Prueba de contrastación de hipótesis

Early transfection studies demonstrated that plasmids containing an Ad.2 replication origin could be replicated efficiently when supported with either an Ad.2 or Ad.4 helper virus. However, plasmids containing Ad.4

origin sequences were only found to be efficiently replicated when co­ transfected with Ad.4 helper virus genome.By comparison co­

transfection with Ad.2 helper virus gave around a 20-fold reduction in replication (Hay et al., 1985b). Sequence analysis of the replication

origins of Ad.2 and Ad.4 revealed that whilst the 1-18 "core" domain of the origin is identical in both serotypes, Ad.4 possesses an A/T rich domain in place of an NF-I binding site. Furthermore it was demonstrated that Ad.4, in contrast to Ad.2, requires only the terminal 18bp of the viral

genome for fully efficient DNA replication in vivo.Subsequent in vitro replication studies confirmed the in vivo observations when it was

demonstrated using a crude Ad.4 infected Hela cell extract that linearised plasmid containing only the terminal 18bp of an adenovirus ITR could support initiation of DNA replication (pTP-dCMP complex formation) in vitro as efficiently as a template containing a complete AD.4 ITR (Harris and Hay, 1988). Recently, a combination of more extensive sequence analysis (Temperley et al., 1991) and in vitro replication studies using a

purified Ad.4 infected Hela cell extract (Temperley and Hay, 1991) has identified two distinct regions within the adenovirus that are required for efficient DNA replication. Temperley et al., (1991) examined the effect of single base changes in positions 9 to 18 of the Ad.4 origin on DNA replication in vitro. Changes in the bases between 12 to 16 had little effect, whereas alterations at positions 9,10,11,17 and 18 all reduced

the efficiency of initiation of DNA replication by between 50 and 90%. It was concluded that the region from 9 to 18 bp contained two sets of bases (separated by 5base-pairs) essential for DNA replication. The region between 9-18bp is thought to be part of the recognition site on Ad.4 for

the pTP-pol heterodimer. Strong proof for this theory has come from studies on Ad.2 when Temperley and Hay, (1992) demonstrated that the region between 8 to 17bp was the recognition site for the Ad.2 pTP-pol

heterodimer. The basis of the difference in origin sequence requirements

between Ad2/5 and Ad4 is that Ad4 appears to be able to replicate its DNA without the need for auxiliary stimulatory functions provided by the

binding of NF-I and NF-III. Ad.4 does not possess an NF-I recognition site although it does have a strong consensus binding site for NF-III

located at the same position as in the Ad2/5 origin of replication. However, it has been unequivocally demonstrated that neither cellular factor stimulates Ad4 DNA replication even if their cognate binding sites

are present in a reconstituted origin of replication (Hay et al., 1988). Since it appears that NF-I and NF-III play auxiliary roles in Ad2/5 DNA replication, whereby the function of the viral replication proteins are enhanced or aided by their binding at the origin, the probability is that

Ad.4 is able to replicate its DNA without them because its own replication proteins alone can perform the required functions. Ad.4

therefore potentially provides a more simplified system in which to

further study the molecular details of the mechanism of adenovirus DNA replication.