Regional assignment of the genes for TK1, GALK, ALDC, and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer

Summary A panel of clones of mink-Chinese hamster somatic cell hybrids was analysed to obtain data for assigning the genes for thymidine kinase-1 (TK1), galactokinase (GALK), subunit C of aldolase (ALDC), and esterase D (ESD) to specific mink chromosomes. The results demonstrate that the genes for TK1, GALK, ALDC and ESD are syntenic and located on mink chromosome 8. Prometaphase analysis of transformed mouse cells obtained by transfer of mink genes by means of metaphase chromosomes demonstrated the presence of mink chromosome 8 fragments of different sizes in some of the independent transformants. Segregation analysis of these fragments and mink TK1, GALK, ALDC and ESD allowed us to assign the genes for TK1 and GALK to 8p24, ALDC to pter-8p25, and ESD to 8q24-8qter.     

Cotransfer and phenotypic stabilisation of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer

Summary By means of metaphase chromosomes, the genes for mink thymidine kinase (TK) and hypoxanthine-phosphoribosyltransferase (HPRT) were transferred to mutant mouse cells, LMTK^-, A9 (HPRT^-) and teratocarcinoma cells, PCC4-aza 1 (HPRT^-). Eighteen colonies were isolated from LMTK^- (series A), 9 from A9 (series B) and none from PCC4-aza 1. The transformed clones contained mink TK or HPRT. Analysis of syntenic markers in series B demonstrated that one clone contained mink glucose-6-phosphate dehydrogenase (G6PD) and the other alpha-galactosidase; in series A, nine clones contained mink galactokinase (GALK) and six mink aldolase C (ALDC). Analysis of 12 asyntenic markers located in ten mink chromosomes showed the presence of only aconitase-1 (ACON1) (the marker of mink chromosome 12) in three clones of series A. The clones lost mink ACON1 between the fifth to tenth passages. Cytogenetic analysis established the presence of a fragment of mink chromosome 8 in eight clones of series A, but not in series B. The clones of series A lost mink TK together with mink GALK and ALDC during back-selection; in B, back-selection retained mink G6PD. No stable TK⁺ phenotype was detected in clones with a visible fragment of mink chromosome 8. Stability analysis demonstrated that about half of the clones of series B have stable HPRT⁺ phenotype whereas only three clones of series A have stable TK⁺ phenotype. It is suggested that the recipient cells, LMTK^- and A9, differ in their competence for genetic transformation and integration of foreign genes.     

DNA-mediated cotransfer of unlinked mammalian cell markers into mouse L cells

Purified DNA from three different types of mammalian cells was precipitated with calcium phosphate and added to mouse L cells deficient in thymidine kinase (TK). Donor DNA was prepared from three cell lines: (a) mouse cells transfected with UV-inactivated herpes simplex virus (HSV) type 1, or a purified fragment of HSV carrying the TK gene (b) human HeLa cells, and (c( CHO, a cell line derived from Chinese hamster ovaries. Several hypoxanthine-aminopterin-thymidine resistant colonies were isolated from each experiment. The origin of the TK that is expressed in these cells was studied by polyacrylamide gel electrohporesis, isoelectric focusing, or heat stability. The TK in all instances was of the donor origin. To determine the extent of gene transfer we have assayed the CHO and HeLa DNA transfectants for galactokinase (GALK), a marker closely linked to TK, and 25 other isozymes representing a large number of different chromosomes. No cotransfer of GALK was observed, indicating that the size of the transferred DNA segment is limited. We observed that, in one instance, esterase-D, an unlinked marker of Chinese hamster origin, was transferred along with TK. These experiments indicate that nonselected markers can be transferred by this method, although at a low efficiency.     

Transformation depending on intermolecular homologous recombination is stimulated by UV damage in transfected DNA

DNA-mediated gene transfer (DMGT) was performed in DNA repair-proficient and UV-hypersensitive, repair-deficient Chinese hamster ovary (CHO) cell lines using the UV-irradiated thymidine kinase gene from herpes simplex virus (HSV-TK). Transformation frequencies in repair-deficient CHO cell lines declined relative to repair-proficient cells with increasing UV damage in transfected DNA; approximately 3-fold higher UV fluence was required to inactivate 50% of irradiated HSV-TK plasmid molecules in repair-proficient cells. In cotransfection experiments performed with pairs of HSV-TK plasmids containing linker insertion mutations in TK coding sequences, moderate UV damage in plasmid DNA enhanced the yield of TK+ transformants resulting from homologous recombination between HSV-TK sequences up to 4-fold. These results suggest that UV damage in DNA can stimulate transformation of mammalian cells dependent on intermolecular DNA homology.     

Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells

Summary Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT^-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10^-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.     

Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine,Hoechst 33258, and visible light

The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK^– mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK⁺ cells by a factor of 10⁵ without affecting the viability of TK^– mutants. This procedure was used to isolate H4IIEC3-derived rat hepatoma cells deficient in TK activity. The properties of several TK^– hepatoma clones are discussed.The valuable technical assistance of J. M. Courvall, F. R. Parker, and M. M. Smith is acknowledged. These studies were supported by grant GM26449 from the National Institutes of Health. A.M.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division. T.G.L. is a Leukemia Society of America postdoctoral fellow. R.E.K.F. is the recipient of an American Cancer Society Faculty Research Award.     

