Pilot Study

The use of FISH to assay replication time was still relatively new when this study began. Because of this, the analytical precision (reproducibility) of the FISH approach needed to be established.

Three separately prepared AHB cultures were used. The same cosmid (11421) from the MHC class I region was hybridized separately to two slides from each culture and the number of S-phase nuclei with the SS, SD or DD patterns were scored in 250 consecutive nuclei (i.e. 500 chromosomes). Different investigators have scored different numbers of nuclei, from 100 (Kitsberg et aL, 1993a), through 100-200 (Selig et aL, 1992), to >200 (Bickmore & Carothers, 1995). Dr Peter Sasieni of the ICRF’s Mathematics, Statistics and Epidemiology laboratory suggested 500 chromosomes per slide. He also devised a simple formula for estimating the mean replication time of a locus. The number of replication events that had occurred (i.e. the number of doublets, D) was divided by the number of potential sites for replication (i.e. the total number of S-phase chromosomes observed):

SD+2DD 2(SS-kSD+DD)

This estimates the probability of observing a doublet on a randomly chosen S-phase chromosome. Whilst this formula assumes that replication at a given locus can be thought of as instantaneous, it recognizes that it may not be synchronous on both the homologues.

Table 3.1 (below) shows good precision for cosmid 11421 under these experimental conditions. Based on our samples of 250 nuclei, the greatest intra-culture range was only 6%, seen in culture A. The two most extreme inter-culture estimates were 0.54 and 0.60, again a difference of only 6%. This probe showed very httle variation across different

slid es and d iffe ren t cultures, therefore, 500 ch ro m o so m es w as sufficient to give a precise

in d icatio n o f w hen in S -phase a locus replicates. T h e ch i-sq u a red test fo r the independence

o f values in the sh aded ro w s and colu m n s o f table 3 .1 , yielded a p -v alu e 0 .0 5 7 . G iv en

then th at the difference betw een the slides is not statistically significant, w e can reaso n ab ly

a ssu m e that this approach to assaying replication tim e yields data from w h ic h w e can

estim ate the m ean replication tim e o f a locus.

C ulture A C ulture B C u ltu re C T otals

S lide 1 Slide 2 Slide 1 Slide 2 S lide 1 S lid e 2

SS N uclei 70 _{. i 7h} 81 ? 84 481 S D N uclei .. 60 5 9 "^ 72 M 53^ 347 D D Nuclei 105 121 100 128 105 113 ; 672 T otal N uclei 25 0 250 250 2 5 0 2 5 0 V 250 ^ 1500 P ro p o rtio n o f D oublet C h ro m o so m e s 0 .5 4 0 .6 0 0 .5 4 0 .5 9 0 .5 5 0 .5 6 0 .5 6

Table 3 .1 . N uclei counts and estim ates o f the probability o f o b serv in g a dou b let signal fo r c o sm id 11421 in B -cells.

3.5.1. D oub lets w ith ou t rep lication

O ccasio n ally , som e B rd U -n eg ativ e nuclei ap p ealed to contain d o u b lets. T h e q u estio n had

first b een raised by B ick m o re & C arothers (1995): are d o u b let signals alw a y s indicative o f

replication? On m etaphase ch ro m o so m es, w h ere chrom atid n u m b ers can b e a s s e ss e d ,

close exam ination reveals a sm all percentage o f ch ro m atid s w ith 'd o u b le t' F IS H sig n als.

T hese false p o sitiv es are th o u g h t to be due to either spatial separation o f the d enatured

c h ro m a tin strands o r chrom atin d e-condensation (H o u se al & K lin g er, 1994). T o estim ate

the extent o f this 'd o u b le t' p h en o m en o n on interphase ch ro m a tin , I re tu rn ed to the slides

nuclei (non-S-phase nuclei with clear doublets on both homologues were not scored as these were considered to be G2 nuclei). The results are shown in Table 3.2.

Culture One Culture Two Culture Three

Slide 1 Slide 2 Slide 1 Slide 2 Slide 1 Slide 2

BrdU 4-ve nuclei 0.54 0.60 0.54 0.59 0.55 0.56

BrdU -ve nuclei 0.07 0.07 0.08 0.05 0.07 0.06

Table 3.2. The proportion of doublet signals observed in BrdU +ve and BrdU -ve nuclei using probe cl 1421.

The above table illustrates that a small percentage (from 6% to 8%) of doublets can be seen without replication. Some replication may have taken place before cytologically detectable amounts of BrdU had been incorporated, but the fact that many BrdU-positive nuclei have no replication on either homologue would argue against this. This phenomenon is most likely caused by over-denaturation of the chromosomal DNA. Indeed, in the early stages of this work, some experiments were uncountable because of multiple signals in all nuclei. This occurred with freshly prepared, ‘young’ slides in which the chromosomal DNA is more easily and extensively denatured, so much so that the individual strands of the DNA duplex can be spatially resolved. I found artificially ageing the slides in a microwave the most effective way of reducing this problem.

3.5.2. Replication without doublets

Observing 'doublets' in non S-phase nuclei prompted the reciprocal question: Does a replicated locus always give a discernible doublet? Without the aid of confocal microscopy, the nuclei can only be examined in two of their three dimensions. Consequently, signals that are separated in three-dimensional space may be superimposed when viewed from a single perspective down the microscope.

To estimate the percentage of doublets that might appear as singlets, I captured ten DD nuclei (20 doublet loci), with the spots in each doublet at varying distances apart. All images were captured as originals (i.e. not pseudo-coloured) at xlOOO magnification then transferred to Adobe Photoshop from where they were printed at the same size. The distances between the centres of each spot in the doublet pairs was measured.

The observed distance between the centres of the spots in each doublet is, at most, 2-3 times the diameter of one of the spots. Spots whose centres are less than one radius apart may not, therefore, appear as a doublet but as a figure of eight. Using these data Dr. Sasieni calculated that if their spots are only two diameters apart then, at most, 16% of doublets could appear as singlets (asin(l/4) = 0.16*p/2). However, since this is assumed to be a random process it should occur to the same extent for all probes and should not affect the assessment of their relative replication times.