DISEÑO HMI

Southw estern analysis using n uclear p ro te in isolated from NG108-15 cells detected two protein/DNA interactions in the -203bp to +3 Ib p region of the B2 b radykinin receptor proximal prom oter. The sizes of the proteins were found to be llS k D a an d 106kDa (figure 3.20). Two p ro tein /D N A interactions were also detected using nuclear protein extracted from CHO cells and the proteins were found to be the same m olecular weight as those detected using NG108-15 nuclear protein (ie. llS k D a and lOGkDa).

The id en tity of the higher m olecular w eight p ro tein (llS k D a) th at interacts with the 234bp fragm ent (-203bp to +3 Ibp) is unknown. There is evidence th at the lower molecular weight p ro tein is Sp-1 as a p ro tein of the same m olecular weight, th a t is running at 106kDa, was detected by an Sp-1 antibody (Wood et al. 1996). Furtherm ore, th ere is a highly conserved Sp-1 site positioned at -25bp (figures 3.12 and 3.13) and less conserved sites at -154bp and -152bp. This implies

th a t the larger isoform of Sp-1, an im portant trans-activator during cell development and differentiation may bind to the B2 bradykinin recep to r proxim al prom oter. However, th ere is evidence from the results of the electrom obility shift assay (EMSA) (section 4.5) to suggest th a t Sp-1 is not the protein binding to this region (-203bp to +3 Ibp) of the B2 bradykinin receptor prom oter. There is also the possibility th a t this is a p ro tein of the same m olecular weight as the 106kDa Sp-1 isoform.

Ultimately, further characterisation is required to confirm the identity of b o th the llS k D a and 106kDa proteins. The ambiguity as to w hether Sp-1 is binding to the 234bp fragm ent m ay be more rigorously checked by using Sp-1 p ro tein and exam ining its ability to in teract w ith this region (using a similar protocol to th at of the EMSA).

One of the lim itations of using cell lines to stu d y protein/D N A in teractio n s is th a t cells w hich have b een transform ed express distinct genes to non-transform ed cells and so may not reflect accurately the physiological phenotype. Therefore to prove th at a protein binding to DNA is present in non-transform ed cells, it is necessary to use nuclear protein extracted from prim ary cells.

An im portant point to also consider is th at in this study the proteins isolated and investigated were specifically nuclear proteins. However, there are m any cases of proteins usually present in the cytoplasm which following a particular stimulus tran slo ca te to th e nucleus and effect tra n sc rip tio n . The tran scrip tio n factor NFkE» is p resent in an inactive form in m any tissues, where it is associated with an inhibitory protein

nam ed inhibitor-icB ( IkB ) . Phosphorylation of IkB results in

dissociation of NFkB>, allowing it to dimerize and translocate to the nucleus where it is able to bind to its DNA recognition site and so regulate gene transcription (Baeuerle and Baltimore

1988a,b).

NFkl?> was originally detected using EMSA, w here it was

developm ental stage for light chain expression an d was suggested to play a critical role in the B cell-specific activity of

the e n h a n cer of the im m unoglobulin k light chain gene

(Lenardo et al. 1987; Atchison and Perry 1987). However, NFki?> b in d in g ac tiv ity (a cco m p an ied by th e a c tiv a tio n of

im m unoglobulin k light chain gene expression) was shown

following the treatm ent of pre-B cells with lipopolysaccharide suggesting its presence in an inactive form (Atchison and Perry 1987). Furtherm ore, NFkF> binding activity was fo u n d in cytosolic extracts from uninduced cells treated with formamide and deoxycholate, both of which are dissociating agents (Baeuerle and Baltimore 1988a). The com bined evidence suggested th a t NFkF» is p resen t in an inactive form in the cytoplasm and w hen induced translocates to th e nucleus. Subsequent analysis of non-B cells treated with phorbol esters,

which modify IkB releasing NFkl?», has dem onstrated NFkF> to be

p re se n t in a wide variety of cell types and w hen induced regulates the expression of several genes involved in the immune, inflam m atory and acute-phase responses (Liptay et al. 1994).

The p re ced in g analyses have id e n tifie d th e gene structure of the B2 bradykinin receptor gene, dem onstrated the presence of a promoter, shown that the -162bp to +3 Ib p region of the prom oter contains regulatory elem ents which drive expression of this gene in NG108-15 cells, as well as providing evidence of at least three protein/DNA interactions within the -203bp to +3 Ib p region of the proximal prom oter.