9 Limitantes y oportunidades
9.4 De la inversión en ACTi para el sector
the Hind\\\ site, incubated with 0, 5,10 or 20 pi of affinity purified EBPI and digested with DNase I as
described in “Materials and Methods". DNA was isolated, fractionated by electrophoresis in 8% denaturing polyacrylamide gels and the cleavage products visualised by autoradiography. C+T (Py) and G+A (Pu) specific cleavage reactions of the labelled fragment were electrophoresed in parallel as markers.
B. The aforementioned labelled fragments were incubated with 20 pi of affinity purified EBPI and, after binding reactions had reached equilibrium, free DNA (F) was separated from EBPI-DNA complexes (B) by electrophoresis in a native polyacrylamide gel. Cleavage with OP/Cu+ was carried out within the acrylamide matrix and DNA eluted as described in “Materials and Methods”. DNA was fractionated, and products visualised as described in A. Deoxyribose residues within the bracketed region are protected from cleavage by OP/Cu+.
0. DNA sequence of the IRE containing the EBPI binding site. Phosphate bonds within the large brackets are protected from DNase I cleavage broken lines depict potential protected bonds). Deoxyribose residues within the small bracketed regions are protected from OP/Cu+ cleavage in the presence of EBPI. Bottom Top Bottom
[ -
I [
c. 5 ' -GAGAATGAAAGTGGGAAATTCCTCTGAATAGAGAGAGGACC-3' 3 ' -CTCTTAÇTTTCACCCTTTAAGGAGACTTATCTCTCTCCTGG-5' - 7 0j ______________L i I j - M -40protection extends from the phosphate bond 3' of base -51 to the phosphate bond 3' of base -69. The boundary of the DNase I footprint on the top strand Is not so well defined as DNase I cuts poorly in these regions. DNase I cleavage patterns are normally staggered by 2 bp which suggests that the protected region extends from the phosphate bond 3' of position -50 to the bond 3' of position -68.
To determine which deoxyribose residues in the DNA backbone are in the proximity of bound EBPI, we have subjected the EBPI-DNA complex to cleavage by hydroxyl radicals generated by the 1,10 orthophenanthroline/copper ion (OP/Cu+) complex. Restriction enzyme fragments p2p]_ labelled on either the top or bottom strand of the IRE were incubated with purified EBPI and E B P I- DNA complexes resolved from free DNA by electrophoresis in a non-denaturing polyacrylamide gel. OP/Cu+ cleavage was conducted in situ (Kuwabara and Sigman, 1987), DNA extracted from the gel and the cleaved products fractionated by denaturing polyacrylamide gel
Of-v Hva-^^àA-bc7vc-'iidi3-=>rv<E_
electrophoresis. On each strand a total of eight deoxyribose jyvc>ida^y|are protected from OP/Cu+ cleavage by EBPI (Figure 3.2B). The region of protection extends from the deoxyribose residue attached to base -57 to base -64 on the bottom strand, and from -56 to -63 on the top strand. A summary of the DNase I and OP-Cu+ protection data is presented in Figure 3.2C.
3.3 Binding of EBPI to the human immunodeficiency virus (HiV) enhancer
The human immunodeficiency virus (HIV) can express its gene products when transfected into a number of cell lines, including HeLa cells (Levy et ai, 1985), suggesting that the virus is able to use general cellular transcription factors for expression of its genes. Genetic analysis of the HIV long terminal repeat (LTR) has revealed several cis -acting regions important for viral gene expression including a negative regulatory region, an enhancer element, SP1 ( Dynan and Tjlan, 1983; Gidoni et ai., 1985; Briggs et ai., 1986) binding sites, TATA and untranslated regions (reviewed by Wu, et ai., 1988). Within the enhancer region are two direct repeats of a DNA sequence, -GGGAGTTTCC-, similar to that present in the "core" region of the SV40 enhancer (Nabel and Baltimore, 1987). Competition analysis (Figure 3.1 A and B) has demonstrated that EBPI binds to both of these sequences present in the HIV enhancer. To determine how EBPI bound to the HIV enhancer , the protein was incubated with a restriction enzyme fragment, [32.p]-iabelled on either the top or bottom strand of the HIV enhancer, digested with DNase I and
Figure 3.3 DNase I protection of the HIV enhancer In the presence of EBPI
A Hind\\\ to Sac I fragment, or Eco RI to Pst I fragment, from pHIVEn was 3-end labelled at the Hind\\\
(Bottom: A) or Eco RI (Top; B) site, incubated in the absence (0) or presence of 5,10 or 20 pi of affinity purified EBPI and digested with DNase I as described in “Materials and Methods”. DNA was isolated and fractionated by electrophoresis in 8 % denaturing polyacrylamide gels and the cleavage products visualised by autoradiography.
