The Locus for Human Adenine Phosphoribosyltransferase on Chromosome No. 16

Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de novo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of No. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAP-resistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16.     

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.     

Genetic control of tumorigenicity in interspecific mammalian cell hybrids.

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.     

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells.

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.     

Absence of demonstrable linkage of human genes for enzymes of the purine and pyrimidine salvage pathways in human-mouse somatic cell hybrids

A number of different reduced human-mouse hybrids have been analyzed for the presence of human enzymes of the purine and pyrimidine salvage pathways. Homologous mouse and human enzymes were characterized by isoelectric fractionation or gel electrophoresis, and the species of origin of the enzyme in hybrid clones was determined. Hybrids selected for one of the human enzymes, thymidine kinase, adenine phosphoribosyltransferase, or hypoxanthine phosphoribosyltransferase, were each found to contain the selected enzyme but not the other two. Neither human adenosine kinase nor human deoxycytidine (cytidine) deaminase was present in any of the hybrid clones. A human 5-nucleotidase was present in two hybrid clones containing human hypoxanthine phosphoribosyltransferase, but the genes for the two enzymes are not linked. The genes for the purine and pyrimidine salvage enzymes appear to be dispersed in the human genome.These investigations were aided by a grant from the National Cancer Institute.     

Human mitochondrial thymidine kinase is coded for by a gene on chromosome 16 of the nucleus

The expression of human mitochondrial thymidine kinase (mt TK) was investigated by polyacrylamide electrophoresis in 19 independent human-mouse somatic cell hybrids which allowed all human chromosomes to be analyzed. In 8 hybrid clones the presence of this enzymatic activity could be demonstrated. Human mt TK segregated concordantly with human adenine phosphoribosyltransferase (APRT) and human chromosome 16. Discordant segregation with all other human chromosomes was demonstrated by karyotype and isozyme analyses. These results suggest that human mt TK is coded for by a gene on chromosome 16 of the nucleus. Thus human mt TK is genetically different from human cytosol thymidine kinase which is coded for by a gene on chromosome 17. The appearance of one heteropolymer band after electrophoretic separation of human and murine mt TK supports the notion that both enzymes have dimeric structures.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Quantitation of the viral DNA present in somatic cell hybrids between mouse and SV40-transformed human cells

Recent studies of somatic cell hybrids between mouse cells and SV40-transformed human cells have demonstrated a correlation between the expression of SV40 T-antigen and the presence of human chromosome 7. We have used two types of nucleic acid hybridization procedures to detect and quantitate the presence of viral DNA sequences in the DNA of the hybrid cell clones. Results of reassociation kinetics as well as hybridization with a single-strand probe indicate that SV40 DNA is present only in those hybrid clones which both contain human chromosome 7 and express the SV40 T-antigen. SV40 DNA was not detectable either in the clones which had lost human chromosome 7, or in the rare clones which retain human chromosome 7 but which do not express T-antigen. We have thus extended the correlation between human chromosome 7 and the SV40 T-antigen to the presence of integrated SV40 DNA in somatic cell hybrid clones.     

Preferential retention of the human chromosome C-7 in human-(thymidine kinase deficient) mouse hybrid cells

On karyotyping human—mouse hybrid cells derived from various parental crosses, we found that if the mouse parental cells were thymidine-kinase deficient, the hybrid clones retained not only the human chromosome E-17 containing the thymidinekinase gene, but a high proportion (82%) also contained the human C-7 chromosome. Other human chromosomes were also found in these clones.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.     

Confirmation of the synteny of the human genes for mannose phosphate isomerase and pyruvate kinase and of their assignment to chromosome 15.

The synteny of human mannose phosphate isomerase and pyruvate kinase and the assignment of the genes for these two enzymes to chromosome 15 were confirmed by analysis of 43 independently derived human-mouse hybrid clones. Hybrids between mouse cells deficient in hypoxanthine-guanine phosphoribosyltransferase and human fibroblasts carrying an X/15 chromosome translocation were also included in this study.     

Expression of the ASV src gene in hybrids between normal and virally transformed cells: Specific suppression occurs in some hybrids but not others

Somatic cell hybrids have been made between clones of rat cells transformed by avian sarcoma virus and rat or mouse cells that are untransformed. Intraspecies hybrids were either predominantly morphologically normal or predominantly transformed, some clones that formed transformed intraspecies hybrids yielding normal interspecies hybrids. Untransformed hybrids usually showed no detectable alteration in the structure or location of the integrated provirus, but viral RNA and pp60^src kinase activities were much reduced. No decrease in viral gene expression was seen in transformed hybrids. Thus hybrid suppression of viral transformation, mediated in trans by the untransformed parent, is a specific event that depends on both untransformed and transformed parental parameters.     

Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP, and PGP in mink: comparison with man and mouse

Segregation of mink biochemical markers uridine 5'-monophosphate phosphohydrolase-2 (UMPH2), adenine phosphoribosyltransferase (APRT), phosphoserine phosphatase (PSP), phosphoglycolate phosphatase (PGP), peptidases D (PEPD) and S (PEPS), as well as mink chromosomes, was investigated in a set of mink x mouse hybrid clones. The results obtained allowed us to make the following mink gene assignments: UMPH2, chromosome 8; PEPD and APRT, chromosome 7; PEPS, chromosome 6; and PSP and PGP, chromosome 14. The latter two genes are the first known markers for mink chromosome 14. For regional mapping, UMPH2 was analyzed in mouse cell clones transformed by means of mink metaphase chromosomes (Gradov et al., 1985) and also in mink x mouse hybrid clones carrying fragments of mink chromosome 8 of different sizes. Based on the data obtained, the gene for UMPH2 was assigned to the region 8pter----p26 of mink chromosome 8. The present data is compared with that previously established for man and mouse with reference to the conservation of syntenic gene groups and G-band homoeologies of chromosomes in mammals.     

Introduction of new genetic markers on human chromosomes

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.     

Segregation of rat chromosomes in somatic cell hybrids between rat cells and HT 1080 human fibrosarcoma cells

Summary We produced somatic cell hybrids between HT 1080-6TG human fibrosarcoma cells and either rat white blood cells (WBC) or cells directly derived from rat spleen. Karyologic and isozyme analyses of hybrid cells indicated that they preferentially lose rat chromosomes. Hypoxanthine-aminopterine thymidine-selected hybrid clones expressing rat hypoxanthine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and phosphoglycerate kinase (PGK) and containing the rat X chromosome were counterselected in a medium containing 30 g/ml of 6-thioguanine. Concordant loss of the rat X chromosome and of the expression of rat HPRT and G6PD was observed in the hybrid clones.     

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.     

Synthesis of ribosomal RNA in synkaryons and heterokaryons formed between human and rodent cells.

A study has been made of the ribosomal RNA and chromosome constitution of man-mouse hybrid cells. Previous work has shown that no human 28s rRNA is detectable in man-mouse synkaryons. In general human chromosomes are lost from such hybrids. With a recently developed method for distinguishing mouse from human chromosomes, an analysis of various man-mouse hybrid cell lines has been made. This indicates that not all the human chromosomes bearing nucleolar organizers are lost in the hybrid cells and such loss cannot alone explain the absence of human 28s rRNA. An examination of the 28s rRNA synthesized by heterokaryons formed from several different parent cells has revealed that both parental types of 28s rRNA are present in heterokaryons. The control of rRNA synthesis in hybrid cells is discussed.     

Somatic cell hybrids of mouse myeloma cells and B lymphocytes from a patient with agammaglobulinemia: failure to secrete human immunoglobulin.

Somatic cell hybrid clones were isolated from the fusion of RPC5.4 mouse myeloma cells and B lymphocytes from a patient with common varied agammaglobuinemia. The patient has B lymphocytes that synthesize immunoglobulin but fail to secrete immunoglobulin. The hybrid character of the six clones was established by examination of metaphase chromosome spreads. Most of the hybrid clones expressed mouse and human surface immunoglobulin. All of the clones synthesized immunoglobulin of mouse and human parental origin. Mouse parental immunoglobulin was secreted, whereas the human parental immunoglobulin was not secreted. Human light chain molecules were secreted as part of hybrid H2L2 molecules formed with mouse heavy chains. Human heavy chains had a reduced m.w. in comparison to the mouse heavy chains. Kinetic experiments indicated that human Ig was synthesized in amounts that were comparable to the mouse Ig. Pulse-chase experiments showed that that the intracellular human Ig was removed from the cytoplasm, probably by degradation. These experiments demonstrate that the hybrid cells are an in vitro model of naturally occurring failure of immunoglobulin secretion from agammaglobulinemia. The failure of fusion with mouse myeloma cells to complement the secretion defect suggests that these B cells produce an altered immunoglobulin molecule that is not programmed for secretion.