Transfer of mink genes into mouse cells by means of isolated lipid-encapsulated nuclei

Summary A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK^- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9x10^-5. Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK⁺ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

Genetic linkage but independent expression of functional HSV-1 tk and mammalian aprt genes after cotransfer to L cells

DNA-mediated gene transformation of mouse Ltk-aprt-hprt-cells was used to obtain stable, doubly selected transformants simultaneously expressing herpes virus thymidine kinase (TK) and mammalian adenine phosphoribosyltransferase (APRT). Cotransformants occurred at a frequency of 5 X 10(-6), a similar frequency for the transfer of the aprt marker has been previously observed. Isozyme and Southern blot analysis show that the TK and APRT expressed in these transformants resulted from gene transfer. For one stable cotransformant, [3H]thymidine [( 3H]TdR) selection against TK activity resulted in the loss of APRT activity as well, suggesting that these genes had become genetically linked together. Similarly selection against APRT expression resulted in the loss of a subset of the transferred herpes simplex virus tk genes. 5-Bromodeoxyuridine (BUdR) selected TK- variants differed from [3H]TdR selected TK- variants, in that they retained tk genes. However, BUdR-selected variants expressed full levels of APRT. Therefore, even though the transferred tk and aprt genes had become genetically linked together, they were, in this case, independently expressed since these cells were phenotypically TK- and APRT+.     

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).     

DNA-mediated gene transfer without carrier DNA

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.     

The African Swine Fever Virus Thymidine Kinase Gene Is Required for Efficient Replication in Swine Macrophages and for Virulence in Swine

African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter–β-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK⁻ ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK⁺ revertant virus. Swine surviving TK⁻ ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.     

Development of new methods in human gene mapping: selection for fragments of the human Y chromosome after chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to generate fragments of human chromosomes and chromosomal maps can be constructed using these fragments. In previous experiments CMGT techniques have been limited to those regions of the genome which encode biochemically selectable markers. We have extended the regions of the human genome which can be subjected to CMGT methods by employing a cell surface antigen as a selectable marker. These experiments have been facilitated by the discovery that co-transformation of chromosomes with a plasmid bearing a biochemically selectable marker followed by selection for the marker pre-selects for cells which have incorporated chromosomal fragments. The plasmid may also integrate into the donor chromosomes and this provides, in some cases, an additional selectable marker in the chromosome fragment of interest. Using these methods we have isolated for the first time cells containing varying portions of the human Y chromosome.     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.     

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK^– (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice × M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts × LMTK^– cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.     

Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK^+/+). TK-deficient (TK^?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK^+/? revertants arising in TK^?/? cultures should possess TK enzyme identical with one of those present in the original TK^+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK^+/? lines, each derived in turn from separate TK^?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) K_m′s; and (4) ATP-Mg²⁺ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.     

Transfection of mouse cells with thymidine kinase gene of herpes simplex virus

A mouse cell line (LP1-1) was established from the murine L cells deficient in thymidine kinase (L-M(TK ^–)) by prolonged selective culture on the hypoxanthine-aminopterine-thymidine (HAT) medium following transfection with the thymidine kinase gene of herpes simplex virus type-I (HSVTK). Southern blot analysis has shown that the viral TK gene was integrated into one of the chromosomal loci by a single copy. From this established cell line, the 5-bromo-2-deoxyuridine (BrdU) resistant revertant was brought out at a frequency of 1×10^–6 and from these BrdU resistant revertants (LP1BU), one out of 1×10⁵ cells could return to the HAT-resistant phenotype. The established LP1-1 cell line showed a typical biphasic nature of DNA synthesis as determined by the ³H-thymidine incorporation test. The activity of thymidine kinase was shown to be equivalent to that of the DNA polymerase- when the whole nuclear fraction or the nuclear matrix were used for examination. These results indicate that the transfected viral TK gene can be expressed under the normal cell-cycle regulation and its gene product can act as a component of the multienzyme complex which is responsible for DNA replication.     

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.     

人体胸苷激酶(TK)基因在共转染过程中结构改变的研究

用磷酸钙沉淀法,我们把带有人体TK基因片段的重组噬菌体DNA共转染小鼠Ltk~-细胞,得到TK~+转化细胞克隆。同时用HeLa细胞DNA转染Ltk~-细胞,得到第一代TK~+转化细胞,再进行第二轮、第三轮转染,得到第二代、第三代TK~+转化细胞。比较其转化效率,结果基因组DNA转化率大于基因两个片段的共转化率,更大于不加携带者DNA的共转化率。限制性内切酶消化各种TK~+转化细胞的DNA,与TK基因探针作Southern印迹杂交,结果表明两个TK基因片段共转染Ltk~-细胞时,它们可以在受体细胞里重建成一个具有完整功能的遗传单位,但在连接过程中结构可以发生改变。当用HeLa纽胞DNA转染Ltk~-细胞时,虽然连续三代转染,每一代TK~+转化细胞中人TK基因的结构未发现变化。但也不能排除基因结构改变的频率很低未能有效检出的可能性。