C. DNA sequence of the HIV enhancer containing the EBPI binding sites (as depicted by the horizontal bars). Phosphate bonds within the bracketed regions are protected from DNase I cleavage in the presence of EBPI.
BOTTOM ^ -7 5 -106
r
5 ' -TCTACAAGGGACTTTCCGCTGGGGACTTTCCAGG-3
3'-AGATGTTCCCTGAAAGGCGACCCCTGAAAGGTCC-5'
i _________________________________________________ f 1the cleavage products fractionated on a denaturing polyacrylamide gel. On both the top and bottom strands a region of 34 bp of the HIV enhancer (Figure 3.3A and B) is protected from DNase 1 digestion by EBPI. This region contains both the-GGGACTTTCC-direct repeats. On the bottom strand the region of protection extends from the phosphate bond 3' of base -75 to the phosphate bond 3' of base -108, and from the phosphate bond 3' of position -74 to the bond 3' of position -107 on the top strand. A summary of the DNase I data is presented In Figure 3.3C with the positions of the -GGGACTTTCC- repeats indicated by the horizontal bars.
Chapter 4. Mutational analysis of the EBPI binding site
4.1 DNA binding specificity of purified EBPI
To determine the bases involved in sequence-specific recognition by EBPI on the DNA, a detailed mutational analysis of sites present in the SV40 enhancer and IRE was carried out. Results of DNase I protection competition experiments revealed that guanines at positions 244 and 245, and cytosines at positions 236 and 237 (Figure 3.1 B, lanes 2 and 3) were an important component in the recognition event. This was also found to be the case by analysing the interaction of the purified protein with [32p]-iabelled SV1 double stranded oligonucleotide in the standard gel electrophoresis DNA-binding assay (Figure 4.1). Whereas unlabelled SV1 oligonucleotide could efficiently compete for binding of the purified factor , the SV1.M1 oligonucleotide (with G to C changes at positions 244 and 245) was unable to do so. This was also true of the SV1.M3 oligonucleotide which contained C to G alterations at position 237 and 238 (Figure 4.1). Thus the purified protein requires interactions within the GT-I motif, at positions 244 and 245, and in the TC-ll motif at positions 237 and 238 for sequence-specific recognition, as determined by DNase I footprinting (Figure 3.1 B) and gel electrophoresis DNA binding assays (Figure 4.1)
4.2 Mutational analysis of the EBPi binding site in the IRE
Comparison of the naturally occurring binding sites for EBPI (Figure 3.10) suggest which base pairs within the recognition site are important for EBPI binding. To evaluate the contribution of individual base pairs to EBPI binding we have made use of a series of point mutants (Figure 4.2B) which span its binding site in the IRE (Goodbourn and Maniatis, 1988). [32p]-iabelled restriction
Figure 4.1 DNA binding specificity of affinity purified EBP
Competition analysis of EBPI binding. Reactions contained 0.5 ng [32p].|abelled double stranded SV1 oligonucleotide, 1.0 pg of unlabeled poly [d(A-T)]: poly [d(C-G)] and 1.0 pi of affinity purified EBPI (lane (-) contained no EBPI). In addition, reactions contained 25 (lanes 1), 50 (lanes 2) or 100 ng (lanes 3) of unlabelled double stranded SV1, SV1 .Ml or SV1 .M3 oligonucleotides. Reaction products were analysed on 6% polyacrylamide gels. The nucleotide sequence of the SV1
oligonucleotide is shown - the corresponding changes incorporated into SV1.M1 and SV1 .M3 are indicated.
SV1
+ 1 2 3
1 2 3SV1.M1
1 2 3SV1.M3
SV1
Ml
M3
CC
GG
tl
It ,
5-AGGGTGTGGAAAGTCCC-3
Figure 4.2 Mutational analysis of the EBPI binding site present in the human