An amplified insect dihydrofolate reductase gene contains a single intron

We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.     

Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.     

Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.     

The establishment of IL-2 producing cells by genetic engineering

Expression plasmids containing human interleukin-2(IL-2) cDNA under the control of viral promoters (SV40 early region, MuLV LTR, HTLV-I LTR, and ASV (Y73) LTR) were introduced into TK- mouse L cells and human FL cells to establish IL-2 producing cells. The highest levels of IL-2 producing clones were obtained in TK+ mouse L cells transformed with a recombinant plasmid having MuLV LTR as a promoter, whereas transformed cells of human FL cells (G418r) were revealed to produce IL-2 at the highest level when the cells were transfected with a plasmid containing HTLV LTR as a promoter. These results suggest that these promoter/enhancer regions possess different cell specificities in gene expression. To obtain higher levels of IL-2 production using gene amplification, the hybrid plasmids containing the hamster DHFR and human IL-2 genes were constructed and transfected into DHFR- CHO cells. DHFR+ colonies produced IL-2 at about the same level as that produced by TK+ L cells transformed with the recombinants containing MuLV LTR. Selection of methotrexate-resistant cells resulted in a 5- to 30-fold increase of IL-2 production. These cells produced IL-2 stably for at least 3 months, even in the absence of methotrexate.     

Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance.

Transformed Drosophila Kc cell lines, resistant to methotrexate, an inhibitor of de novo purine and pyrimidine synthesis, have been obtained by calcium phosphate transfection of plasmids containing a sequence coding for a methotrexate-resistant dihydrofolate reductase enzyme (DHFR). The introduced DNA is stably maintained in the cells as head-to-tail multimeric structures of the initial DNA sequence even after several months of culture in the presence of the selective agent. The introduced sequences are present at a high copy number in the transformed cells and express cytoplasmic RNAs transcribed from the DHFR gene.     

黄瓜花叶病毒cDNA载体在烟草细胞中表达外源基因

为了探讨利用黄瓜花叶病毒(CMV)构建表达载体的可行性,分离了山东株(SD) CMV RNA 3的全长cDNA。测定其全序列后,采用定点突变的方法在衣壳蛋白(CP)基因起始密码子处改造出一个NsiⅠ位点,可将外源基因引入NsiⅠ位点和CP基因终止密码子上游附近的XhoⅠ或SalⅠ位点而置换掉CP基因。分别用绿色荧光蛋白(GFP)基因、β-葡糖醛酸酶(GUS)基因以及小鼠二氢叶酸还原酶(DHFR)基因3种报告基因置换CP基因。将Fny株CMV RNA 1、RNA 2和SDCMV嵌合RNA 3的cDNA分别插入pCass载体的35S启动子和终止子之间,将构建的置换型载体直接以质粒的方式转染烟草原生质体,表达了3种报告基因。     

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.     

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.     

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.     

Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.     

Construction of a dominant selectable marker using a novel dihydrofolate reductase.

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.     

Similarities between human ataxia fibroblasts and murine SCID cells: High sensitivity to γ rays and high frequency of methotrexate-induced DHFR gene amplification,but normal radiosensitivity to densely ionizing α particles

Two-ray hypersensitive cell lines, human ataxia telangiectasia (AT) and murine severe combined immune deficiency (SCID) cells, proved to be very competent in amplifying their dihydrofolate reductase (DHFR) gene under methotrexate selection stress. Over a period of months, methotrexate-resistant clones were obtained which were able to grow in progressively increasing methotrexate concentrations up to 1 mM. By then methotrexate-resistant AT and SCID cells had amplified their DHFR gene 6- and 30-fold, respectively, and showed very high DHFR mRNA expression. In contrast, related cells with normal radiosensitivity (human GM637 and mouse BALB/c fibroblasts) did not show DHFR gene amplification under comparable conditions. This correlation of the capacity of DHFR gene amplification and-ray hypersensitivity in AT and SCID cells suggests that gene amplification may have a mechanism(s) in common with those involved in repair of-radiation-induced damage. No difference in cell killing could be observed following exposure to densely ionizing particles: AT and SCID cells exhibited comparable survival rates to GM637 and BALB/c cells, respectively.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.