Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations

A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.     

DNA distribution and respiratory activity of Spodoptera frugiperda populations infected with wild-type and recombinant Autographa californica nuclear polyhedrosis virus.

Spodoptera frugiperda cells were infected with a wild-type Autographa californica nuclear polyhedrosis virus and with a recombinant Autographa californica nuclear polyhedrosis virus. The recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. The changes in cell size, cell growth, viability, DNA distribution, and respiratory activity were followed through the time course of the infection. The DNA content as measured by flow cytometry of infected cells increased to approximately 1.8 times the value of uninfected cells and the distributions of single-cell DNA content of the infected cells were strongly deformed. Early in the infection the respiratory activity passed through a maximum. The mitochondrial activity based on Rhodamine 123 labelling of cells infected with the recombinant virus, as determined by flow cytometry, also passed through a maximum at 24 h post infection while the mitochondrial activity of cells infected with the wild-type virus continued to increase. Evolution of single-cell mitochondrial activity was different in uninfected populations and in populations infected with wild-type and with recombinant virus. In all experiments performed, the recombinant virus influenced cell behavior and the measured parameters earlier than the wild-type virus. The influence of the multiplicity of infection was stronger for the wild-type virus than for the recombinant virus.     

An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice.

We have obtained transgenic mice expressing nuclearly targeted beta-galactosidase (nls-beta-gal) under the control of a chicken acetylcholine receptor alpha-subunit promoter. The expression of the transgene was detected in early somites, starting before embryonic day 9.5. In 13-day embryos, the expression pattern of the transgene closely paralleled that of the endogenous mouse alpha-subunit gene, assessed by in situ hybridization. Our results illustrate, with single-cell resolution, the tissue specificity of this alpha-subunit promoter during embryogenesis. After birth, the overall beta-galactosidase activity rapidly decreased with age. However, in diaphragms of newborn animals, beta-galactosidase activity selectively persisted in nuclei underlying the motor endplates. The latter were revealed by an acetylcholinesterase stain. Nls-beta-gal was also visualized by indirect immunofluorescence, while endplates were labelled with fluorescent alpha-bungarotoxin. Confocal microscopy unambiguously identified the more intensely stained nuclei as synaptic 'fundamental nuclei', and allowed estimates of relative staining levels. Thus an 842 bp acetylcholine receptor gene promoter confers preferential synaptic expression to a reporter gene within myofibres in vivo.     

Phenotypic diversity of cloned lines of Leishmania major promastigotes

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.     

A segregated model for plasmid content and product synthesis in unstable binary fission recombinant organisms

Plasmid propagation in populations of unstable, binary fission recombinant organisms has been studied using a segregated, population balance mathematical model. Segregated models have the advantage of direct incorporation of basic information on mechanisms and kinetics of plasmid replication and segregation at the single-cell level. The distribution of cellular plasmid content and specific rates of plasmid gene expression have been obtained for several single-cell models of plasmid replication, partition, and gene expression. Plasmid replication kinetics during cell growth significantly influence the plasmid content distribution. In the case of transient growth of plasmid-containing and plasmid-free cells in partially selective medium, the degree of selection required for stable maintenance of plasmid-containing cells has been determined. Guidelines are presented for applicability of simpler, nonsegregated models and for evaluation of the parameters in these models based on single-cell mechanisms and associated parameters.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Structure of the beta-galactosidase gene from Streptococcus diacetylactis 144 and its expression in Escherichia coli and Streptococcus diacetylactis cells

The ability of industrial strains of mesophylic Streptococcus diacetylactis to synthesize the enzyme beta-galactosidase has been studied. Among the 22 studied strains 8 were found to synthesize the enzyme. Plasmid DNA was isolated from the Streptococcus diacetylactis strain 144 possessing the highest level of beta-galactosidase activity. The cells of the strain harbour the 35, 40 and 60 kb plasmids. The alpha-galactosidase genes from this strain was cloned in Escherichia coli cells. The gene is located on the BglIII DNA fragment of the total plasmid DNA from Streptococcus diacetylactis the size of 2.8 kb. Following the Sau3A restriction endonuclease digestion the gene was subcloned on a birepliconed vector plasmid pCB20. The latter is capable of replication in the Gram-negative as well as Gram-positive microorganisms. The pCB20 derivatives carrying the different length fragments with the beta-galactosidase gene were isolated. DNA of an obtained plasmid was used for transformation of Streptococcus diacetylactis cells. The presence of the recombinant plasmid in streptococcus strain 144 results in the 1.8 fold increase in beta-galactosidase production.     

Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.     

Isolation and characterization of the yeast 3-phosphoglycerokinase gene (PGK) by an immunological screening technique

An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.     

Mathematical modeling of a single-cell enzyme assay

A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.     

Preservation of cells sorted individually onto microscope slides with a fluorescence-activated cell sorter

Fluorescence-activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one-hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.     

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.     

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.     

The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells

Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TM antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.