2.2 General materials and methods
2.2.1 Cell culture, radioactive labelling and irradiation 2.2.2 N on-denaturing filter elution
2.3 Results and discussion of preliminary non
denaturing filter elution experiments
2.3.1 Radioactive labelling
2.3.2 Com position of the lysis and eluting solutions 2.3.3 Proteolytic enzym es
2.3.4 The effect of pH
2.3.5 D etergents an d lysis tem perature
2.1 Introduction
The cell culture an d radioactive labelling protocols used throughout th is project are described in th is chapter. W here individual experim ents differed from th e g en eral m eth o d s d escrib ed h ere, the d etails are provided u n d e r th e M aterials an d m eth o d s sectio n of the individual c h ap ter.
M ost im portantly th is ch ap ter co n tain s a description of th e filter e lu tio n tec h n iq u e perform ed u n d e r n o n -d e n a tu rin g conditions, the tech n iq u e w hich form s the backbone of th is project. The m ethodology followed w as basically th a t of B radley an d Kohn (1979). A literatu re se arc h soon revealed th a t m o st investigators u se m ore or less the sam e m ethodology, b u t th a t very little attention h ad been paid to standardising th e d e tails of th e ex p erim en tal p rotocols. The lack of co n sen su s re g a rd in g th e ex p erim e n tal p ro to co ls is p o ssib ly due to v ario u s investigators who hold differing views as to the factors affecting elution, a s m entioned previously in ch ap ter 1 (section 1.6).
M ost w orkers agree th a t th e lysis and eluting conditions should be chosen to achieve DNA th a t is ideally free of all bound proteins and hence h a s lo st all hig h er-o rd er ch ro m atin stru c tu re , w hich could otherw ise m a sk DNA dam age (Radford 1988). A uthors how ever disagree on how th is should be achieved and th e experim ental protocols u n d er contention are; (a) th e pH a t w hich th e technique sh o u ld be perform ed, (b) the chem ical com position of th e lysis and eluting solutions, (c) the u se of p ro teo ly tic enzym es in th e lysis so lu tio n an d (d) th e type an d co n cen tratio n of deterg en t u sed in the lysis solu tio n an d (e) th e lysis tem p eratu re, (a) to (e) are discussed in greater detail below.
(a) The effect o f pH
B radley and Kohn (1979) found th a t the DNA of cells th a t h ad been exposed X -rays or restric tio n en d o n u cleases exhibited m ore extensive elution a t pH 9.6 th a n a t pH 7.4, and they attrib u ted this to more effective rem oval of proteins or interfering cellular m aterial from the DNA a t the h igher pH. This point of view is also held by Radford as explained in his re c e n t review article (Radford 1988). On th e o th er h a n d Tilby e t al.
(1984) and Evans et al. (1986) proposed th a t th e greater elutability a t pH 9 .6 w as due to hydrolysis of alkali-labile bonds in the dam aged DNA w hich w ould n o t occur a t norm al intracellular pH. The recent resu lts of Flick et
al. (1989) suggest th a t the additional dsb a t pH 9.6 are in fact the product of existing ssb an d pH 9.6-labile sites in close proxim ity on the opposite stra n d s. This issue, regarding the pH a t w hich the no n -d en atu rin g filter elution assay should be perform ed, rem ains largely unresolved and hence m an y investigators tend to rep o rt th eir findings a t b o th pH values {e.g.
S igdestad et a l 1987; V an A nkeren and Meyn 1987; Koval and K azm ar 1988a; Rowley an d Kort 1988).
(b) Composition o f lysis and eluting solutions
As described in th e original p ap er of B radley an d Kohn (1979), identical solutions for the lysis and elution steps were used except for the addition of 0.5 m g /m l proteinase K to the lysis solution ju s t prior to use. T his solution consisted of 0.05 mol/1 tris, 0.05 mol/1 glycine, 0.025 mol/1 NagEDTA and 0.07 mol/1 SDS (2 %) adjusted to either pH 9.6 or 7.4. Tris (tris(hydroxym ethyl)m ethylam ine) an d glycine w ere u se d in a buffering capacity while th e Ca++ chelating action of EDTA (ethylenediam ine tetra- acetic acid) in h ib ited th e actio n of n u cleases w hich w ould otherw ise degrade th e DNA. SD S (sodium dodecyl su lp h a te or sodium lauryl
sulphate), a strong detergent, w as u sed to lyse the cells and to free the DNA of proteins.
Shortly afterw ards R oss and B radley (1981) reported using a lysis so lu tio n of above m entioned com position b u t su b stitu tin g th e eluting solution w ith a buffer containing 0 .06 mol/1 TPAH (tetrapropylam m onium hydroxide) and 2 % SDS. W oods (1981) had u sed sim ilar lysis conditions b u t a n elution buffer of 0.02 m ol/1 EDTA (free acid form) and sufficient TPAH to give a pH of 9.6. S u b seq u en t stu d ies {e.g. Kohn et al. 1980; Zw elling et al. 1981; Iliakis an d O k ay asu 1988) have u se d v arious com binations of the above reagents in the eluting solution. O ther th a n in a stu d y by Koval and K azm ar (1988b), n o t m uch attention h as been given to th ese variations in the com position of the eluting solution. They in fact found th a t the choice of eluting solution (tris v ersu s TPAH) did affect the resu lts obtained with the n eu tral elution assay a t both pH values.
(c) Use o f proteolytic en zym es
Proteinase K, the enzym e m o st widely u sed in th e n eu tral elution
■| a ssa y is isolated from th e fu n g u s Tritirachium album Lim ber (Ebeling e t |
al. 1974) and is know n to have a broad and pow erful proteolytic action j
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w hich frees DNA of RNA, proteins an d degrading enzym es (Gross-Bellard ]
I
e t al. 1973). Proteinase K w as found to have a pH -optim um in the range 3
i
of 7.5-12.0 and to be very active in th e presence of th e detergent SDS and 3
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m etal-chelating EDTA (G ross-Bellard et al. 1973), w hich m akes it an ideal |
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proteolytic enzyme for u se in th e non-denaturing filter elution technique. 4 Pronase is a proteolytic enzyme th a t had been u sed in the rigorous |