1. PROBLEMA DE LA INVESTIGACIÓN
1.2. Planteamiento del problema
2.2.2. Estados Financieros
A poaalble crlticiaa of chaae experiment# carried out in the way deacribed in thia chapter i.e. an initial period of hi#^ intenaity labelling followed by a period of further growth in unlabelled medium# la that wh«i ^(t-TdH enter# the cell it paaaea into an already exiating pool of MIA precuraor*. Depending on the rate of entry and pool aiae# the apeclflc activity of DMA ayntheaiaed at the beginning of labelling would be expected to riae# aloviy or rapidly# until the apeclfic activity of precuraor mole cule* had rlaea to equilibrium. Thia effect would appear in autoradiograph*
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ma a grain gradient at the b e ^ ruling of labelling# and might be miacon- atrued a# a tail coaaequant upon the chaae. That thia aource of error la not aerioua la evident in certain track# from chamed material to be aeen in many labelled aectlona ahown in Flatea 8 to 22. Crigiaa which initiated replication aome time before labelling atarted# and whieh therefore ahow up la autoradiograph# a# gap# flanked by heavily labelled traoka# have well defined diatal taila but their proximal heavily labelled région# be*iii abruptly. It follow# that tlm aim# of the peel of thymidylat# molecules meat be auffioleatly email# at leaat after FDdK treatment# to allow rapid awamplng by the influx of ^R-Tdk at the beginning of the pulae. Chi the other hand the pool nuat be aufficiently lar#» to allow the autoradiographic detection of the progreaaive dilution of t)ie remaining ^B-TdK during the later chaae.
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k growing fork haa been defined am any one aite on a parental double atranded JMk molecule where replication haa reaulted in the production of two daughter atranda of DNA. If two divergently arranged growing fork# are produced from an initiation aite# then two polymeraae moleculea muat
clamp onto the initiation aite a# a pair and atart replication aimultaaeoualy. It therefore aeena reaaonable that initiation ai tea might be region# along the DHA where there are reveraed complementary baae aequanoea i.e.
palindromaa# the two polymeraae moleculea clamping omto the initiation aite at the border between the two complementary eequenoea.
Huberman A kigga DMA fibre autoradiographic atudiea on Chiaeee hamater oella in 1966 ahowed central région# of hi^^ grain denaity bounded at both end# by gradient# of declining grain denaity when an initial pulae
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and la tar chaaa procedure# aimilar to that uaed in the present study# vas employed. They were led to propose that synthesis of the DMA had been proceeding bidirecttonally from a common origin in the great majority of oases, fty own evidence for chicken somatic cells confirms thia pattern.
Lark# at ml. (1971 ) again working with FDdA-treated Chinese hamater cells but using a step-up labelling procedure i.e. a pulae of low specific aoitvity label# 5.1 Ci/aNol for 30 min followed by a pulae of hi#^ specific activity# 11.9 Cl/mNol for 45 min# claimed that the majority of the labelled
sections they examined showed unidirectional replication. When however -j they did pulse-chaae experiments of the type reported by Huberman A Higgs
and myself# nearly all the fibres they examined showed bidirectional repli cation. They do not attempt to answer this anomaly.
In reference to their first experiment it will be recalled that once replication haa progressed aome distance from an initiation point in the absence of label# any single fork will necessarily appear to be replicating one-way! Again# if the difference in specific activities of the Low - Ei^ pulae-chase protocol was not sufficient to produce a demonstrable increase in grain density during the chase period# their conclusion as to
the direction of replication based on their interpretations of which labelled sections represented tail regions# must be considered unsound. Thia haa also recently been investigated by Huberman A Tsai (l973)# In the lig^t of
Lark et al#s evidence they re-examined their proposal for bidirectional replication# and repeated DMA fibre autoradiographic experiments using various labelling protocols# including an exact repeat of Lark et al*a first experiment. They concluded that about 90^ of their unambiguous autoradiographic patterns could be explained by bidirectional replication, not by unidirectional replication. They also found that in autoradiographic experiments using two different specific activities of ^B-TdH, obvious
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differences in grain density were obtained only when the difference in Bpecific activity was threefold or more. The two-fold difference in
specific activity employed in experiments by Lark et ai. referred to earlier was therefore not adequate to allow a difference in grain density to be
observed. In addition to the evidence from the present study and that presented by Huberman and co-workers# DNA autoradiographic patterns consis tent with bidirectional replication in eukaryotes have also been observed by Callan (l972, 1973), using both the pulse-chase and the step-up labelling procedure (see notably Pig. C, Callan, 1973) and by Hand & Tamm (l973)
and Amaldl et al. (l972) using a pulse-chase protocol. Furthermore Weintraub (1972a) detected bidirectional replication in chicken somatic cells by an entirely different technique based on specific breakage of 5-bromouracil
(BrdU) - containing DBA by ultra violet light. Synchronized cells were labelled with BrdU for 5 min and then with ^H-TdR for 15 mln. Similar
14
control cells were labelled for 20 min with C-thymidine. DMA was extracted from both and centrifuged throu^ an alkaline sucrose gradient after exposure to ultra violet li^^t. After 10 minutes irradiation the DMA containing
BrdU was reduced to about one-half the molecular weié^t of the marker
14C-DNA which was unaffected by irradiation. Prom these experiments Weintraub concluded that the BrdU segment was in the middle of the pulse labelled DNA and that replication was bidirectional at each replication unit.
