Assignment of the Gene for Adenine Phosphoribosyltransferase to Human Chromosome 16 by Mouse-Human Somatic Cell Hybridization

A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16.     

Inactive X chromosome has the highest concentration of unmethylated Hha I sites.

A procedure enabling the highly sensitive detection of accessible restriction endonuclease sites on metaphase chromosomes is described. The procedure is based on the following: (i) a terminal deoxynucleotidyltransferase is used to add a biotinylated nucleotide (Bio-11-dUTP) tail to the 3' hydroxyl terminus generated by the action of a restriction enzyme and (ii) the biotinylated oligonucleotide is detected by a peroxidase-based immunocytochemical method. When used with the 5-methylcytosine-sensitive enzyme Hha I, it gives rise to a pattern close to R and T banding on autosomes. In addition, the staining of one X chromosome in females appears very unusual by its pattern and its strong intensity. This procedure, as applied on a case with a polysomy X chromosome, provides direct evidence of an overall hypomethylation of the inactive X chromosomes.     

Active and Inactive Renins in Human Urine

Urinary excretions of active and inactive renin were studied in normal subjects and in patients with hypertensive or renal disease. Excessive excretion of active and inactive renins was observed in some patients with no significant correlation to their plasma levels or the degree of proteinuria. Inactive renin excretion correlated to active renin excretion, but the clearance was lower than that of the active form. No correlation was found between the urinary kallikrein excretion and the active/inactive renin ratio in the urine or the plasma. Urinary renin activity was increased by acidification and by trypsin treatment, but not by cold exposure. Both active and inactive renins in the urine showed multiple peaks corresponding to molecular weights between 45,000 and 64,000 by gel filtration.     

Tumor-Host Cell Hybrids in Radiochimeras

F(1) hybrid mice syngeneic or semiallogeneic with respect to the relevant tumor were lethally irradiated and then reconstituted with hemopoietic cells from strain CBAT6T6 mice. After chimerism had been established, the animals were inoculated with solid or ascites tumors. Tumor-host cell hybrids were selected from enzyme-deficient solid tumors by explanting the tumor cell suspension into hypoxanthine-amethopterin-thymidine containing medium. The selection of hybrid cells from ascites tumors was achieved by exploiting the difference between the ascites tumor cells and hybrid cells in their ability to adhere to the surface of culture vessels. T6T6 chromosomal and H-2 antigenic markers served to distinguish between the hemopoietic cells derived from the donor graft and the cells of the host. All solid tumors tested fused with cells of the irradiated host, whereas ascites tumors fused with repopulating cells of hemopoietic origin.     

Expression of Differentiated Functions in Hepatoma Cell Hybrids, I. Tyrosine Aminotransferase in Hepatoma-Fibroblast Hybrids

The inducible enzyme tyrosine aminotransferase (TAT) has been investigated in hybrids between rat hepatoma cells and mouse fibroblasts. In the latter the TAT baseline activity is low, and in the presence of steroids does not change. By contrast, the hepatoma cells have high TAT activity, and this activity increases by a factor of 4-6 in the presence of steroids. The hybrid cells, like the fibroblasts, have low TAT activity and are not inducible. Heat inactivation curves demonstrate that the hybrid cells contain both parental forms of TAT, and therefore contain the parental genes specifying the enzyme. The presence in the hybrids of detectable rat TAT and the total absence of its inducibility suggest that a second gene is involved in the regulation of TAT inducibility, and that this gene is not expressed in the hybrids.     

Mitotic Separation of Two Human X-Linked Genes in Man—Mouse Somatic Cell Hybrids

SIX INTERSPECIFIC SOMATIC HYBRID CELL LINES WERE DERIVED FROM A MOUSE LINE DEFICIENT IN HYPOXANTHINE: guanine phosphoribosyltransferase (HGPRT) and human diploid cells with normal enzyme activity. Human HGPRT was present in all six hybrids and the clones derived from them. However, in two of the six, and in some clones from another two, human glucose-6-phosphate dehydrogenase (G6PD) was absent. Since the structural loci for both these enzymes are X-linked in man, these findings suggest that these two loci have separated quite frequently through chromosome breakage and that they must be rather far apart on the X chromosome.     

Inactive X chromosome DNA does not function in DNA-mediated cell transformation for the hypoxanthine phosphoribosyltransferase gene.

The molecular nature of the X chromosome inactivation process has been investigated by utilizing the techniques of DNA-mediated cell transformation of the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus. The findings indicate that purified DNA from the inactive X chromosome of a near-euploid mouse cell line is not functional in transformation for HPRT, but the DNA from its "homologous" active X readily elicits transformation for HPRT in the same hamster cell recipient. These findings suggest that there is a difference between the DNA, per se, of the active and inactive X at (or near) the HPRT locus and that this difference could account, at least in part, for its inactivation.     

