins are cell membrane, oligomeric metalloendo-peptidases composed of two distinct but evolutionarily related subunits, α and β. The structural genes for the meprin subunits, Mep-1α and Mep-1β, have been previously mapped to chromosomes 17 and 18, respectively, of the mouse genome. We now report the localization of MEP1A and MEP1B in the human genome. MEP1A mapped to the short arm of chromosome 6 by the use of radiation and somatic cell hybrids. More specifically, it is localized between the centromere and GSTA2 in 6p11–p12. MEP1B mapped to chromosome 18, by the use of somatic cell hybrids, in 18q12.2–q12.3, proximal to the TTR/PALB gene. As in the mouse genome, the two homologous human structural genes for α and β (50% identical on the cDNA level) are unlinked. These new markers on human chromosomes 6 and 18 extend the region of known linkage homology with mouse chromosomes 17 and 18, respectively, and provide new molecular access to regions of the human genome.

Use of Somatic Cell Hybrids and Fluorescence in Situ Hybridization to Localize the Functional Serum Amyloid A (SAA) Genes to Chromosome 11p15.4-p15.1 and the Entire SAA Superfamily to Chromosome 11p15

The serum amyloid A (SAA) superfamily consists of two acute phase genes, SAA1 and SAA2; a pseudogene, SAA3; and a constitutively expressed gene, SAA4. The SAA proteins, which are found associated with high-density lipoprotein, are believed to have an essential function. Chronic infection, inflammation, or trauma causes very high levels of the acute phase SAA proteins. This may result in the potentially fatal condition, amyloidosis, in which amyloid fibrils are deposited in the essential organs. Somatic cell hybrids have been used by several groups to map one or more of the SAA genes to chromosome 11p. We used FISH analysis and PCR amplification of DNA from 17 somatic cell hybrids carrying all or part of chromosome 11 as their only human component to fine map systematically the chromosomal location of the entire SAA superfamily. We demonstrate by these methods that the location of the entire SAA superfamily is at 11p15. Furthermore, we have demonstrated that SAA1, SAA2, and SAA4, i.e., all of the functional genes of the superfamily, map within this region to chromosome 11p15.4-p15.1.

Genomic organization and chromosomal localization of the human CTP synthetase gene ( CTPS)

To elucidate the organization of the human genomic sequences encoding CTP synthetase (CTPS), fragments homologous to the cDNA were isolated from genomic λ libraries. The fragments cloned were overlapping and cover over 40 kb. Cotransfection of the DNAs into CTPS-deficient, cytidine-requiring CHO mutants can transform them to cytidine-independent growth, indicating that the complete structural gene has been isolated. Direct sequencing and enzymatic amplification of the cloned genomic fragments revealed that the coding sequences are distributed to 19 exons covering about 35 kb. Multiple transcriptional start sites were detected by primer extension in a G+C-rich 5′ flanking sequence that is separated from the translational start by an ∼3-kb intron. A panel of human-rodent somatic cell hybrids and the CTPS cDNA were used to assign the structural gene to the short arm of human chromosome 1. This assignment was further refined through the use of somatic cell hybrids bearing fragments of the short arm of the chromosome, allowing localization to 1p36.11-p31, a region notable for its disruption in many types of tumors.

Characterization of a set of variable number of tandem repeat markers conserved in Bovidae

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.

Use of somatic cell hybrids in gene mapping and expression studies: Non isotopic ISH and PCR-technique.

Use of somatic cell hybrids in gene mapping and expression studies: Non isotopic ISH and PCR-technique.

Homologs of eleven loci on human chromosome 11 assigned to two owl monkey chromosomes

We localized 11 loci mapped to human chromosome 11 to two chromosomes, 4 and 19 of owl monkey karyotype VI (2n = 49/50), by the use of somatic cell hybrids. Furthermore, using in situ hybridization to chromosomes of two owl monkey karyotypes, the HSTF1 oncogene locus was precisely localized on homologs 19q (K-VI) and 2q (K-II). Comparative analysis of available gene-mapping data among human, mouse, and owl monkey chromosomes revealed a pattern of evolutionary change in a syntenic group on human chromosome 11. These structural changes could be explained as having derived from a pericentric inversion of human chromosome region 11cen----q13 and a translocation involving human region 11q22----qter during primate evolution.

Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.

Biochemical and genetic analysis of the OKa blood group antigen.

