Using nucleoproteins (transcription complexes) from Adenovirus-2 (Ad-2) infected cells, we have recently developed the first soluble system in which the major late Ad-2 transcription unit is transcribed and terminated with fidelity, polyadenylated faithfully and spliced specifically. We now propose to extend and continue this in vitro system to study the basic mechanism of RNA transcription and processing. Specifically, the aims are: 1. To investigate transcriptional or processing controls in the biogenesis of Ad-2 mRNAs by studying Ad-2 transcription complexes and soluble extracts separately and in combination in vitro. We propose: a. To further map polyadenylation sites used in vitro by Sl nuclease mapping, fingerprinting and sequence analysis; to identify factors involved in site specific polyadenylation; and to measure the frequency of usage of the poly(A) sites in vitro. b. To determine the relationships of polyadenylation, splicing and internal methylation in vitro. c. To identify template-associated proteins which are correlated with the differential expression of the early and late genes during different stages of lytic infection. d. To investigate the role of termination of transcription during early to late switch. e. To optimize initiation of transcription in vitro. 2. A long-term goal is to apply this system to studying the transcription and RNA processing of immunoglobulin genes by first constructing adenovirus-immunoglobulin heavy chain recombinant viruses. These recombinants will then be used for investigating the expression of the Cmu gene in vivo in the recombinant infected cells, including lymphocytic cells of various stages of differentiation and in vitro as transcription complexes.
| Lavery, D; Fu, S M; Lufkin, T et al. (1987) Productive infection of cultured human lymphoid cells by adenovirus. J Virol 61:1466-72 |
| Ruether, J E; Maderious, A; Lavery, D et al. (1986) Cell-type-specific synthesis of murine immunoglobulin mu RNA from an adenovirus vector. Mol Cell Biol 6:123-33 |
