Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.     

Molecular cloning of Chinese hamster dihydrofolate reductase-specific cDNA and the identification of multiple dihydrofolate reductase mRNAs in antifolate-resistant Chinese hamster lung fibroblasts.

ds cDNA from antifolate-resistant Chinese hamster lung fibroblast subline DC-3F/MQ19 was ligated to Eco RI and Sal I oligonucleotide linkers and cloned into Eco RI and Sal I digested pBR322. Transformed colonies containing dihydrofolate reductase (DHFR)-specific recombinant plasmid were identified by Grunstein Hogness assay using a Chinese hamster DHFR-specific cDNA probe. A recombinant plasmid, pDHFR6, containing a 650 bp HFR insert was isolated and analyzed. This plasmid was used as a molecular probe in a Northern blot analysis of both cytoplasmic and polysomal DHFR, poly A+ mRNAs of the DC-3F/MQ19 subline, which over-produces a 20,000d DHFR 150-fold, and DC-3F/A3 subline, which over-produces a 21,000d DHFR 170-fold. This analysis revealed the presence of three DHFR mRNA species of 1350, 2200, and 3300 nucleotides in both independently-derived cell lines. The relative abundance of each species however varied strikingly between the two cell lines.     

Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.     

Deoxyhypusine/hypusine formation on a 21,000-dalton cellular protein in a Neurospora crassa mutant in vivo and in vitro

Hypusine formation on an 18,000-dalton cellular protein is a unique spermidine-dependent, post-translational modification that appears to be ubiquitous in mammalian cells. To determine whether this modification also exists in lower eukaryotes, we examined possible labeling in vitro and in vivo of cellular protein(s) by [3H]spermidine in a mutant strain of Neurospora crassa (arge-12 ota aga) in which ornithine and polyamine synthesis could be nutritionally manipulated. Because of poor uptake of polyamines in this organism, [3H]ornithine, the immediate precursor of polyamines, was used for the in vivo labeling experiment. Both in vitro and in vivo labeling resulted in a specific labeling of a 21,000-dalton protein. Radioactive hypusine was recovered from radiolabeled 21,000-dalton protein following acid hydrolysis. The in vitro labeling of the 21,000-dalton protein was dramatically stimulated by NAD+ and NADP+, but not by FMN or FAD, suggesting that an NAD+/NADP(+)-dependent oxidative cleavage of spermidine is involved in deoxyhypusine formation. Isoelectric focusing/sodium dodecyl sulfate two-dimensional gel analysis revealed three isoforms of the in vitro labeled 21,000-dalton protein, with pI values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo gave only one spot with a pI value of approx. 3.5.     

Surfactant protein C precursor is palmitoylated and associates with subcellular membranes.

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by proteolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of Mr = 21,000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.     

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.     

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.     

Creatine phosphokinase: isoenzymes in Torpedo marmorata

Creatine phosphokinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) is the major constituent of the "low-salt-soluble" proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43 000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti-(electric organ creatine phosphokinase) antibodies are specific for the muscle-type enzyme and do not cross-react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine-receptor-enriched membranes show that this enzyme is as the "low-salt-soluble" electric organ enzyme of the muscle-specific type. In vitro translation of electric organ poly(A)-rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain-type polypeptides are synthesized. The subunits of the brain-specific enzyme differ in molecular mass (Mr approximately equal to 42000) and isoelectric properties (pI approximately equal to 7.0-7.2). The unexpected finding that the brain forms are more basic than the muscle-specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion-exchange chromatography properties.     

Identification of amino acids required for the functional up-regulation of human dihydrofolate reductase protein in response to antifolate Treatment

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.     

Purification and characterization of major extracellular proteinases from Trichophyton rubrum.

Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).     

Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor

In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.     

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.