Eutanasia: entre la responsabilidad y la autonomía

ensyme on thin ntruoture# Fig$42 was taken 3 minutes after the application of M a e o solution. The onsyme ha@ begun to act upon

the loop matrix and two granules, attached to one another by a thin fibril have come adrift, T!&e loop axis can be seen at the thick end of one loop. In Pig#43, taken 2 minutes later, more loop matrix has gone into solution, granules are being scattered by Brownian motion, and the loop axis fa clearly visible.

Fig.44 was taken 6 minutes after thq start of RNcae action# The loop axis fma been stripped of its Amtrix and granuloe for

over half its length# "Stripping" progreseee towards the thick end of the loop, leaving the axi# bare and limp, but unbroken# In the long run, RNaee aeeme to reimove less material from normal loop» and from landmark» than doee trypsin, pepnin or pan-protea»e (cf. Fig»,46 and 12). The giant loop» of chromosome» X, XI, and XII Of carnifex first become increasingly vacuolated; later theyeynw.i, mi. \ ^ ^ ^ ^ slowly shrink to about half their original volume». Am normal

loop ABtrice» are dissolved by RNase, loop axe» are exposed;

these contract and increase in refractility. There in also some contraction of the mm in axes of chromosome», but no less of

oiiromomeric pattern and no disruption of fusions even if pH is subsequently lowered to 2. The linear continuity of lampbrush chromosomes remains undiq^urbed by RNaae, and is equally

-41-

Thé rat© of RNaso action on lamphrush chromosome»

appears roughly proportional to tho concentration at lAich tho en»yme io used. Tho final state reached by the chromoaomoe is tho same over a range of RNose concentrations from 0#25mg. to 0.OOOOBSmg,/ml. $ome ovidence of îlîla s © action can be aeon even

ê * at a concentration as low as 10 mg,/ml#

In accordance with expectation, testa made with RNaae which had been boiled for 18 minntea showed that enaymatic activity

ia retained#

O. Deoxyribonncleaae

In the preceding eha%)tera we have described ho%^ trypain, lion-protenae, pepsin and ribonucleaae affect lateral loop matrices, apherea and other landmarlta, Wo have also demonstrated that none of these enaymes break the axes of lampbrnsh chromosomes, no

matter how the en&ymoa are applied. Taken by Itself, this is evidence that the linear continuity of lampbrush chromosomes does not depend either on protein, on RNA, or on a combination of those materials. The other k n o w constituent of lampbrush chromosomes is DNA, and we will now present the complementary ovidence which demonstrates that it is this substance wiiich holds lampbrush chromosomes together.

-42-

DNase was used at effective oonoentrations of 0&128 or 0*0128 Rig/ml dissolved in C medium at pH 6.2* In some experimontB % $ 0 ^ was diaaolved in the enayme solution at 0.008% to function as activator, but its presence made no significant difference to the résulta. DNase was used at lower concentrations than the other enaymes in order to alow down its action and thus allow its dramatic effects to be recorded photograph ically *

A few minutoa after application of DNase solution lampbrueh chromosome» break at several places along their axes, each break occurring aoroas the slender fibril connecting

adjacent chromomeno» (Figs*49 and GO). The first break» generally occur in accidentally stretched regions. As time passes more and more axial breaks occw, but soon breaks in the lateral loops also become evident* Lateral loops are

rapidly broken into smaller and smaller fragments, each fragment retaining, for a time at least, its original fine morphology

(Figs.48 and 61)# Brownian motion so scatters the products of this continuing disruption that after 16 to 20 minutes the entire chromosome group is no longer recognisable.

For purposes of comparison we have followed the action of DNase on the giant granular loop of chromosome XII of cristatus. Besides breedcing the axis of this loop, DNase mimics the action of ribonuclease or a proteolytic ensyme in that the matrix of the loop is dissolved and the granules released (Figs.62 to 66).

This led urn to wonder whether o%ir sample of DNase im» perlmps contaminated with other engymee. DNase solution after being boiled for 18 minutes proved entirely inactive, hence we can rule out the possibility of RNase contamination. We cannot absolutely rule out the poanibility of contamination by a proteolytic onnyme, hence the "stripping" action of our

sample of DNase on the giant granular loop of chromosome XII of cristatus romaine an inconclusive obeorvation.

The crucial result of our test with DNase, however, is the discovery that this ensyme breaks the main axes and the lateral loops of lampbruah chromoaomea. Poeeible

contamination with proteolytic enaymea ia here inconsequential, since we have already shown that proteolytic onaymea are

unable to prod%:ce such breaks.

II, Vera one

In a paper published in 1084, Mania described the results of certain experiments on grasshopper mitotic and meiotic chromosomes and on salivary gland chromosomes of

ila. from td&ich he concluded that these chromosomes may consist of particulate ":oacromolecular complexes of nucleic acid and proteins ,,,, linked together by bridges of divalent ions". The basis for Mhsia^s claim was that in material frosen by solid COm, extracted in distilled water, and then either treated with