Cultured Monkey Cells Research Articles

Peroxidase-labeled primary antibody method for detection of pestivirus contamination in cell cultures

Contaminating bovine viral diarrhea (BVD) virus in cell cultures and in fetal bovine serum (FBS) is a well recognized problem. This study describes a direct peroxidase (DP) labeled primary antibody method for detection of pestivirus antigens in cell culture that is simple and reliable. Using this method, most bovine and porcine cell cultures, a cat lung, four mosquito and two monkey cell cultures were found contaminated with BVD virus. The rodent and human cell cultures tested were negative by this method for BVD virus.

Translation Initiation at Non-Aug Triplets in Mammalian Cells

In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S. (1987) J. Biol. Chem. 262, 11847-11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [35S]tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.

Leader length and secondary structure modulate mRNA function under conditions of stress.

Simian virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonucleotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.

Leader length and secondary structure modulate mRNA function under conditions of stress.

Simian virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonucleotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.

Evidence for Drosophila P element transposase activity in mammalian cells and yeast

Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

Translation initiation at an ACG triplet in mammalian cells.

The initiator AUC of the mouse dihydrofolate reductase gene (dhfr) was converted to ACG by site-directed mutagenesis and assayed for expression in cultured monkey cells using an SV40 recombinant called SVGT5dhfr26m2. Synthesis of apparently full-length dihydrofolate reductase (DHFR) protein was significantly reduced compared to wild-type, but not entirely abolished, suggesting that the ACG triplet was being utilized for translation initiation. In addition, a truncated form of DHFR was produced, apparently by initiation at the next in-frame AUG downstream. This result was confirmed in vitro. Transcripts of the dhfr sequence were produced by SP6 RNA polymerase in the presence of m7GpppG and translated in vitro using reticulocyte lysates and wheat germ extracts. The results paralleled those observed in vivo. Synthesis of full-length DHFR was reduced, but not eliminated, and a new species was produced by initiation at an internal site. Amino acid sequence analysis of the products of in vitro translation demonstrated that translation does indeed initiate at the ACG triplet and that it initiates with methionine. Additional mutations were introduced which altered the sequence context of the ACG triplet. Mutation of the translation initiation consensus sequence by substitution of the A residue at position -3, or of the G at +4 resulted in a significant decrease in initiation at the ACG and an increase in the level of the internal initiation product. Thus, translation initiation at a non-AUG triplet depends on a favorable sequence context.

Isolation of a protein antigen from Coxiella burnetii

Antigens prepared from Coxiella burnetii, strain Frankfurt, phase II, propagated in persistently infected Buffalo Green Monkey (BGM) cell cultures were purified by guanidinium hydrochloride treatment and chloroform/methanol extraction. By ELISA analysis, chloroform/methanol residues (CMR) proved to be free of host cell antigens. The CMR were sensitive to trypsin, pronase E and proteinase K, as determined by absorption-kinetics of CMR suspensions at 600 nm and release of protein. Coomassie blue stained SDS Polyacrylamide gels of proteinase hydrolysates from CMR revealed only a single component of apparently 27,000 D. Silver stained gels, however, showed a second component of apparently 12,000 D. In contrast, from untreated native C. burnetii a large variety of proteins, most of them protease-sensitive, were released by detergents at low temperatures, but the 27,000 D component was only solubilized at 60–100°C. The 27,000 D component was obviously the major protein of CMR as well as of whole cells. Antigenicity of this 27,000 D protein could be demonstrated by agargel precipitation test, ELISA and immunoperoxidase techniques applying antisera raised against whole cells and against the extracted component. The component was also recognized in a dot immunobinding assay by sera from guinea pigs infected with cloned C. burnetii strain Nine Mile, phase I, thus indicating an important role of this antigen in C. burnetii specific immune response.

Repair of single-stranded loops in heteroduplex DNA transfected into mammalian cells.

Repair of heteroduplex DNA, generated between two interacting DNA molecules during homologous recombination, has been implicated as a contributing factor in the process of gene conversion. To assess patterns of heteroduplex repair in mammalian cells, we constructed 13 different heteroduplexes from simian virus 40 wild-type and deletion mutant DNAs. Each heteroduplex contained one or multiple single-stranded loops in the intron of the gene for large tumor antigen, which is not essential during lytic infection. After transfection into cultured monkey cells, cellular repair was evaluated by restriction analysis of the amplified viral progeny from 1123 individual plaques, each representing the clonal expansion of a single repair event. Single-stranded loops were corrected prior to replication with an overall efficiency of 90%. At the position of a loop, one of the two heteroduplex strands served as a template for accurate repair 98% of the time. Repair of single-stranded loops was biased nearly 2 to 1 in favor of the strand without the loop. The efficiency, accuracy, and strand bias of repair were unaffected by loop size within the tested range, which was 25-247 nucleotides. The excision tract associated with repair of single-stranded loops rarely exceeds 200-400 nucleotides in length.

Aquatic toxicity evaluated using human and monkey cell culture assays.

A new bioassay using cultured mammalian cells was reported. In this report, the author describes the use of a cell culture assay using growing cells as a screen for aquatic toxicity. Cadmium, copper and zinc toxicity were measured.

Cell-cycle-dependent repair of damage in alpha and bulk DNA on monkey cells

Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B 1 (AFB 1) and N-acetoxy-2-acetylaminofluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially and exponentially growing cultures of monkey cells. Proficient removal of AFB 1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G 1 or late S/G 2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.

Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells.

Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis.

A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus.

Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.

Nucleotide sequence, evolution, and expression of the fetal globin gene of the spider monkey Ateles geoffroyi.

The single gamma-globin gene of the New World spider monkey Ateles geoffroyi is similar to other primate genes of the beta-globin gene cluster ("beta-like" globin genes). The number of nonsynonymous nucleotide substitutions between the coding regions of Ateles and other primate gamma-globin genes suggests that the Platyrrhine and Catarrhine evolutionary lines diverged approximately equal to 40 million years ago, an estimate reasonably consistent with the fossil record. However, the number of synonymous coding region and noncoding base differences is much smaller than predicted by various molecular "clocks." This suggests that the rate of synonymous coding and noncoding base substitution has not been constant per absolute time in primate lineages. Expression of the cloned Ateles gamma-globin gene in cultured monkey cells showed that the sequence AAUAAA near the mRNA 3' terminus is not sufficient to define the site of transcript polyadenylylation.

The minimum amount of homology required for homologous recombination in mammalian cells.

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells.

Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 Mr virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus fiber mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus.