Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.     

Modular PH and C2 domains in membrane attachment and other functions.

The pleckstrin homology and C2 domains are modular protein structures involved in mediating intermolecular interactions. Although they represent distinct domains, there are several parallels regarding their function and type of interactions in which they participate. Both domains are stable structural entities that incorporate variable regions which, in different proteins, can be adapted to perform a specific function through binding to membrane phospholipids or specific protein ligands. A number of recent examples illustrate the function of some of these domains in regulated membrane attachment, with an important role in many cellular signalling pathways.     

Functional equivalence of the uridine turn and the hairpin as building blocks of tertiary structure in the Neurospora VS ribozyme.

Mutational, kinetic, and chemical modification experiments show that one of the three-way helical junctions in the Neurospora VS ribozyme contains a uridine turn that is important for organizing the functional three-dimensional structure of this junction. Disruption of the uridine turn disrupts the structure of the junction and decreases the self-cleavage activity of the ribozyme; however, substitution of the uridine turn with a variety of different hairpins, thereby transforming the three-way junction into a four-way junction, maintains catalytic activity. Chemical modification structure probing reveals that both the native junction and the hairpin-containing junction support the same tertiary interactions required elsewhere in the ribozyme for catalysis. These observations show that functionally equivalent three-dimensional RNA structures can be built from different secondary structure elements.     

Interaction of RNA with transformed glucocorticoid receptor. I. Isolation and purification of the RNA

The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.     

Effects of cholinergic drugs on longitudinal muscle contractions of Fasciola hepatica

Acetylcholine, cholinergic agonists and acetylcholinesterase inhibitors significantly decrease the amplitude and frequency of spontaneous longitudinal muscle contractions in Fasciola hepatica. In order of their effects on the inhibition of muscle contractions, the cholinergic agonists can be ranked as nicotine greater than carbachol greater than acetylcholine. High calcium ion concentration also causes a significant inhibition of contractions. Atropine, a cholinergic antagonist that acts on muscarinic receptors, significantly increases the amplitude and frequency of spontaneous contractions and completely reverses the effects of cholinomimetic drugs, including nicotine. In adult F. hepatica, the levels of acetylcholine and its precursor choline are 3.14 +/- 0.55 and 13.75 +/- 3.72 pmol/mg wet weight, respectively. The activities of choline acetyltransferase, specific acetylcholinesterase and the nonspecific cholinesterase are 1.25 +/- 0.19, 238.0 +/- 13.0, and 83.0 +/- 33.0 nmol/hr/mg protein, respectively.     

An adaptor strategy to subclone entire cDNA libraries as single insert recombinants

The current methods for subcloning entire cDNA libraries usually result in a significant portion of recombinants containing multiple inserts, since in most instances the inserts derived from the library to be subcloned are released as a bulk of self-ligatable DNA fragments. By use of an adaptor strategy, a method is presented to confer noncompatible ends to primarily self-ligatable inserts, resulting in efficient subcloning of entire libraries as single insert recombinants.     

Hormonal regulation of skeletal muscle glycogen synthase through covalent phosphorylation

Studies have been initiated to determine the hormonal regulation of glycogen synthase in rabbit skeletal muscle. It was found that glycogen synthase purified from control animals was quite highly phosphorylated (2.35 mol phosphate/mol synthase subunit) with 40% of the phosphate in the trypsin-sensitive or COOH-terminal domain, and 60% in the trypsin-insensitive or NH2-terminal domain. The phosphorylation state of synthase was elevated (3.9 mol/mol) by epinephrine injection and in the diabetic condition. With epinephrine, about 76% of the additional phosphate was incorporated in the trypsin-sensitive domain, which strongly supports the contention that this hormone acts through the cyclic AMP (cAMP)-dependent protein kinase. In the synthase purified from diabetic rabbits, 90% of the additional phosphate was in the trypsin-insensitive domain. Insulin treatment of the diabetics resulted in specific dephosphorylation of the trypsin-insensitive domain. These results indicate that in this system insulin is not acting by inhibition of the cAMP-dependent protein kinase.     

Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.