Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene

We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.     

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.     

Antibodies to peptide determinants in transforming growth factor beta and their applications

Polyclonal antibodies have been raised to a series of synthetic peptides which correspond to essentially all regions of the transforming growth factor beta 1 (TGF-beta 1) molecule. All antisera were evaluated for their abilities to react with TGF-beta 1 and TGF-beta 2 in either the native or reduced form in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. While all antisera demonstrated some ability to recognize TGF-beta 1 in these systems, there was limited cross-reactivity with TGF-beta 2, suggesting that substantial sequence or conformational differences exist between the two growth factors. On Western blots 5-10 ng of purified human platelet TGF-beta 1 could be detected when probed with affinity-purified peptide antisera generated against peptides corresponding to residues 48-77, 50-75, and 78-109 of the 112 amino acid TGF-beta 1 monomer. Antisera raised against peptides 50-75 and 78-109 were most effective in immunoprecipitating reduced and native 125I-TGF-beta 1, respectively. The antisera also were tested for their effectiveness in blocking the binding of 125I-TGF-beta 1 to its receptor. Anti-peptide 78-109 and anti-peptide 50-75 blocked 80% and 40% of the binding, respectively, while antibodies against amino-terminal peptides were without effect. These data suggest that the carboxyl-terminal region of TGF-beta 1 may play a significant role in the binding of the native ligand to its receptor.     

Identification of a pterin derivative in Escherichia coli DNA photolyase

DNA photolyase from Escherichia coli contains reduced flavin adenine dinucleotide plus a second chromophore, partially characterized in previous studies. Both chromophores function as sensitizers in catalysis. The second chromophore has been identified as a 6-substituted pterin derivative. The compound is oxidized with permanganate to yield 6-carboxypterin or reduced with sodium cyanoborohydride to yield a 5,6,7,8-tetrahydropterin derivative. The second chromophore exhibits spectral properties (lambda max = 360, 255 nm, pH 2) similar to that observed for 7,8-dihydropterin cations. The compound does not exhibit a spectrally detectable pKa around 4 but is converted to a dication (lambda max = 346, 255 nm) in strong acid (pKa approximately 1). Similar ionization behavior is observed with 7,8-dihydropterin derivatives that are alkylated at N(5). The instability of the second chromophore in weakly alkaline solution is due to a fully reversible conversion to a labile bleached form. As compared with other pterin derivatives, the hydrolytic instability is unusual but is very similar to that observed for 5,6-dialkyl-7,8-dihydropterinium salts. It is proposed that the second chromophore is a 7,8-dihydropterin with substituents at positions 5 and 6. The discovery that a pterin derivative functions as a photosensitizer in DNA repair is apparently the first example of a photobiological function for pterins.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Altered plasma membrane ultrastructure in multidrug-resistant cells

Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.     

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin

We have examined the ability of various forms of activin and inhibin, which are structurally related to transforming growth factor-beta (TGF-beta), to interact with various types of cell surface TGF-beta binding sites. Activin AB, inhibin A, and inhibin B were unable to compete with 125I-TGF-beta 1 for binding to the TGF-beta receptor types I, II, or III that coexist in human skin fibroblasts, rat liver epithelial cells, and mink lung epithelial cells. In contrast, activins and inhibins effectively competed for TGF-beta 1 binding to GH3 rat pituitary tumor cells. Binding of TGF-beta 1 to GH3 cells was mediated by about 2700 sites/cell with a Kd = 90 pM. Affinity labeling of these GH3 binding sites by cross-linking to 125I-TGF-beta 1 yielded 70-74-kDa labeled complexes distinct from previously identified TGF-beta binding components. Labeling of these 70-74-kDa components with 125I-TGF-beta 1 was inhibited by TGF-beta 1, TGF-beta 2, activin AB, and inhibin B at concentrations in the high picomolar to low nanomolar range, but it was not significantly affected by other polypeptide hormones and growth factors tested. The 70-74-kDa labeled GH3 components represent a novel type of cell surface TGF-beta binding protein that is unique in its ability to recognize various other members of the TGF-beta family of bioactive polypeptides.