Human β-D-N-Acetylhexosaminidases A and B: Expression and Linkage Relationships in Somatic Cell Hybrids

Knowledge of the genetic relationships between beta-D-N-acetylhexosaminidases A and B (EC 3.2.1.30) may help in understanding the hexosaminidase deficiency associated with GM(2) gangliosidosis, a fatal lipid storage disease in man. Through the use of man-mouse somatic cell hybrids we have found that a gene involved in hexosaminidase A expression was linked to the genes coding for mannosephosphate isomerase and pyruvate kinase-3. The gene coding for hexosaminidase B was not linked to any of the genes coding for 25 enzyme markers tested. A combination of immunological and electrophoretic techniques was employed to identify human hexosaminidases A and B with certainty in cell hybrids. Discordant segregation of hexosaminidase A and hexosaminidase B in 60 clones indicated that the genes coding for their expression were not linked. However, hexosaminidase A was never expressed in cell hybrids in the absence of hexosaminidase B. This suggests that the gene responsible for the hexosaminidase A phenotype, linked to mannosephosphate isomerase and pyruvate kinase-3, requires the presence of the gene coding for hexosaminidase B for the expression of hexosaminidase A. These observations offer a genetic explanation for the biochemical and immunological relationships between hexosaminidases A and B and provide the framework for identifying the basic genetic defects responsible for GM(2) gangliosidosis.     

Lambda-Chain Production in Human Lymphoblast-Mouse Fibroblast Hybrids

Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity were hybridized with thymidine kinase (EC 2.7.1.21)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (lambda) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human lambda chain, and continued to do so after prolonged culture.As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or alpha(1)-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive lambda-chain reactivity, suggesting that the kinase and lambda-chain loci are not linked.The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system.     

In Vitro Generation of Inactive Renin in Hypertensive Human Plasma

When plasma from normal and hypertensive human subjects is incubated at pH 7.4 in the absence of angiotensinase inhibitors, a significant decline of active plasma renin occurs. The fall of active renin was more pronounced in the plasma from normotensive controls and was accompanied by a fall of total renin without change of inactive renin. in hypertensive subjects total renin levels (trypsin treatment) did not change. The fall of active renin in plasma from hypertensive subjects was due therefore to its in vitro conversion to an inactive form of renin which could be reactivated by trypsin. If reversible inactivation of active plasma renin occurs in vivo, inactivated active renin may contribute to a portion of the plasma pool of inactive renin. This form of inactive renin which may be activable in vivo would not therefore represent a renin precursor or “prorenin” of renal origin.     

Cytological Mapping of Human X-Linked Genes by Use of Somatic Cell Hybrids Involving an X-Autosome Translocation

Man-mouse and man-Syrian hamster somatic hybrid cell lines were prepared by fusion of mouse A9 or hamster TG2 cells, which are deficient in hypoxanthine-guanine phosphoribosyl transferase, with cells of a diploid fibroblastic strain, KOP-1, derived from a woman heterozygous for an X-autosome translocation. 61 clones were derived in nonselective medium and 85 sublines of these were derived in selective media: 53 in hypoxanthine-aminopterine-thymidine and 32 in 8-azaguanine. All three human X-linked markers studied, i.e., hypoxanthineguanine phosphoribosyl transferase (EC 2.4.2.8), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and phosphoglycerate kinase (EC 2.7.2.3), were present together, or absent together, in most of these clones and sublines. However, loss or retention of only phosphoglycerate kinase was occasionally observed, even in the absence of selective growth, while no evidence of separation of hypoxanthine-guanine phosphoribosyl transferase from glucose-6-phosphate dehydrogenase occurred. Cytological examination of eight man-hamster clonal lines by the quinacrine fluorescent technique showed that human phosphoglycerate kinase was only present when the translocation chromosome carrying most of the long arm of the X chromosome was present. The presence of human glucose-6-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyl transferase was not related to the presence or absence of this chromosome, but appeared to be correlated with the presence of the other translocation chromosome.     

Human Kidney Cathepsins B and H Activate and Lower the Molecular Weight of Human Inactive Renin

Cathepsins B, H, and D, extracted from human kidneys, activate human inactive renin. Inactive renin, obtained from human kidneys, contains two components of Mr 53,000 and 50,000. Upon incubation with 0.5 μM cathepsin B, the Mr of the larger component decreased progressively to 45,000 (similar to the Mr of active renin) without appreciable loss of renin activity. Cathepsin H also activated and decreased the Mr of kidney inactive renin. Plasma inactive renin was activated by the thiol proteases, cathepsins B and H, with less reduction in Mr than that observed in kidney renin.     