The monoclonal antibody TRA-1-85 recognizes a cell surface antigen which is expressed by all human cell types tested, including red blood cells (RBCs), but not by mouse cells. All the human RBCs tested were TRA-1-85 positive except those with the rare phenotype Ok(a-). Oka is a blood group antigen of very high frequency and only three unrelated Ok(a-) people are known. The red cells of all three propositi were negative with the TRA-1-85 antibody. To confirm the relationship between the TRA-1-85 antibody and anti-Oka, the immune antibody found in the serum of Ok(a-) individuals, Western blot analysis was used: the TRA-1-85 antibody and anti-Oka gave identical but complex patterns of reactivity in Western blot analysis of human cell lysates or membranes. This suggests that the anti-Oka and TRA-1-85 antibodies recognize the same cell-surface determinant and implies that Oka is not restricted in its expression to the surface of RBCs but is expressed on white blood cells (WBCs) of Ok(a+) individuals and all human cell lines tested to date. WBCs from one of the Ok(a-) propositi were tested and found to be negative with the TRA-1-85 antibody. Finally, the species specificity of the TRA-1-85 antibody has been exploited by the use of somatic cell hybrids and DNA transfection techniques to examine the genetic control of the Oka antigen defined by the TRA-1-85 antibody. We report that the determinant is controlled by a single gene OK present on human chromosome 19.

Characterization and use of somatic cell hybrids with interspecific translocations involving the human X chromosome.

Two hybrid cell lines, whose only human material was a portion of the X translocated on to a mouse chromosome, have been characterized by cytogenetics, in situ hybridization and Southern blotting. In one hybrid (HORL911R8B) the region Xpter to Xq2(2-4) was identified. In the other (PIP) the single human fragment was found to contain sequences from two separate X chromosomal regions (corresponding approximately to Xp11.4-Xp22.1 and Xq26-Xqter). These two hybrids in combination with a third (WAG 8) retaining Xqter to Xp21 as a human X-autosome translocation chromosome, form a mapping panel for rapid subregional assignments to the human X chromosome. This mapping panel has been used to provide information about the order of DNA sequences derived from the X chromosome and to provide an assignment for an anonymous DNA segment, M201 gamma, to Xp11.4-Xp21.1.

Measurement of low levels of x-ray mutagenesis in relation to human disease.

We previously demonstrated that conventional methods for measurement of mutagenesis in mammalian cells are subject to serious error that causes underestimation of environmental contributions to cancer and genetic disease. This error has been corrected by use of somatic cell hybrids containing a single human chromosome on which the marker genes are carried and by using doses of mutagenic agents so low that little cell killing occurs. This method permits direct measurement of the effects of low doses of radiation and other mutagens without resort to the controversial extrapolation procedure customarily used to estimate effects of doses in the neighborhood of actual human exposures. The new data demonstrate that the true mutagenesis efficiency at the low doses of ionizing radiation that approximate human exposures is more than 200 times greater than those obtained with conventional methods. This methodology also permits evaluation of localized mutations, large and small chromosomal deletions, and nondisjunctional processes and can be used for mutagens that need metabolic activation as well as for cooperatively acting agents. The two opposing classical views that in mammalian cells extrapolation to low doses of x-radiation is linear, on the one hand, or involves a threshold, on the other, are both demonstrated to be incorrect at least for the conditions here considered. The actual curve exhibits a downward concavity so that the mutational efficiency is maximal at low doses. These data may have important implications for human health.

The gene for human transforming growth factor alpha is on the short arm of chromosome 2.

Transforming growth factor alpha is a polypeptide growth factor that participates in the reversible transformation of cells in vitro and is secreted by many transformed cell lines. It also shares sequence and functional homologies with epidermal growth factor. Working with a cloned cDNA probe (lambda hTGF1-10) and derivatives, we have mapped this gene (TGFA) to 2p13 with the use of somatic cell hybrids and in situ hybridization. This is the same region involved in the 2;8 translocations of Burkitt lymphoma. Such a rearrangement could orient c-myc (8q24) adjacent to TGFA, resulting in activation of one or both of these genes.

50] Genetic mapping of apolipoprotein genes in the human genome by somatic cell hybrids

Publisher Summary The development of recombinant DNA technology has also made important contributions to gene mapping, especially by combining cell hybrids and cloned gene probes for chromosomal and regional assignment of many structural genes that may not be expressed in cultured cells or cell hybrids. This chapter discusses the use of somatic cell hybrids in apolipoprotein gene mapping. Although many assays can be performed to identify particular genetic markers that have been assigned to particular human chromosomes, the following two analyses are most commonly and conveniently used, especially when large numbers of cell hybrids will have to be analyzed for each of the 24 different human chromosomes. The chapter indicates that DNA sequence markers can be effectively used for fine structure mapping of specific regions of the chromosome in which various deletions produced by chromosomal breaks and localized in a small region may not be resolvable by current cytogenetic or in situ hybridization techniques.

Molecular Cloning and Chromosomal Assignment of the Human Homolog of int-1, a Mouse Gene Implicated in Mammary Tumorigenesis

Viral mammary tumorigenesis in mice is frequently initiated by proviral activation of a highly conserved cellular gene called int-1. We have cloned the human homolog of this putative mammary oncogene and compared its structure to that of the mouse gene by heteroduplex analysis. The human int-1 gene was localized on chromosome 12 by use of somatic cell hybrids.

Use of somatic cell hybrids for quantitation of mutagenesis: reduction in background mutants by fluorescence-activated cell sorting (FACS).

Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human-Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells. The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence-activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100-fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.