Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins.

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined.

D-ribose-5-phosphate isomerase from spinach: heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme.

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C.

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions.

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis.

Identification of a catalytic aspartyl residue of D-ribulose 5-phosphate 3-epimerase by site-directed mutagenesis.

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants.

Structure-function analysis of a conserved aromatic cluster in the N-terminal domain of human epidermal growth factor.

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue.

Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography.

Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment.

D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme.

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol.

Mutations in the murine fitness 1 gene result in defective hematopoiesis.

Identification and characterization of mutations that disrupt normal hematopoiesis are essential for understanding the genetic pathways that control the development and regulation of the mammalian hematopoietic system. Previously, the fitness 1 gene was identified by five, independent mutations in N-ethyl-N-nitrosourea (ENU) saturation mutagenesis experiments within the albino (c) region of mouse chromosome 7 (MMU7). We report here that fit1 mutants are anemic, display numerous peripheral blood defects, and are deficient in early hematopoietic progenitor cell populations.

Inherited somatic mosaicism caused by an intracisternal A particle insertion in the mouse tyrosinase gene.

A recessive, fully penetrant mutation (c(m1OR)) at the mouse albino locus that results in coat-color mottling has been characterized at the molecular level. Restriction mapping and DNA sequencing analyses provide evidence that mutants carry a 5.4-kb intracisternal A particle (IAP) element insertion upstream of the tyrosinase (Tyr) promoter.

An oligopeptide transport gene from Candida albicans.

A Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S.

Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.

The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77.

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: correlation of stain and phosphorus.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.

Large-scale production of palindrome DNA fragments.

Our structural studies of nucleosomes necessitated the production of over 100 mg of a 146-bp perfect palindrome DNA for use in the reconstitution of perfectly symmetrical nucleosome core particles for detailed X-ray crystallographic analysis. The propagation of palindromic DNA DNA sequences by bacterial culture is hindered by the instability of these sequences during bacterial replication and recombination. While the loss of some palindrome sequences can be eliminated by the use of sbcB or sbcC mutants of Escherichia coli, not all palindrome-containing plasmids are faithfully maintained by these strains.

A signature of the oxygenase intermediate in catalysis by ribulose-bisphosphate carboxylase/oxygenase as provided by a site-directed mutant.

An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M.

Genetic and physical mapping of the fitness 1 (fit1) locus within the Fes-Hbb region of mouse chromosome 7.

Mutations at the fit1 locus affect normal pre- and post-natal development by retarding growth and reducing viability. We report mapping of the fit1 locus, by trans-complementation crosses to mice carrying deletions of the albino (c) locus in Chromosome (Chr) 7, to a subregion of the c-deletion complex within the Mod2-sh1 interval. The fit1 locus, which is currently defined by five N-ethyl-N-nitrosourea (ENU)-induced mutations, was found to map in a subregion between the eed and exed loci.

Modeling the 3-D RNA distribution in the Balbiani ring granule.

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA.

Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.

Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere.

Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA.

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme.

Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF.

Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule).

Critical factors affecting the permeabilization of Drosophila embryos by alkanes.

Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum.

Murine fumarylacetoacetate hydrolase (Fah) gene is disrupted by a neonatally lethal albino deletion that defines the hepatocyte-specific developmental regulation 1 (hsdr-1) locus.

Homozygous deletion of the hepatocyte-specific developmental regulation 1 (hsdr-1) locus in mouse chromosome 7 results in perinatal death and a pleiotropic syndrome characterized by ultrastructural abnormalities of the liver and kidney, failure of induction of a number of specific transcription units in the liver and kidney during late gestation, and marked overexpression of an enzyme that defends against oxidative stress. Previously, the breakpoints of two albino (c) deletions (c14CoS and c1FAFyh) that genetically define hsdr-1 were localized, on a long-range map, in the vicinity of the distal breakpoint of a viable albino deletion (c24R75M) that breaks proximally within the c locus. Here we report the use of a probe derived from a deletion breakpoint fusion fragment cloned from c24R75M/c24R75M DNA to clone a breakpoint fusion fragment caused by the c14CoS deletion.

Characteristics and kinetics of subzero chilling injury in Drosophila embryos.

Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and -25 degrees C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, -9, and -15 degrees C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h.