Fate of Mitochondrial DNA in Human-Mouse Somatic Cell Hybrids

Several hybrid lines between human and mouse somatic cells, containing one or two complements of mouse chromosomes and a reduced complement of human chromosomes, have been examined for the presence of mouse and human mitochondrial DNAs. For this analysis, advantage was taken of the fact that these two types of mitochondrial DNA have a buoyant density difference in CsCl gradients of 0.008 g/cm(3). In all the hybrid clones analyzed, which retained an average number of human chromosomes estimated conservatively to vary from 5 to 23, only mitochondrial DNA of mouse character was detected. It seems likely that either repression of relevant human genes by the mouse genome or loss of human chromosomes is responsible for these results. If the latter explanation is true, since chromosome loss under the conditions used here was substantially a random process, one would have to assume that the activity of nuclear genes distributed in many chromosomes is required for the survival of mitochondrial DNA.     

Genetics of Human-Mouse Somatic Cell Hybrids: Linkage of Human Genes for Lactate Dehydrogenase-A and Esterase-A4

By use of human-mouse somatic cell hybrids, an autosomal gene linkage has been determined for the human loci controlling the phenotypes of lactate dehydrogenase-A (EC 1.1.1.27) and esterase-A(4) (EC 3.1.1.2). These structural loci were not linked to the lactate dehydrogenase-B, peptidase-B linkage, the X chromosome, the E(17) chromosome, or to nine other enzyme phenotypes examined.     

Quantitation of Inactive Renin in Human and Dog Plasma: Techniques for Activation

For human samples quantitation of inactive renin can be carried out by incubation with trypsin under defined conditions, followed by RIA of the activated renin. for dog samples we were unable to obtain evidence for the presence of inactive renin in the plasma by using trypsin, acid or cold to activate. Increases in angiotensin generation did occur with trypsin and acid but they both changed renin substrate such that the rate of angiotensin generation by exogenous renin was increased at pH 7.4, but not at pH 5.7; also following trypsin or acid treatment angiotensin I was cleaved from renin substrate by a plasma acid protease that normally does not cleave renin substrate in plasma. Therefore, for dog samples, it is important to demonstrate that an increase in the rate of angiotensin generation is indeed due to activation of inactive renin and not to changes in pH optimum of renin with angiotensinogen or to the effect of another enzyme.     

Human Regulatory Gene for Inducible Tyrosine Aminotransferase in Rat-Human Hybrids

The inducibility of tyrosine aminotransferase (EC 2.6.1.5) by corticosteroid hormones in rat-human hybrid clones was studied. The presence of human X chromosome activity in the cells was always associated with the suppression of tyrosine aminotransferase inducibility in all the clones examined. Negative correlation between the human X chromosome and inducibility of the enzyme was clearly established. Corticosteroid receptor was present to the same extent in hybrid cell clones that either contained or lost the human X chromosome. The human repressor for inducible tyrosine aminotransferase has a linkage relationship with glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and hypoxanthine-guanine-phosphoribosyltransferase (EC 2.4.2.8) and, therefore, can be assigned to the X chromosome.     

Expression of Differentiated Functions in Hepatoma Cell Hybrids: Induction of Mouse Albumin Production in Rat Hepatoma-Mouse Fibroblast Hybrids

The synthesis of serum albumin has been studied in hybrids between well-differentiated rat hepatoma cells, which synthesize serum albumin, and mouse fibroblasts (3T3) that do not synthesize albumin. By immunodiffusion techniques with noncrossreacting antisera, the production of both rat and mouse albumin by the hybrids has been examined. Karyologically identified hybrids were produced between 3T3 cells and cells of a 1s hepatoma (Fu5) clone, and of a 2s hepatoma (2s Fu5-5cl.lE) clone. Each of the 3T3 x Fu5 hybrids produces only rat albumin. Among five 3T3 x 2s Fu5-5cl.lE hybrid clones isolated, one produces both rat and mouse albumin, two produce only mouse albumin, and two do not produce rat or mouse albumin.     

Genes for Neuronal Properties Expressed in Neuroblastoma × L Cell Hybrids

Neuroblastoma cells with electrically excitable membranes were fused with electrically passive L cells having a hitherto undescribed electrical marker. Hybrid cells, examined 10-40 generations after fusion, were found to be electrically excitable. The results show that at least a part of the genetic information for neuron differentiation can be functionally expressed in N x L hybrid cells. Evidence for the regulation of action potential components was also found.