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Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.
Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for adrenodoxin binding which is electrostatic in nature.
Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.
Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.
Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.
A unique cAMP regulatory sequence, -129/-96 base pairs (bp), associated with the gene encoding human cytochrome P450C21 (CYP21B) binds a nuclear protein designated ASP, as described previously (Kagawa, N., and Waterman, M. R. (1991) J. Biol. Chem. 266, 11199-11204). This putative transcription factor required for cAMP-dependent transcription of the human CYP21B gene has been purified from the nuclear extracts of mouse Y1 cells by using sequence-specific DNA-affinity chromatography. The purified ASP is 78 kDa as estimated by SDS-polyacrylamide gel electrophoresis and binds to its specific recognition site, -126/-113-bp CACTCTGTGGGCGG, which has been demonstrated to be the minimum cAMP regulatory sequence of the human CYP21B gene. To characterize ASP more precisely, an antibody was raised against the 78-kDa protein. This antibody led to a supershift of the DNA.ASP complex on gel shift analysis and inhibition of in vitro transcription promoted by the ASP binding sequence, thereby indicating that ASP is a 78-kDa transcription factor. Upon DNase I footprinting experiments, ASP showed a characteristic footprint which very closely resembles but is distinct from that of Sp1 which also occupies a binding site within -129/-96 bp. Furthermore, the addition of purified ASP enhanced the mRNA synthesis promoted by the minimum cAMP regulatory sequence in a cell-free transcription system using HeLa cell extracts, whereas added Sp1 does not. These results indicate that ASP is a primary transcription factor for the cAMP-dependent regulation of the human CYP21B gene.
In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)
The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported. Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones. The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions. No helper VSV was required. Replication occurred at high levels and was assayed by direct biochemical means. An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.
Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.
Respiratory syncytial virus (RSV) is the most important cause, throughout the world, of severe viral lower respiratory tract illness in young children. Antibodies are known to mediate resistance to RSV infection and illness. We have isolated a number of human monoclonal Fab fragments to RSV F glycoprotein from a combinatorial antibody library expressed on the surface of phage. One of these neutralized a wide range of virus isolates, 10 subgroup A and 9 subgroup B isolates, with a titer (60% neutralization) of approximately 0.1-1.0 micrograms/ml. Another Fab neutralized diverse isolates at a concentration somewhat higher. These human Fab fragments show great promise for use in the prophylaxis or therapy of serious RSV lower respiratory tract disease. For intramuscular or intravenous administration, whole antibodies will be required, whereas for aerosol application, F(ab')2 or Fab fragments may suffice.