M.J. Welsh and A.E. Smith, RAC Application 
promoter. This is known to be expressed at moderate levels in a wide variety of cells. 
Termination/polyadenylation occurs at the site normally used by the El b and protein IX 
transcripts. An endogenous 84 bp intron is present between the coding sequences for 
CFTR and protein IX. 
The regulation of the E la promoter has been studied extensively, since it was one of the 
earliest and most readily available eukaryotic transcriptional control regions (108-110, 
reviewed in 111). The transcription factors that bind the E la promoter are well 
characterized (reviewed in 1 12,1 13). The promoter has been transferred into a wide variety 
of cells and shown to be active in transcription of marker genes. Promiscuous expression 
perhaps reflects the role of E la in the early stages of viral replication where it might 
encounter different host cells in various stages of proliferation (77). 
Adenovirus DNA replication has been extensively studied (reviewed in 75 and in 114) and 
in vitro systems for replication of adenovirus DNA have been developed (1 15,1 16). These 
studies have allowed the dissection of the adenoviral DNA replication process and have 
identified virally encoded proteins of the E2 transcriptional unit involved in DNA 
replication as well as a number of host cell encoded factors involved in the process. In 
addition, the viral DNA sequences within the terminal repeats which are required for DNA 
replication have been defined for Ad2, Ad5, and Ad4 (1 17,1 18). The relative permissivity 
of a specific cell to adenovirus DNA replication is thus a result of complex interactions 
between host-encoded nuclear factors, virally encoded replication machinery and specific 
sequences within the terminal repeats. 
In addition, in quiescent cells, replication of viral DNA is dependent upon the ability of the 
virus to stimulate the infected cell into a proliferative state able to support efficient viral 
replication (77). It has been proposed that one of the functions of the adenovirus Ela-12S 
protein is to induce infected cells to enter such a proliferative state by displacing 
transcription factor E2F from the retinoblastoma gene product, thereby leading to activation 
of genes required for efficient DNA synthesis as well as viral transactivation (119). E4 
gene products also play a role in activation of E2F (120). Thus, as for DNA synthesis, 
adenovirus gene expression is a complex series of cooperative reactions involving both host 
cell proteins and multiple viral gene products. Cells that are already in a proliferative state 
may be capable of supporting limited adenovirus replication in the absence of El a 
transactivation. Therefore, experiments with proliferating cells in tissue culture may not 
accurately reflect the replicative ability of an E la-deleted virus upon infection of a 
quiescent cell. 
In add ition to the elements expected to be involved in the expression and replication of the 
Ad2/CFTR-1 vector, many other regulatory sequences are present within the remainder of 
the Ad2 geno me. E xpression of these sequences is expected to be low and to have little 
influence on CFTR expression. 
B.l.a.(3) Describe the steps used to derive the DNA construct. 
Construction of the recombinant Ad2/CFTR-1 virus was accomplished as follows (Fig. 3 
and 4). The CFTR cDNA was excised from the plasmid pCMV-CFTR-936C using 
restriction enzymes Spel and Eel 1361. pCM V -CFTR-936C consists of a minimal CFTR 
cDNA encompassing nucleotides 123-4622 of the published CFTR sequence cloned into 
the multiple cloning site of pRC/CMV (Invitrogen Corp.) using synthetic linkers. The 
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Recombinant DNA Research, Volume 16 
