Excess information at bacteriophage T7 genomic promoters detected by a random cloning technique.

In our previous analysis of the information at binding sites on nucleic acids, we found that most of the sites examined contain the amount of information expected from their frequency in the genome. The sequences at bacteriophage T7 promoters are an exception, because they are far more conserved (35 bits of information content) than should be necessary to distinguish them from the background of the Escherichia coli genome (17 bits). To determine the information actually used by the T7 RNA polymerase, promoters were chemically synthesized with many variations and those that function well in an in vivo assay were sequenced. Our analysis shows that the polymerase uses 18 bits of information, so the sequences at phage genomic promoters have significantly more information than the polymerase needs. The excess may represent the binding site of another protein.     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli

Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids. Transformed E. coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF. The two growth factors were purified from E. coli lysates by cation exchange and heparin-Sepharose affinity chromatography. The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea.     

Preliminary crystallographic data for transketolase from yeast

Crystals of the vitamin B1-dependent enzyme transketolase from baker's yeast have been grown from the apo- and the holoform of the enzyme, using PEG as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell constants a = 76.3 A, b = 114.2 A, and c = 163.5 A. The crystals are stable in the x-ray beam and diffract to at least 2.2 A on a conventional x-ray source. The enzyme is a dimer of identical subunits, and a Vm value of 2.2 A/dalton indicates that the asymmetric unit contains a dimer. Rotation function calculations using native data (10-5 A) revealed a local 2-fold rotation axis with phi = 0 degree and omega = 20 degrees.     

Crystal structure of the complex of ribulose-1,5-bisphosphate carboxylase and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate

The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.     

Phenolic substances as allelopathic agents arising during the degradation of rye (Secale cereale) tissues

Rye seedlings, tillering plants and crop residues were allowed to decompose in model incubation experiments. Young tissues gave rise to high concentrations of allelochemicals, whereas crop residues did not produce inhibitors. Seven phenolic acids were identified in the investigated materials; p-hydroxybenzoic protocatechuic, gallic, vanillic, syringic, p-coumaric, ferulic as well as benzoic acid. However, neither the level of these acids nor the total content of phenolic compounds corresponded to the level of phytotoxicity determined in bioassays. This demonstrated that, apart from phenolics, other unidentified water-soluble organic compounds were also responsible for the toxicity of rye decomposition products. The study was conducted within program CPBP 04.10.03. The study was conducted within program CPBP 04.10.03.     

Heavy ion effects on yeast: inhibition of ribosomal RNA synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.     

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.