Identification of chick thymidine kinase determinant in somatic cell hybrids of chick erythrocytes and thymidine kinase-deficient mouse cells

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses of chick-mouse somatic cell hybrids [LM(TK⁻)/CRB]isolated from fusion mixtures of chick erythrocytes and thymidine (TdR) kinase-deficient mouse [LM(TK⁻)]cells have demonstrated that the somatic cell hybrids contain only chick cytosol TdR kinase F and mouse mitochondrial TdR kinase A activities. Karyotypes were analysed by the method which sequentially reveals Q- and C-bands. Four hybrid clones contained the full complement of mouse chromosomes and 1 to 3 chick micro-chromosomes. Counterselection of the LM(TK⁻)/CRB hybrids in 5-bromodeoxyuridine (BUdR) medium resulted in the loss of chick cytosol TdR kinase F activity and at least one of the chick chromosomes, but mouse mitochondrial TdR kinase A activity was unaffected. Unlike the LM(TK⁻)/CRB somatic cell hybrids, the BUdR-resistant clones could not grow in HATG (hypoxanthine-aminopte-rin-thymidine-glycine) medium. The results demonstrate that: (1) the chick cytosol TdR kinase F gene is on a member of the micro-chromosomes; and (2) selection in HATG- and BUdR-containing medium involves only cytosol TdR kinase F.     

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.     

The effects of glucocorticoids on thymidine kinase and nucleoside phosphotransferase during development of chicken embryo retina

Thymidine kinase in chick embryo retina reaches its highest values on the 8–10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 × 10^?6M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8–18-day-old chick embryo and, except on the 18th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine kinase during development. Brief periods of exposure to steriods increase nucleoside phosphotransferase activity in isolated chick embryo retinas. When the exposure was longer than 3 h this activity was also clearly decreased. We conclude that other factors are responsible for the natural increment which occurs for this activity during development.     

Nucleoside phosphotransferase of chick embryo

Summary This paper describes a purification procedure and some properties of a nonspecific nucleoside phosphotransferase of chick embryo, an activity which catalyzes the transfer of the phosphate ester from a deoxyribonucleotide or a pyrimidine ribonucleotide to a deoxyribonucleoside acceptor.The enzyme is very unstable to heat, dilution and dialysis and it is almost entirely inactivated by DEAE-cellulose chromatography or gel filtration. A marked enhancement in its stability is caused by numerous nucleotides. In these experiments at least 920-fold purification was obtained by using dTTP (50m) as nucleotide protector.The enzyme, purified in presence of dTTP, has a molecular weight about 270 000, an isoelectric point of 6.27, a pH optimum of 8.8 and is stable at 37 °C at least for 10 min.In absence of nucleotide protector, nucleoside phosphotransferase is converted at 37 °C or by gel filtration in a very small active form with a lower molecular weight (about 30 000) and a pH optimum of 7.6.     

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.     

Expression of thymidine kinase activity in hybrids between human leukemic cells and a TK-mouse cell line.

A series of actively proliferating clones have been isolated after PEG-induced fusion of a thymidine kinase deficient murine line and white blood cells from two leukemic patients. Their hybrid nature was proved both cytologically and biochemically. All the hybrids tested showed levels of TK activity significantly higher than the TK- mouse parental cell line and comparable to those exhibited by replicating TK+ cells.     

Characterization of beta-D-N-acetylhexosaminidase isoenzymes in man-Chinese hamster somatic cell hybrids.

A series of man-Chinese hamster hybrids were investigated with the use of an anti-Chinese hamster hexosaminidase serum, a specific anti-human hex A serum and an anti-human hex B serum. The expression of human hex A was found to be dependent on the presence of hex B. A heteropolymeric molecule is formed independently of hex B, which consists of Chinese hamster and specific hex A moieties. It has an electrophoretic mobility nearly identical to hex A. A relationship between the absence and presence of the heteropolymeric molecule, mannosephosphate isomerase (MPI), and pyruvate kinase (PK-3), assigned to chromosome 15, was established. With respect to the two locus subunit model, the gene coding for the alpha subunit, specific for hex A, has been localized on chromosome 15.     

