Recombinant simian virus 40 viruses carrying rabbit ,-globin cDNA failed to express the 0-globin sequence unless an intron was included in the transcription unit. The addition of either j-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable 0-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2-to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.The occurrence of intervening sequences (or introns) within eucaryotic genes poses many interesting questions regarding their role in gene expression. The ability to splice RNA transcripts differentially permits great flexibility in the design and expression of eucaryotic genes. Thus, adenovirus expresses a battery of proteins that is involved in capsid formation from a single viral promoter by splicing a common mRNA leader to each of the coding sequences (63). Slightly different patterns of splicing are used to express some cellular genes in different cell types (11,29,48). In this way, closely related proteins are produced that differ only in a specific region. In some instances, intervening sequences are the sites where other regulatory elements reside. For example, sequence elements that direct specific DNA rearrangements (52) or that regulate transcription (2, 15, 34, 46) occur in introns.Some of the earliest studies of eucaryotic vectors uncovered another aspect of introns which still remains unresolved: an apparent necessity for RNA splicing in the production of stable RNA. The first simian virus 40 (SV40) recombinant viruses, which were created before the discovery of splicing, gave somewhat disappointing results in that the foreign sequences were not expressed (17,18,25). This failure was later correlated with the absence of introns in these constructions, and it was shown that the deficiency could be corrected by including an intervening sequence from either a viral or cellular gene (23,24,41). At that time, it was proposed that splicing is generally required for gene expression (22,24). Since then, numerous eucaryotic genes have been found that lack intervening sequences (1, 28, 42). Furthermore, while attempting to engineer the expression of different sequences in animal cells, many investigators achieved adequate lev...

During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.At present, the molecular mechanisms controlling gene expression in higher eucaryotes are only poorly understood. One of the best characterized systems which has been used to approach this problem is the early region of simian virus 40 (SV40) (1, 24, 56). The early region codes for two known proteins, large T antigen and small t antigen. Although the function of small t antigen remains obscure, large T antigen is known to be required for the initiation of viral DNA synthesis. These proteins are translated from differentially spliced RNAs which are transcribed, using the same promoter and polyadenylation signals. The SV40 early-region promoter is contained within a set of repeated segments adjacent to the origin of viral DNA replication (ori) (Fig. 1). Extensive analysis of this region has demonstrated the presence of three separate elements involved in SV40 early transcription (5, 16). At least one copy of a set of six short guanine-cytosine-rich repeats is necessary for early transcription. In addition, a sequence within the 72-base-pair (bp) repeat has been shown to be an essential element. Finally, a Goldberg-Hogness or TATA box sequence (19; M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978), imbedded within a larger 17-bp adenine-thymine (AT)-rich block, is responsible for positioning the 5' ends of early RNA.The SV40 early region is also one of the best characterized examples of regulation mediated by a defined protein. Large T antigen regulates its own synthesis by controlling the level of early-region transcription (33, 53, 57). Furthermore, T antigen binds to SV40 DNA at three adjacent sites overlapping ori and the early promoter (Fig. 1) that T-antigen binding a...