Cell-type-specific synthesis of murine immunoglobulin mu RNA from an adenovirus vector.

The mouse immunoglobulin heavy-chain mu constant region gene was cloned into the early region 1B of an adenovirus type 5 vector to allow reproducible kinetics of expression of the mu gene in the presence of continuous host protein synthesis after infection by the recombinant. The immunoglobulin-adenovirus recombinant is helper independent in infecting human fibroblastic and B- and T-cell lines and expresses mu in a cell-type-specific manner. By Northern blot analysis, correctly polyadenylated and spliced E1B-mu S and E1B-mu m mRNAs are found to be equally abundant at steady state in fibroblasts. In contrast, and appropriately, only E1B-mu S mRNAs accumulate in a lambda light-chain-secreting myeloma cell line. Analysis of nascent transcripts pulse labeled in isolated nuclei demonstrates equimolar polymerase loading throughout the mu region in all cell types infected by mu-Ad. Thus, correct polyadenylation and splicing of E1B-mu S and E1B-mu m in fibroblasts does not require transcription termination in the region separating the mu S and mu m polyadenylation sites. Furthermore, differential expression of mu transcripts in the background of myeloma cells is regulated at the level of RNA processing and does not require the presence of the immunoglobulin heavy-chain enhancer or promoter element.

Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.

Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit.

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

A genetic factor controlling morphogenesis of the laryngotracheo-esophageal complex the mouse.

A new autosomal, recessive lethal mutation in the mouse cause failure of division of the embryonic foregut into totally separate digestive and respiratory ducts. The newborn homozygote has a cleft that extends between the laryngotracheal and esophageal tubes, allowing excessive air to pass into the stomach and subsequently into the peritoneal cavity. This mutation has been designated lec (laryngotracheo-esophageal cleft). The common region shared by the respiratory and digestive tubes has features characteristic of both structures. Development of cartilage around the laryngotracheo-esophageal complex is also affected in the mutant.