Pluripotent teratocarcinoma-thymus somatic cell hybrids.

We have produced a series of somatic cell hybrids by fusing pluripotent PCC4aza1 embryonal carcinoma ("teretocarcinoma") cells with thymocytes from young adult mice. When these hybrids form tumors in nu/nu or syngeneic mice, all the tumors contain a range of differentiated tissues, as well as embryonal carcinoma-like tissues. Some of the tumors produce alpha-fetoprotein. These results show that pluripotency in embryonal carcinoma cells need not to be abolished by the introduction of a complete diploid genome from a differentiated cell.     

Phosphorylation and protein kinase activities of chromosomal non histone proteins from chick embryo fibroblasts.

Confluent chick embryo fibroblasts were cultured in vitro in (i) medium which prevented the cells from dividing, (ii) medium which stimulated the cells to divide synchronously, (iii) medium without lysine in which the cells were blocked in G1. Chromosomal non histone proteins (NHP) were extracted from cells pulse labelled with 32P phosphate, and the radioactivity analyzed by acrylamide gel electrophoresis. Several radioactive peaks were found all along the gel in the NHP from confluent and stimulated cells. The highest phosphorylation was found in the fast moving proteins, but the stimulation of the cells increases the phosphorylation of the slower moving proteins. In the NHP from cells cultured in the medium without lysine only the slow migrating proteins were phosphorylated. NHP were extracted from unlabelled cell cultures in the three different media, incubated with [gamma-32P] ATP and analyzed by acrylamide gel electrophoresis. Highly labelled peaks were observed in the fast moving proteins from stimulated cells and from cells cultured in a medium deprived from lysine. By comparing in vivo and in vitro phosphorylation, it can be concluded that in confluent cells the turnover of bound phosphate is slow. In stimulated cells there is a fast turnover of the phosphate bound to fast turnover of the phosphate bound to a small group of fast migrating proteins and very little turnover of the phosphate bound to slow migrating proteins. The cells were incubated with labelled lysine and NHP analyzed by gel electrophoresis. The radioactivity of individual NHP varied with the culture conditions, but in all cases, there was little radioactivity in the fast moving proteins. The phosphate groups submitted to a fast turnover are bound to stable proteins. Phosvitin and casein kinase activities were measured in the NHP fractions. Nine-ten peaks of activities were observed with each substrate. Some variations were observed which apparently correlate with the culture conditions.     

Transfer of the human genes coding for thymidine kinase and galactokinase to Chinese hamster cells and human-Chinese hamster cell hybrids.

Cotransfer of two linked human genes, coding for the enzymes thymidine kinase (TK) and galactokinase (Gak) was demonstrated following incubation of Chinese hamster TK-deficient cells with isolated human chromosomes. The 5 colonies which were isolated all expressed a stable TK-positive phenotype. Cotransfer of the human genes coding for TK and Gak has also been observed in experiments in which isolated human chromosomes were incubated with TK-deficient human-Chinese hamster cell hybrids. These receipient hybrids had lost all human chromosomes at the time of incubation. From these experiments, four colonies were isolated, all expressing an unstable TK-positive phenotype. Using chromosome staining techniques, the presence of human chromosomes could not be demonstrated in either of the transformed clonal lines obtained with the Chinese hamster and the hybrid recipient cells. This indicates that incorporation of only the fragment of the human chromosome 17, bearing the genes for TK and Gak, has occurred in the recipient cells.     

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells.

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

TAM selection of Drosophila somatic cell hybrids.

The selection of MDR3, an adenine-salvage-deficient variant of the Kc line, is described. It is resistant to methylpurine and to diaminopurine and is TAM (thymidine, adenine, methotrexate) sensitive. Two wild-type (TAM-resistant) cell lines, Schneider's line 3 (S3) and Dübendorfer's line 1 (D1), due to their different nutritional requirements, are unable to proliferate in medium ZH1% used for line MDR3. This allowed the selection of hybrids between MDR3 and either D1 or S3 in TAM cloning medium after treatment with polyethyleneglycol. Hybrids were identified by the isoenzyme pattern of NADP-dependent isocitrate dehydrogenase.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.