Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site.

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.

Assembly of the nicotinic acetylcholine receptor. The first transmembrane domains of truncated alpha and delta subunits are required for heterodimer formation in vivo.

To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal fragments of the alpha and delta subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and to form a ligand-binding site. Truncated fragments of the alpha subunit that include all, part, or none of the first transmembrane domain (M1) folded to acquire alpha-bungarotoxin binding activity. Neither the full-length alpha subunit nor any of the fragments were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into the medium. When coexpressed with the delta subunit, the alpha subunit fragment possessing M1 formed a heterodimer containing a ligand-binding site, but shorter fragments, which lack transmembrane segments, did not associate with the delta subunit. N-terminal delta subunit fragments gave similar results. An N-terminal delta subunit fragment that contains M1 associated with the alpha subunit to form a heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association of truncated N-terminal alpha and delta subunits into a heterodimer with high affinity ligand binding activity.

Transduction of primitive human hematopoietic cells with recombinant adenovirus vectors.

We have examined the ability of recombinant adenoviral vectors to transduce human hematopoietic cells. Our findings indicate that adenovirus readily infects a large proportion of CD34+ cells. Using adenovirus vectors that transduce either a lacZ or an alkaline phosphatase reporter gene, we observed up to 45% of total CD34+ cells infected. Upon more detailed analysis, we observed comparable levels of transduction for CD34+/CD38- cells and for CD34+ cells in G(zero) phase of the cell cycle. Importantly, exposure to adenovirus resulted in negligible levels of toxicity as assayed by propidium iodide staining and colony-forming ability. Using adenovirus vectors, we also describe a model system for regulated gene expression in early hematopoietic tissues. CD34+ cells were simultaneously infected with two viruses, one carrying a TetR/VP16 transactivator (tTA) and the second carrying a tTA-dependent lacZ reporter gene. Using this approach, beta-gal expression was only observed upon coinfection with the transactivator vector. In addition, as shown previously (Gossen and Bujard, Proc Natl Acad Sci USA 89:5547, 1992), tetracycline was able to inhibit tTA mediated induction, thereby providing an effective means to regulate expression of the reporter gene. We conclude that recombinant adenovirus is an effective vehicle for transiently expressing genes in primitive human hematopoietic cells.

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

Cofactor requirements of splicing of purified messenger RNA precursors.

The origin and functions of introns in protein coding genes is one of the enigmas of molecular biology. Splicing processes that remove intervening sequences from precursor RNAs must have either predated or co-evolved with introns. Inferences about the origin of introns and the possible modes of regulation of splicing should emerge from an understanding of the biochemical mechanisms of splicing. The biochemistry of splicing of tRNA and rRNA precursors has rapidly advanced with the development of in vitro reactions containing soluble components that duplicate in vivo reactions. We have recently shown that accurate splicing of an adenovirus mRNA precursor occurs during a coupled transcription/splicing reaction in a soluble whole cell extract. We now report that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells. ATP and Mg2+ are essential cofactors for the reaction. The time course of splicing is unusual; a lag of 45 min is observed before the appearance of splicing product.

Splicing of adenovirus RNA in a cell-free transcription system.

A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

Use of dodecyl-beta-D-maltoside in the purification and stabilization of RNA polymerase from brome mosaic virus-infected barley.

The activity and specificity of RNA-dependent RNA polymerase (replicase) isolated from brome mosaic virus-infected barley was enhanced by extraction with the nonionic detergent dodecyl-beta-d-maltoside. The enzyme was stable for at least 8 weeks when stored at -70 degrees . A further 100-fold purification was obtained by centrifugation through sucrose in the presence of detergent. The polymerase activity was associated with the pellet fraction; the template dependence and specificity were similar to those of the enzyme before sucrose purification. SDS-PAGE analysis revealed a 110-kd protein in the purified pellet fraction from infected leaves that was absent from a similar fraction from healthy leaves. The protein had an identical electrophoretic mobility to that of protein la, the product of brome mosaic virus RNA 1 translation in vitro, and the profile of its tryptic polypeptides was very similar to that of protein 1a. These results support data obtained by inoculation of protoplasts with separated BMV RNA components (Kiberstis, et al. (1981), Virology 112, 804-808) that are consistent with the notion that RNA 1 codes for the viral replicase, or a subunit thereof.

Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus.

The extraction of a template-dependent and template-specific RNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from a eukaryotic source is described. The enzyme, extracted from barley leaves infected with brome mosaic virus (BMV), is capable of incorporating high levels of radioactivity into trichloroacetic acid-insoluble products. The purification procedure included solubilization with nonionic detergent and precipitation with polyethylene glycol. The enzyme was more than 50 times more active than was a comparable preparation from mock-inoculated leaves and was stimulated more than 15-fold by the addition of BMV RNA to the reaction. Other viral RNA templates were less than 25% as efficient as was BMV RNA in stimulating UMP incorporation; poly(A), tRNA, and mRNA gave little stimulation and rRNA was inactive. Autoradiographic analysis after electrophoretic separation of the radioactive products from reaction mixtures containing BMV RNA template revealed prominent bands that coelectrophoresed with replicative forms of BMV RNAs. When BMV RNA template was enriched in RNA3 or RNA4, larger proportions of the products were replicative forms of RNA3 or RNA4, respectively.