Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of chemical selectivity in micellar electrokinetic chromatography. VI. Effects of surfactant counter-ion.

Linear solvation energy relationships and free energy of transfer data were used to evaluate the influence of the surfactant counter-ion on selectivity in micellar electrokinetic chromatography. It was determined that selectivity differences are dependent on the valency of the counter-ion but not the type of counter-ion. Monovalent surfactants, sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate, have nearly identical selectivity behavior. The divalent surfactants, magnesium didodecyl sulfate and copper didodecyl sulfate also show very similar behavior. However, when the divalent counter-ion species is compared to SDS under similar conditions, significant differences are observed. Most notably, the utilization of divalent counter-ion species of dodecyl sulfate surfactants causes the micelles to become more hydrophobic and a weaker hydrogen bond donating pseudo-stationary phases. It is believed that the divalent counter-ions reduce the electrostatic repulsion between the surfactant head groups and therefore, increase the chain packing of the monomers in the micelle aggregates. This reduces the degree of hydration of the micellar palisade layer leading to a decreased ability of the micelle to participate in polar/polarizable and hydrogen bonding interactions with solute molecules.

Affinity chromatography of an S-adenosylmethionine-dependent methyltransferase using immobilized S-adenosylhomocysteine. Purification of the indolethylamine N-methyltransferases of phalaris tuberosa.

In the cases that have been studied so far, S-adenosylhomocysteine (SAH) is a powerful inhibitor of S-adenosylmethionine (SAM) binding to SAM-dependent methyltransferases. We deduced, from the available data on the binding of SAM and SAH analogues to SAM dependent methyltransferases, that linkage of SAH through the carboxyl group to an immobilized support would lead to a more general affinity adsorbent for SAM-dependent methyltransferases than linkage through other functional groups. This paper describes the synthesis of this affinity adsorbent and its use to purify the two indolethylamine N-methyltransferases of Phalaris tuberosa.

A general method of curve fitting and error analysis using a spreadsheet: determination of the binding constants of tight binding ligands in variable volume assays.

A generalized iterative method of curve fitting and error estimation is described, using a spreadsheet with Monte Carlo simulations. The method is demonstrated on an equilibrium binding experiment with reverse transcriptase of human immunodeficiency virus-1, in which the concentration of all reactants, including enzyme, can vary arbitrarily and any number of ligands can bind tightly.

N,N-Dimethyltryptamine Production in Phalaris aquatica Seedlings: A Mathematical Model for its Synthesis.

The activities of three enzymes and the concentration of intermediates involved in the synthesis of N,N-dimethyltryptamine (DMT) from endogenous tryptophan (TRP) have been measured in vitro in seedlings of Phalaris aquatica L. cv Australian Commercial over 16 days after planting. The activities of tryptophan decarboxylase and the two N-methyl-transferases increased rapidly to maximal rates of substrate conversion at day 5 of 95, 1000, and 2200 micromoles per hour per milliliter, respectively. After these maximal rates, the activities decreased rapidly. The concentration of intermediates increased rapidly from zero in the seeds to maximal values of 25 and 53 micromolar at day 5 for tryptamine (T) and N-methyltryptamine (MT), respectively, 1000 micromolar at day 6 for TRP, and 650 micromolar at day 8 for DMT. The concentration of DMT and of all the intermediates in its synthesis declined rapidly after the maximal value had been reached. A mathematical model of the pathway from TRP to DMT using these enzymes correctly predicts the concentrations of T and MT, intermediates whose concentration is determined only by the pathway, and confirms that these three enzymes are responsible for the in vivo synthesis of DMT. Kinetic studies are reported for these enzymes. Tryptophan decarboxylase uses pyridoxal phosphate (PALP) as a coenzyme and has the following kinetic constants: K(m) (PALP) = 2.5 micromolar, K(m) (TRP) = 200 micromolar, K(i) (MT) = 5 millimolar, and K(i) (DMT) = 4 millimolar. The N-methyltransferases use S-adenosylmethionine (SAM) as substrate; S-adenosylhomocysteine (SAH) is assumed to be the product. The mechanism of secondary indolethylamine-N-methyltransferase, determined by initial velocity studies, is rapid equilibrium random with formation of both dead end complexes. Secondary indolethylamine-N-methyltransferase methylates both MT and 5-methoxy-N-methyltryptamine (5MeOMT). The kinetic constants for the methylation of MT are: K(MT) = 40 +/- 6, K(SAM) = 55 +/- 15, K(DMT) = 60, K(SAH) = 4.3 +/- 0.4 micromolar with unity interaction factors. The kinetic constants for the conversion of 5MeOMT to 5-methoxy-N,N-dimethyltryptamine (5MeODMT) are K(5MeOMT) = 40 +/- 10, K(SAM) = 90 +/- 40, and K(SAH) = 2.9 +/- 0.3 micromolar with unity interaction factors, except for SAM-5MeODMT = 2.0 +/- 0.9 and SAH-5MeOMT = 0.45 +/- 0.25. The kinetic constants for primary indolethylamine N-methyltransferase are K(m) (T) = 20, K(m) (SAM) = 40, K(i) (DMT) = 450 micromolar with the substrates binding independently.

Microbial film development in a trickling filter.

The transmission and scanning electron microscopes were employed to visualize the sequence of the biofilm development in the trickling wastewater filter. After the deposit of a small amount of debris upon a hard surface, the bacterial cells attach and develop the matrix on which the biofilm is formed. Protozoa invade the basic layer where they feed on the bacteria. The algae are seeded upon the bacterial matrix and grow so profusely that the bacteria must develop aerial colonies in the competition for food and oxygen. Destruction of the bacteria in the matrix and the weight and hydraulic pressure cause detachment of the biofilm and a new matrix must be developed.

Retroviral integrase domains: DNA binding and the recognition of LTR sequences.

Integration of retroviral DNA into the host chromosome requires a virus-encoded integrase (IN). IN recognizes, cuts and then joins specific viral DNA sequences (LTR ends) to essentially random sites in host DNA. We have used computer-assisted protein alignments and mutagenesis in an attempt to localize these functions within the avian retroviral IN protein. A comparison of the deduced amino acid sequences for 80 retroviral/retrotransposon IN proteins reveals strong conservation of an HHCC N-terminal 'Zn finger'-like domain, and a central D(35)E region which exhibits striking similarities with sequences deduced for bacterial IS elements. We demonstrate that the HHCC region is not required for DNA binding, but contributes to specific recognition of viral LTRs in the cutting and joining reactions. Deletions which extend into the D(35)E region destroy the ability of IN to bind DNA. Thus, we propose that the D(35)E region may specify a DNA-binding/cutting domain that is conserved throughout evolution in enzymes with similar functions.

A covalent complex between retroviral integrase and nicked substrate DNA.

Purified retroviral integrase (IN) from avian sarcoma-leukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequence-independent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.

Requirement for a conserved serine in both processing and joining activities of retroviral integrase.

Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.