Calcium Phosphate Precipitation Technique Research Articles

90Sr, 210Pb, 210Po and Ra isotopes in marine macroalgae and mussel Mytilus galloprovincialis from the Bulgarian Black Sea zone

90Sr, 210 Pb, 210Po and Ra isotopes were determined in macroalgae Ceramium rubrum, Cystoseira crinita, Ulva rigida, Cladophora vagabunda, Enteromorpha intestinalis and in mussels Mytilus galloprovincialis collected from the Bulgarian Black Sea coast. Radiochemical separation was made by a fast and reproducible procedure based on a calcium phosphate precipitation technique. The obtained results for macroalgae vary in the interval 0.23–0.85 Bq kg−1 for 90Sr, 2.2–10.6 Bq kg−1 for 210Pb, 1.2–9.7 Bq kg−1 for 210Po and 1.9–13.2 Bq kg−1 for Ra isotopes. Data obtained for mussels demonstrate similar abilities in the accumulation of 90Sr, 210Pb and Ra isotopes.

Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys(48)-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230-288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1-430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4(+) T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.

Constitutive c-fos expression in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption.

The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7 +/- 38.5, was significantly higher than 19.0 +/- 10.1 of the control (P < 0.05). The absorption area, 12,394 +/- 3145 mm2, was significantly larger than 1646 +/- 314 mm2 of the control (P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).

Pax-6 and c-Maf Functionally Interact with the α-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

Establishment and characterization of a tumorigenic cell line from areca quid and tobacco smoke-associated buccal carcinoma

A cell line, TW2.6, has been established from the surgically resected specimen of an untreated primary squamous cell carcinoma of the buccal mucosa from a 48-year-old man who was an areca quid chewer and tobacco smoker. TW2.6 cells exhibited morphological features of keratinocytes and replicated rapidly in culture with a doubling time of 24h. The karyotype showed human chromosomes with high hyperdiploidy and complex rearrangements. Western blotting showed pronounced expression of p53 and moderate expression of p21(CIP1). The baseline expressions of p27(KIP1) and p16(INK4a) were barely detectable. Low levels of Bax and Fas were found in TW2.6 cells but Bcl-2 expression was more readily observed. Mutational analysis of p53 gene revealed an A-->G transition at the second base of codon 220, resulting in amino acid substitution from tyrosine to cysteine in the protein. Functional analysis showed that TW2.6 was unable to activate the p53-specific PUMA promoter. Lipofectamine 2000 and calcium phosphate precipitation technique offer good transfection efficiencies for TW2.6 cells and may be used in future transfection experiments. A xenograft-SCID mouse tumor model was established for TW2.6. Histological examination demonstrated that the engrafted tumors maintained the morphological features of a squamous cell carcinoma. It is thought that the establishment of tumorigenic TW2.6 cell line provides a valuable model for AQ and tobacco smoke-associated buccal carcinoma.

Inhibition of Heme Oxygenase-1 Interferes with the Transforming Activity of the Kaposi Sarcoma Herpesvirusencoded G Protein-coupled Receptor

Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Molecular Determinants of Proton Modulation of Glycine Receptors

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.

Activation of the ERK1/2 Signaling Pathway Promotes Phosphorylation and Proteasome-dependent Degradation of the BH3-only Protein, Bim

Both the ERK and phosphatidylinositol 3'-kinase (PI3K) signaling pathways can protect cells from apoptosis following withdrawal of survival factors. We have previously shown that the ERK1/2 pathway acts independently of PI3K to block expression of the BH3-only protein, BimEL, and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced BimEL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of BimEL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum BimEL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote BimEL phosphorylation and that this leads to a substantial increase in turnover of the BimEL protein. ERK1/2-dependent degradation of BimEL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperature-sensitive mutation in the E1 component of the ubiquitin-conjugating system. Finally, co-transfection of BimEL and FLAG-ubiquitin causes the accumulation of polyubiquitinated forms of Bim, and this requires the ERK1/2 pathway. Our findings provide new insights into the regulation of Bim and the role of the ERK pathway in cell survival.

Liposome Mediated Transfer of Marker and Cytokine Genes Into Rat and Human Glioblastoma Cells in Vitro and in Vivo

AbstractGene transfer of Lac Z and TNF-α gene was performed in several glioblastoma cell lines in vitro (A172, F98, U373-MG, HS683, U343-MG, N31, N64, N28, N39 and N65). In comparison to the calcium phosphate precipitation technique (CPPT) the cationic liposomes were more successful measured by marker gene transfer (0.76 or 2.0%, depending on the cell line). In contrast, the amount of released human TNF-α was independed from the chosen transfer technique. Since the vector pWG29del3-hTNF contains a hormone response element it was possible to stimulate the hTNF secretion by dexamethasone up to 18 fold from a basic level of 1.1 ng/ml/24h/5×105 cells. A liposomal mediated marker gene transfer (Lac Z) into a rat glio-blastoma in vivo revealed a three layer area of transfected cells around the stereotactical injection channel.

Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor.

We have examined whether the two cysteine residues (621 and 622) of the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.

Measurement of Cell-Monolayer Adhesion in Cells Transfected with N-CAM cDNAs

To measure the adhesion of cells expressing the neural cell adhesion molecule N-CAM, mouse Lmtk fibroblast cells were transfected by a calcium phosphate precipitation technique with eucaryotic expression vectors encoding N-CAM polypeptides. We obtained cell lines expressing the 140-kDa transmembrane isoform of N-CAM at high levels by several rounds of selection by fluorescence-activated cell sorting and compared the adhesion of these cells to that of untransfected cells using a centrifugal removal assay that measures the centrifugal force required to remove radiolabeled probe cells from a cell monolayer. The adhesion of cells prepared from embryonic chicken neural retinas also was examined. Retinal probe cells remained associated with a retinal cell monolayer with an adhesive force of approximately 5 × 10 -6 dyn/cell, and this force was not reduced by treatment with specific anti-N-CAM antibody fragments. Transfected and untransfected mouse L cells each were dislodged from transfected cell monolayers with a removal force of 5 × 10 -5 dyn/cell and thus did not differ in their adhesion. These results support the hypothesis that N-CAM-mediated homophilic adhesion in retinal cells and transfected fibroblasts is relatively, weak and that the major adhesive interaction involved in N-CAM-mediated cell-cell adhesion is heterophilic.

Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression.

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.

The expression and chromatin structure of the chicken glyceraldehyde-3-phosphate dehydrogenase gene in mouse cells.

Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.

Activation of c-Ki-ras gene in human pancreatic cancer.

DNA isolated from a lymph node with metastasis from pancreatic adenocarcinoma in a Japanese male patient transformed NIH3T3 cells upon transfection by the calcium-phosphate precipitation technique. Analysis of DNA from the transformant revealed the presence of an activated human c-Ki-ras gene, which is considered to be responsible for the transformation of the NIH3T3 cells.

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.

Integration of Ecogpt and SV40 early region sequences into human chromosome 17: a dominant selection system in whole cell and microcell human-mouse hybrids.

The dominant selectable gene, Ecogpt, has been introduced, by the calcium phosphate precipitation technique, into normal human fibroblasts, along with the SV40 early region genes. In one transfectant clone, integration of these sequences into human chromosome 17 was demonstrated by the construction of human-mouse somatic cell hybrids, selected for by growth in medium containing mycophenolic acid and xanthine. A whole cell hybrid, made between the human transfectant and a mouse L cell, was used as donor of the Ecogpt-carrying human chromosome 17 to 'tribrids' growing in suspension, made by whole cell fusion between a mouse thymoma cell line, and to microcell hybrids made with a mouse teratocarcinoma cell line. Two tribrids contained karyotypically normal human chromosomes 17 and a small number of other human chromosomes, while a third tribrid had a portion of the long arm of chromosome 17 translocated to mouse as its only human genetic material. Two independent microcell hybrids contained a normal chromosome 17 and no other human chromosome on a mouse teratocarcinoma background. These experiments demonstrate the ability to construct human-mouse somatic cell hybrids using a dominant selection system. By applying this approach it should be possible to select for a wide range of different human chromosomes in whole cell and microcell hybrids. In particular, transfer of single human chromosomes to mouse teratocarcinoma cells will allow examination of developmentally regulated human gene sequences after differentiation of such hybrids.

Structure and expression of human globin genes introduced into mouse fibroblasts.

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.

Competence for DNA transfer of ouabain resistance and thymidine kinase: clonal variation in mouse L-cell recipients.

We have used the calcium phosphate precipitation technique to study the competence of mammalian cell recipients for transformation with genomic mammalian cell DNA. The transformation efficiency for thymidine kinase (tk) varies 10- to 20-fold (up to 10(-4) transformants/recipient) among different subclones of the LM tk- CL 1D mouse fibroblast cell line. Analysis of this phenotype among second-generation subclones indicates that subclones exhibiting high competence tend to breed true, whereas those with low competence do not. Isolation of Tk- revertants from TK+ transformants results in the selection of cells with a high-competence phenotype as measured by their subsequent transformation for tk. This phenotype appears to be a general characteristic of such cells because recipients more competent for transfer of a second marker, ouabain resistance (ouaR). This codominant marker coding for the Na K+-ATPase can be transferred at frequencies of 10(-5) in the high-competence recipients. These results indicate that competence for DNA-mediated gene transfer can be determined in part by genetic factors.

Transfection with baculovirus DNA

Purified DNA from the nuclear polyhedrosis viruses of Autographa californica (AcM NPV) and Rachiplusia ou (RoMNPV) were found to be infectious in TN-368 cells employing the calcium phosphate precipitation technique (F. L. Graham and A. J. van der Eb, Virology, 52 ,456–467, 1973). Transfection with Ac M NPV produced 3600 PFU/μg DNA compared to 2900 PFU/μg DNA with Ro M NPV. Of eight baculovirus DNAs tested, only Ac M NPV DNA and Ro M NPV DNA could transfect TN-368 cells. The in vitro host range of Ac M NPV DNA was determined to be the same as Ac M NPV extracellular virus. Ac M NPV Form I DNA was fourfold more infectious in TN-368 cells than Form II DNA.