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T B Usdin

Publications and source records attributed to T B Usdin.

5 recordsLinked to original sources
PubMed1992

Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

Animals↗
PubMed1991

Cloning of the cocaine-sensitive bovine dopamine transporter.

A cDNA encoding the dopamine transporter from bovine brain substantia nigra was identified on the basis of its structural homology to other, recently cloned, neurotransmitter transporters. The sequence of the 693-amino acid protein is quite similar to those of the rat gamma-aminobutyric acid, human norepinephrine, and rat serotonin transporters. Dopamine transporter mRNA was detected by in situ hybridization in the substantia nigra but not in the locus coeruleus, raphe, caudate, or other brain areas. [3H]Dopamine accumulation in tissue culture cells transfected with the cDNA was inhibited by amphetamine, cocaine, and specific inhibitors of dopamine transport, including GBR12909.

Amino Acid Sequence↗
PubMed1990

Subtractive hybridization system using single-stranded phagemids with directional inserts.

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

Bacteriophages↗
PubMed1986

Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes.

Acetylcholine receptors (AChRs) are packed in the postsynaptic membrane at neuromuscular junctions at a density of approximately 20,000/micron 2, whereas the density a few micrometers away is less than 20/micron 2. To understand how this remarkable distribution comes about during nerve-muscle synapse formation, we have attempted to isolate factors from neural tissue that can promote the accumulation of AChRs and/or alter their distribution. In this paper we report the purification of a polypeptide from chick brains that can increase the rate of insertion of AChR into membranes of cultured chick myotubes at a concentration of less than 0.5 ng/ml. Based on SDS PAGE and the action of neuraminidase, the acetylcholine receptor-inducing activity (ARIA) appears to be a 42,000-D glycoprotein. ARIA was extracted in a trifluoroacetic acid-containing cocktail and purified to homogeneity by reverse-phase, ion exchange, and size exclusion high pressure liquid chromatography. Dose response curves indicate that the activity has been purified 60,000-fold compared with the starting acid extract and approximately 1,500,000-fold compared with a saline extract prepared from the same batch of brains. Although the ARIA was purified on the basis of its ability to increase receptor incorporation, we found that it increased the number and size of receptor clusters as well. It is not yet clear if the two effects are independent. The 42-kD ARIA is extremely stable: it was not destroyed by exposure to intact myotubes, low pH, organic solvents, or SDS. Its action appears to be selective in that the increase in the rate of receptor insertion was not accompanied by an increase in the rate of protein synthesis. Moreover, there was no change in cellular, surface membrane, or secreted acetylcholinesterase. The effect of ARIA is apparently independent of the state of activity of the target myotubes as its effect on receptor incorporation added to that of maximal concentrations of tetrodotoxin.

Acetylcholinesterase↗