Criticisms that FUdH pre-treatment mi^t be responsible for an unnatural pattern of bidirectional replication, possibly due to a build-up of initiator proteins during PUdR treatment (Lark et al.. 1971; Weintraub, 1972a) were tested in further experiments described in Chapter 4. These experiments demonstrate that labelled sectbns from non-synchronized, non- PUdR treated cells show at least the same degree of unambiguous bidirectional replication as DNA fibre autoradiographs prepared from PUdB-trested cells.
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Finally, further support for the general occurrence of bidirectional replication may be taken from the evidence gained from prokaryotes.
Bidirectional replication has now been found in phage ^ (Schnos St Inman, 1970), L. CQli (Bird et al.. 1972; Masters & Broda, 1971), phage T4 (Delius et al.. 1971), Salmonella tvphimurium (Nishioka & Eisenstork, 1970) phage T7 (Volfson et al.. 1972) and in Bacillus subtilua (Vake, 1972).
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The present study also supports observations from other workers that at any one growing point both newly synthesized strands appear to extend in the same direction alongside the strands of the original duplex. Plates 10, 11 and 20, show the identical pattern of label distribution that is indicative of separated sister strands. That the grain density per unit length of supposed separated sister strands is indeed hedf that present in single labelled tracks from the same tandem series is shown in Table 1.
However, in vitro studies with DNA polymerases from both prokaryote and eukaryote sources have shown these enzymes are only capable of adding nucleotides to a pre-existing nucleotide chain in the 5* to 3* direction. Since the duplex strands of DNA are anti-parallel, how is the apparent concurrent replication of both parental strands from an initiation site to be explained? Recent evidence has accumulated in support of a oK>del circum venting this difficulty first outlined by Okazaki, Okazaki, Sakabe, Sugimoto
St
Suglno in 1968. From experinwntal evidence on E. coli they proposed that DNA is in part replicated by a discontinuous mechanism involving synthesis and joining (’ligasing*) of short deoxynucleotide units, each about 0.4 pm in length. Britten& Davidson (l97l) Davidson et al. (l972) working with
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ÀenùDu* reported olmilmr length# of "nnlqne •equence** DMA eepmmted by •hort piece# of repetition# DMA. Eveneon, et al. (1972) eleo dleoevered eeotlon# of thia l^igth, or multiple# of it, regularly eepamted by ehort region# of lew melting point, in aKA from ühine## hamater cell# end o h lc k
fibrobleete. All thl# evidence point# to the general end oharmcterietic occurrence of t&ieee Wiort eeotiom# of StfA (Okaaakl fragneut#}.
DNA eyntheei# occur# therefore In the 5* to 3* direction along one etrand, eithmr contiaueaaly or dioeontinaotuily, while on the cof^le- mentary atrand DMA i# ayntheaiaed diacoatinuoualy again in 5* to 5* direction but neceaaarily ghAl. From the growing point. The abort chain# are then joined to the growing pelyaucleotide chain by formation of phoaphodieater linkage# oatalyoed by DNA llgaaee. The work of Faaling $ Hamm (19#) euggeata that replication la diacontinuoua aleng both ale ter atranda of DKA. AM atudiea by Eriegatein A Hogneae (1974) ahow that the direction Gt ayntheaia along aim ter atranda ia indeed antiparallel. Small "blip" région# along a atretoh of DMA ropreaent rogiona where MIA replica tion ia 0^ earring. In favourable cireuaataacea a abort length of aingle atranded DMA (repreeentiag tmapirod newly ayntheaiaed DMA) la obeerved at either end of a "blip" region, each extending from the oppoaite daugliter atrand of M(A. Beoent vork by Bugino et ai. (1972) euggeat that Ukaaaki fragmenta are initiated from ahort piece# of USA tranacribed aa "fold-back" point# in the aeparated parental !%A chain#.
The above model ia illuatrated in it# aispleat form in Fig. 11 (l)« Diacontiououa replication ia indicated alorig both complementary atranda. An exteaaion of diia model muat be propoaed to explain bidirectional replication# each a model ia illuatrated in Fig. 11 (b). The ayatheeie of DKA earn onl^ occur in the 5* to 3* direction therefore bidirectional
Fig. 11. üftdeXg iUttOtratiiig g mimple raaolttUom* pgitem #f nui roplioatioa. Short arrewed
lia## r#pr###mt Qk###kl fragment#, 0, rapr— »at# th# poaition of aa initiation ait# or origin and aolid caatianoa# lia## rapraaaat parant palynaclaotida ohaiaa,
(a) Raplioatlan oocurrlag unidlraetianally along
h#n» oaaplaaantary atrand# o i MU., diaoantinaoaaly aaay fram the ialtiatlaa ait# la th# 3* ta 3* diraetioa alaag oaa atrand and tovarda th# iaitiatloa ait#, again in th# 9* t# 3* dirao- tion, aloag tba eaaplæentery atrand.
(h) hidiraatiwal rapliaatian. PKk ia raplieatad dlaoantinaoaaiy aaay from th# initiation ait# in th# 5* to 3* diraotion alwg ooaplamantary atraada. DRd la thaa raplieatad, again diaooa- tinaoaaly in th# 5* ta 3* diraetioa, along hath oa#pla##ntary atraada and toward# tha