Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster overy (CHO) cell line producing recombinant hepatits B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gane copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autroadiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.     

Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells

We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

A foreign dihydrofolate reductase gene in transgenic mice acts as a dominant mutation.

We have produced 17 lines of transgenic mice by microinjecting a full-length cDNA clone of an altered dihydrofolate reductase (dhfr) gene. The protein specified by this gene carries a point mutation which triples its Km for dihydrofolate and reduces substrate turnover 20-fold relative to the wild-type enzyme. Transgenic mice from different pedigrees, several of which carry a single copy of this gene in different integration sites, manifest an array of similar developmental abnormalities including growth stunting, reduced fertility, pigmentation changes, and skeletal defects. These defects appear in animals heterozygous for the foreign gene. RNA analyses demonstrate significant expression of the cDNA in newborn mice and adult tissues. These findings show that the additional dhfr gene exerts its mutational effects in a dominant fashion, and therefore the data indicate that transgenic mice can serve as models for elucidating mechanisms of dominant mutagenesis.     

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.     

Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities.

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.     

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.     

An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.     

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.