In situ localization of galactosyltransferase in surface mucous cells of the rat stomach.

Cryostate sections of the rat stomach fundus were incubated in the presence of uridine 5'-diphosphate-[3H]-galactose. The radioactivity in the surface mucous epithelial cells was shown by autoradiography to be specifically incorporated into a supranuclear area, the area where in these cells the Glogi system is situated. The incorporation lasted only 5-6 min and was Mn++ dependent. Galactose was probably incorporated into a beta-glycosidic bond.     

In vitro incorporation of (3H)threonine and (3H)glucose by the mucous and serous cells of the human bronchial submucosal gland. A quantitative electron microscope study

Incorporation of [3H]threonine and [3H]glucose by the mucous and serous cells of the human bronchial submucosal gland has been studied over 8 h using, for the first time in vitro pulse labeling and electron microscope autoradiography. In assessing the autoradiographs, two methods were compared, the circle analysis and the recently described hypothetical grain analysis. Preliminary studies showed formaldehyde to be the most suitable fixative. Chemical analysis of tissue revealed that [3H]threonine was incorporated into the polypeptide moiety of the bronchial gland product and that metabolites of [3H]-glucose were incorporated into the carbohydrate. Tritiated threonine was first localized in the endoplasmic reticulum of both mucous and serous cells and later migrated to the Golgi apparatus, while metabolites of [3H]glucose localized first mainly in the Golgi apparatus. From here, both radioactive precursors were next identified in vacuoles and, finally, in secretory granules. The mucous cell incorporated strikingly more of both radioactive precursors than the serous cell. Thus, it seems that oligosaccharides of mucous and serous cell glycoproteins are synthesized mainly in the Golgi apparatus and added there to the polypeptide core which is synthesized in the endoplasmic reticulum. The relationship of the mucous cell to the serous cell is discussed. It seems that under "normal" conditions each cell represents a different line but that injury may transform a serous cell into a mucous cell.     

Autoradiographic demonstration of in vivo sialylation of endogenous acceptors at the microvillar surface of intestinal columnar cells after intraluminal administration of CMP-[3H]-sialic acid

To investigate the presence of glycosyltransferase activity at the apical surfaces of columnar cells in small intestine, CMP-[3H]-sialic acid was injected into the lumen of a ligated segment of rat jejunum; 5 min later the tissue was fixed and processed for light microscopic autoradiography. After a 3-6-month exposure, an autoradiographic reaction appeared over the microvillar surfaces of columnar cells, indicating the presence of surface sialyltransferase activity accompanied by endogenous acceptors. When CMP-[3H]-sialic acid was injected into the posterior chamber of rat eye or the lumen of mouse gallbladder, no autoradiographic reaction was observed at the surfaces of the cells facing these cavities. After injection of UDP-[3H]-galactose into the same three sites, an autoradiographic reaction was observed in the Golgi regions of the various epithelial cells, but not along their apical surfaces. Competition experiments using unlabeled galactose indicated that [3H]-galactose had been released from the nucleotide and had entered the cells to be incorporated into the Golgi apparatus.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. II. Differentiating mucous cells

Labeled leucine, serine, galactose, glucosamine and sulphate were administered to rat stomachs in a perfusion system. Sections of the gastric fundus were studied by light microscopic autoradiography. Five categories of mucous cells were distinguished and their glycoprotein synthetic activity was measured in autoradiographs by counting silver grains over each category. During their differentiation, while migrating from the isthmus of the fundic glands to the free luminal surface, the surface mucous cells (SMC) showed an increase in incorporation of all precursors used. Differences between the incorporation patterns of the various precursors, in cells of different ages, suggest that structural development runs ahead of functional activity, and that the latter continues up to the very moment the cell is shed from the surface. Sulphate was incorporated at a considerably lower rate by the SMC of the free surface than by the foveolar SMC, in which by cytochemical staining strongly acidic glycoproteins were shown. Since the mucous neck cells incorporated all precursors at a low rate, these cells apparently do not play an important role in gastric mucus synthesis. They did not incorporate sulphate, which is consistent with histochemical observations.     

Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells

Intact murine L1210 leukemic cells incorporated significant quantities of [³H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[³H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[³H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[³H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.     

Chromatographic measurement of mucin production in cultures of gastric mucous cells.

The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Turnover of the plasma membrane proteins of hepatoma tissue culture cells.

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods--loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucose-labeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using 125I and 131I, or [3H]leucine and [14C]leucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma membrane in which the proteins are incorporated and removed in large structural units.     

