Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coamplification of the transfected sequences. Expression vectors containing the t-PA cDNA gene and dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.     

Structure and expression of the Chinese hamster thymidine kinase gene.

My colleagues and I have cloned a nearly full-length Chinese hamster thymidine kinase (TK) cDNA in a lambda gt10 vector and characterized this cDNA by nucleotide sequencing. The hamster TK protein is encoded in this cDNA by a 702-base-pair open reading frame which specifies a 25,625-dalton protein closely homologous to the previously described human and chicken TK proteins. Using cDNA nucleotide sequence data in conjunction with sequence data derived from selected subclones of the hamster TK gene recombinant phage lambda HaTK.5, we have resolved the structure of the TK gene, finding the 1,219 base pairs of the cDNA sequence to be distributed through 11.2 kilobases of genomic DNA in at least seven exon segments. In addition, we have constructed a variety of Chinese hamster TK minigenes and exonuclease III-S1 derivatives of these genes which have permitted us to define the limits of the Chinese hamster TK gene promoter and demonstrate that efficient TK transformation of Ltk- cells by TK minigenes depends on the presence of both TK intervening sequences and sequences 3' to the site of mRNA polyadenylation.     

Expression of amplified cDNA and genomic sequences encoding human interleukin 2 in Chinese hamster ovary cells

Expression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Transformants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2 mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in Chinese hamster cells.

Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferas (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT+ Chinese hamster or human cells resulted in the appearance of HPRT+ cells. Clones derived from these cells were isolated in HAT medium. Cells in mitosis during incubation with the chromosomes yielded thr-e times more HPRT+ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT+ clones than did the interspecies system using human chromsomes and Chinese hamster recipient cells (5 X 10(-5) and 6 X 10(-6) respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.     

The Chinese hamster cell emetine resistance gene. Analysis of cDNA and genomic sequences encoding ribosomal protein S14

We used an intersecting pool strategy to recognize chimeric plasmids containing Chinese hamster ribosomal protein cDNAs. The screening procedure involved hybridization-selection of messenger RNAs, cell-free translation of selected mRNAs, and electrophoresis of polypeptide products on one- and two-dimensional polyacrylamide gels. The protocol was designed to recognize ribosomal protein S14 cDNAs specifically. Of 500 chimeric plasmids screened, two possessed cDNAs complementary to S14 mRNA and 18 contained sequences complementary to other ribosomal protein messages. Previously we demonstrated that mutations affecting Chinese hamster ovary cell ribosomal protein S14 are responsible for genetic resistance to the translational inhibitor emetine (emt b). Because emetine-resistant mutant and wild type Chinese hamster ovary cells elaborate mRNAs that encode electrophoretically distinguishable forms of S14 protein, we were able to identify S14 cDNA clones unambiguously. The data described here indicate that: 1) clone pCS14-1 contains most, if not all, of the S14 coding sequence as a cDNA; 2) S14 mRNA is approximately 0.01% of a Chinese hamster cell's polyadenylated messenger RNA; and 3) genomic DNA-encoding ribosomal protein S14 is a low, perhaps single, copy sequence with a complex structure, including several, long intervening sequences.     

Nucleotide ectoenzyme activities of human and Chinese hamster fibroblasts in tissue culture

We have previously assigned human ecto-5'-nucleotidase (NT) to chromosome 6 on the basis of conversion of exogenously supplied [14C]AMP to adenosine by whole cells of human and Chinese hamster hybrids carrying chromosome 6. In this paper we demonstrate that the activity on human MRC-5 fibroblasts is typical of previously described and purified ecto-5'-nucleotidases. In contrast to MRC-5 cells, Chinese hamster V79A2 cells weakly express an AMPase activity that is not NT. The cytosolic form of NT in human and hybrid fibroblasts is similar to the ectoenzyme in substrate specificity. Hybrids that lack chromosome 6 express neither the ecto- nor the cytosolic enzyme, suggesting that both forms may be coded by the same gene on chromosome 6. Ecto-ATPase, ecto-ADPase, and ecto-ADP kinase activities are each expressed at similar levels in MRC-5 and V79A2. The ATPase, ADPase and NT activities of MRC-5 cells act sequentially to generate adenosine. A similar cascade acts on V79A2 cells but the lack of NT causes the accumulation of AMP.     

The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen

The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.     

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.     

Transforming potential of the v-myb oncogene from avian myeloblastosis virus: alterations in the oncogene product may reveal a new target specificity

Transfection of brown leghorn chicken embryo fibroblasts by DNA containing v-myb sequences cloned either in a complete AMV proviral DNA or in a retroviral derived vector has led to the isolation of two kinds of transformed cells. A characterization of the proviral sequences retained and expressed in these transformed cells revealed that they contained either new or altered v-myb-related RNA species. The experiments presented in this paper also show that both types of transformants expressed truncated myb-related polypeptides, suggesting that alterations of the v-myb product may result in a new target specificity, leading to the transformation of chicken embryo fibroblasts.     

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes.

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.     

Expression of several amplified genes in an adenylate-deaminase overproducing variant of Chinese hamster fibroblasts

Unstable variants with increasing amounts of adenylate-deaminase (AMPD) have been stepwise recovered from Chinese hamster fibroblasts plated in selective medium containing increasing coformycin concentrations; several polypeptides accumulate in the variants in parallel to AMPD: they are no longer detectable in cells which reverted to the wild-type enzyme level. We report here the molecular cloning of cDNA sequences complementary to mRNAs coding for four such polypeptides. The plasmidic probes have been exploited to characterize their complementary mRNAs and to quantify the copies of these cognate genes in a variant and in two revertant clones. The results show that different mRNAs code for the four polypeptides; their accumulation is accounted for by amplification of their specific genes; these observations suggest that cells overproducing AMPD are characterized by the presence of amplification units comprising several expressed genes.