Oct-1 protein promotes functional transcription complex assembly on the mouse mammary tumor virus promoter.

The ubiquitous transcription factor Oct-1 stimulates basal transcription from the mouse mammary tumor virus (MMTV) promoter by binding to octamer-related sequences present in the proviral long terminal repeat. The mechanism of transcriptional activation by Oct-1 was investigated using in vitro transcription assays with a HeLa cell nuclear extract depleted of endogenous Oct-1. Oct-1-mediated transcriptional activation could be reconstituted by addition of bacterially expressed recombinant Oct-1 protein.

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase.

Both RNA polymerase III and RNA polymerase II accurately initiate transcription from a human U6 promoter in vitro.

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box.

Transcriptional regulation of bioluminesence genes from Vibrio fischeri.

The phenomenon of cell-density-dependent control of gene expression, called autoinduction, has long been a subject of interest and investigation in bioluminescent marine bacteria. It is now becoming clear that many other bacteria, including animal and plant pathogens, use an autoinduction mechanism to regulate a variety of functions. Cell-density-dependent gene expression provides an excellent example of multicellular behaviour in the prokaryotic kingdom where a single cell is able to communicate and sense when a minimal population unit, a 'quorum' of bacteria, is achieved in order for certain behaviour of the population to be performed efficiently.

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues.

High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron.

The 3.6 kb of 5' flanking sequence, leader intron, and 0.7 kb of 3' sequence from the potato sucrose synthase gene Sus4-16 are sufficient to direct high-level expression in developing tubers, in basal tissues of axillary buds and shoots, and in meristems and caps of roots, and to confer sucrose inducibility in leaves.

Sink- and vascular-associated sucrose synthase functions are encoded by different gene classes in potato.

Two differentially regulated classes of sucrose synthase genes, Sus3 and Sus4, were identified in potato. They cannot be classified as Sus1 and Sus2 types based on sequence homology and appear to have evolved after the divergence of the major families of dicotyledonous plants but before the divergence of tomato and potato. The potato sucrose synthase clones Sus3-65 and Sus4-16 share an 87% nucleotide identity in the coding regions, and both are interrupted by 13 introns, including a long leader intron.

Phi X174 lysis requires slyD, a host gene which is related to the FKBP family of peptidyl-prolyl cis-trans isomerases.

Recessive mutations in the slyD (sensitivity to lysis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage phi X174 [1]. The slyD- mutation, transduced into the normal phi X174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues.

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun.

Stimulation of basal transcription from the mouse mammary tumor virus promoter by Oct proteins.

The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif ATGCAAAT. Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by two-to threefold in in vitro transcription assays. Oct-1 protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences, as shown by DNase I footprinting.

Structure-based analysis of inhibitor binding to Ht-d.

A theoretical study was performed on the structure of both the native and inhibited metalloproteinase Ht-d (E.C. 3.

The baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect cell factors.

The transregulatory IE1 protein of Autographa californica nuclear polyhedrosis virus binds to the viral enhancer element hr5. To test whether IE1 binds independently of host cell factors, IE1 was translated in rabbit reticulocyte extracts and tested for DNA binding activity by an electrophoretic mobility shift assay. Complexes with the hr5 probe were detected with translation reaction mixtures primed with ie1 RNA but not with control translation reaction mixtures.

S gene expression and the timing of lysis by bacteriophage lambda.

The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107.

The iron-copper connection: the link to ceruloplasmin grows stronger.

A rare genetic defect in ceruloplasmin biosynthesis has provided the strongest evidence to date that ceruloplasmin is essential for iron metabolism and tissue distribution in humans. A aceruloplasminemia results in massive accumulations of iron in the liver and brain and is associated with retinal degeneration and diabetes.

Control of proliferation activation in quiescent neuroblasts of the Drosophila central nervous system.

Stem cell proliferation is controlled through cell cycle arrest and activation. In the central nervous system of Drosophila melanogaster, neuroblast quiescence and activation takes place in defined spatial and temporal patterns. Two genes have been identified that regulate the pattern of neuroblast quiescence and proliferation.

Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 2. pH and kinetic isotope effects.

pH and kinetic isotope effects on steady-state kinetic parameters have been determined for the flavoprotein tryptophan 2-monooxygenase with tryptophan, phenylalanine, 2-hydrazino-3-propanoic acid, and methionine as substrates. The V/K values of the amino acid substrates show that a residue with an apparent pKa value of 5 must be unprotonated for activity, a residue with a pKa value equal to that of the amino group of the substrate must be protonated, and deprotonation of a residue with pKa value of 10 increases the V/K value. A group in the free enzyme with a pKa value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establishing this as the intrinsic pKa value.

Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 1. Kinetic mechanism.

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indole-3-acetamide, carbon dioxide, and water. The kinetic mechanism of the enzyme has been determined with tryptophan as substrate at pH 8.3.

Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose.

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate.

The soybean vegetative storage proteins, VSP alpha and VSP beta, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp alpha and Vsp beta, was examined and compared to expression of the soybean Vsp genes.

Contribution of cotranslational folding to the rate of formation of native protein structure.

To compare the process of protein folding in the cell with refolding following denaturation in vitro, we have investigated and compared the kinetics of renaturation of a full-length protein upon dilution from concentrated urea with the rate of folding in the course of biosynthesis. Formation of enzymatically active bacterial luciferase, an alpha beta heterodimer, occurred 2 min after completion of beta-subunit synthesis in an Escherichia coli cell-free system. Renaturation of urea-denatured beta subunit, either in the presence of the cell-free protein synthesis system or in buffer solutions, proceeded more slowly.

Purification and characterization of the flavoprotein tryptophan 2-monooxygenase expressed at high levels in Escherichia coli.

Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan. This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein.

Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization.

The telomerase reverse transcriptase uses an essential RNA subunit as a template to direct telomeric DNA synthesis. The 190-nucleotide Oxytricha nova telomerase RNA was identified by using an oligonucleotide probe complementary to the predicted CCCCAAAA template. This RNA displays extensive sequence similarity to the Euplotes crassus telomerase RNA and carries the same 5' CAAAACCCCAAAACC 3' telomeric domain.

A complex that contains proteins binding to the PSE and TATA sites in a human U6 small nuclear RNA promoter.

The proximal promoter of a human U6 small nuclear RNA (snRNA)-encoding gene contains two separate elements, the proximal sequence element (PSE) and the TATA box. We investigated the interaction of the PSE- and TATA-binding proteins (PBP and TBP) with normal and mutant U6 proximal promoters using an electrophoretic mobility shift assay. We detected a complex containing both PBP and TBP bound to the wild-type U6 promoter.

Nucleic acid binding properties of recombinant Zn2 HIV-1 nucleocapsid protein are modulated by COOH-terminal processing.

The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. In a previous report, we showed that recombinant NC protein from HIV-1, a 71-amino-acid protein (NC71), is apparently able to form two types of protein-nucleic acid complexes under low [NaCl], pH 8.3 and 25 degrees C.