Metabolic-morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii)

Summary Light- and electron-microscopic autoradiography were used to obtain a coordinated metabolic-morphologic view of some of the events of cellular differentiation that occur across the epidermis of the Pacific hagfish (Eptatretus stoutii) and which enable this animal to secrete copious amounts of mucus. As judged by epidermal incorporation of [³H]-thymidine in vivo, about 98 % of DNA replication is confined to the basal three layers of the total of 6–8 layers of cells. Small mucous cells (SMC), the most numerous of the three major cell types involved in mucigenesis, show in vitro and in vivo radioincorporation profiles of [³H]-L-lysine and [³H]-D-glucosamine which differ markedly from those of [³H]-L-fucose and [³H]-D-galactose. Time-course incorporation profiles (mean silver grains/cell and percentage of cells with at least one cluster of silver grains) of [³H]-L-lysine and [³H]-D-glucosamine not only reflected the metabolic activities of cell renewal and differentiation in basally-located cells but also the high mucigenic activity in cells near the epidermal surface. By contrast, [³H]-L-fucose and [³H]-D-galactose were mainly incorporated by the more mature SMC in juxtanuclear regions near Golgi complexes and newly formed secretory vesicles. The intensity of [³H]-fucose labeling appeared proportional to the intensity of histochemical staining of the apical cytoplasm. The prominent capsule, within SMC in basal and lateral regions, which arises from a tight intermingling of tonofilaments, appears to restrict secretory vesicles to apical regions while the cell progressively differentiates and migrates to the epidermal surface. The other mucigenic cell types, large mucous cells and thread cells, each show distinctive differentiation and radioincorporation patterns.This study was supported in part by General Research Support Grant, FR5366 of the NIH, Minnesota Medical Foundation Grants, and the Asthmatic Children's Aid of Chicago, Illinois. The authors wish to thank Karen Brintzenhofe and Doreen Fleetwood for their assistance     

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.     

Proliferation of cells undergoing chondrogenesis in vitro

Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [3H] thymidine thymidine [(3H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [3H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [3H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [3H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype. We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.     

The transfer of 5'-methylthioadenosine from B82 cells through the medium to CHW-1102 cells

The Chinese hamster cell line. CHW-1102, which is deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT+), incorporated a [3H]purine metabolite(s) from medium in which B82 cells, but not V79, A9 and BHK cells, had been grown for 24 h with [3H]hypoxanthine. A thin-layer chromatographic comparison of the medium revealed a large radioactive peak that was unique to the B82 medium and co-chromatographed with methylthioadenosine (MTA), but not with most other common purine bases and nucleosides. The addition of either MTA, adenine, or adenosine to B82 medium reduced the amount of radioactive material incorporated by CHW-1102 cells. Methylglyoxal bis(guanylhydrazone) inhibited the production of the [3H]metabolite(s) that were incorporated from B82 medium by CHW-1102 cells. Little MTA phosphorylase activity was detected in the mouse L cell lines, L929, B82, and A9, but activity was present in CHW-1102 cells. These results suggest that one of the metabolites in B82 medium is [3H]MTA, and this is taken up and cleaved by CHW-1102 cells to yield [3H]adenine, which is incorporated into nucleic acids. This accounts for the majority of contact-independent metabolite transfer (CIMT). In cocultures some interactions between B82 and CHW-1102 cells were positive for contact-dependent metabolite transfer (CDMT) or metabolic cooperation.     

Effects of [3H]UdR on the cell-cycle progression of L1210 cells

Tritium-labelled uridine [( 3H]UdR) perturbs progression of L1210 cells through the mitotic cycle. The main effect manifests as a slowdown or arrest of a portion of cells in G2 and is already observed 2 hr after addition of 0.5-5.0 microCi/ml of [3H]UdR into cultures. At 2.5-5.0 microCi/ml of [3H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [3H]UdR for 8-24 hr. A pulse of [3H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G2 slowdown) are observed when the amount of the incorporated [3H]UdR is such that, on average there are fewer than thirty-six [3H] decays per cell which corresponds to approximately 12-19 rads of radiation. The S-phase slowdown is seen at a dose of incorporated [3H]UdR twice as high as that inducing G2 effects. The specific localization of [3H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [3H]UdR and [3H]thymidine. Mathematical modelling of the cell-cycle effects of [3H]UdR is provided.     

Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor.

We report that the cell surface glycoprotein CD4 expressed in HeLa cells can be metabolically labeled with [3H]palmitic acid. Analysis of the 3H-label after hydrolysis of the protein indicated that it was incorporated predominantly as palmitic acid. Comparison of the amount of [3H]palmitate incorporated into CD4 with that incorporated into a protein known to contain one molecule of esterified palmitate suggested that one to two molecules of palmitate were added to CD4. The fatty acid was readily cleaved from CD4 by treatment with weak base suggesting a thioester linkage. Mutations of each of 2 cysteine residues, Cys394 and Cys397, in CD4 at the junction of the transmembrane and cytoplasmic domains reduced labeling with [3H]palmitic acid, and mutation of both cysteines eliminated labeling. These results indicate that both cysteines are esterified to palmitate. Modification with palmitate was not required for expression of CD4 on the cell surface or for binding of p56lck to its cytoplasmic domain.     

Incorporation of yeast tRNA into mouse L 1210 lympholeukemic cells

[32P]tRNA from baker's yeast is incorporated without degradation into lympholeukotic cells of L1210 mice. The tRNA incorporation determined after tRNA hydrolysis on cell surface by RNAase increases linearly with a rise in the initial concentration from 0.5 to 500 micrograms per ml. According to gel electrophoresis of intracellular nucleic acids, after a 3 hour incubation the [32P]tRNA incorporated into the cells by 50% to form tRNA fragments without any conspicuous reutilization. The kinetic curve of tRNA incorporation during the first 60 min demonstrates a severalfold decrease in the initial maximal incorporation of [32P]tRNA into the cells (2 min), with a subsequent restoration of the incorporation within 2-3 hours.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.