41

4,o

-» I1 -

2 H ,rTiijli’.ii;;-

h v i m c a

4o

3s

■ I1-1'! i:n< . !;•(•!!

F * * .» & / .

?,)}< i'vC- ll'.JJ i7.‘l( • f. 5!/

FiffUre 1,41 CorT-ar » ;^Flv.ovoi-vaHt

(5XMix “v

X )o >r

no-cn,

i..cm

X/»

i;»

on. (\I if;'' "C ■ i ) 1 , f , S H U "1/

jj/M 'x i ! i !> it

r • ‘! . , i • I ..

Figure L 4 2 ('}

W t i ' M H v 5- ■ ^rs'tnd iyu : { k ~a )

5-fl’dr inhibits the ORzyuo thy.ai<:ly1nte synthetase, This is the enzyiw which in the production of endogenous thy,«i dine converts dUMP (uridinn Boncphorjphats} to dTMP (thymidine monophosphate)» indicated in Figure l.ffj below. In the noniuil tnctuhol 1c cycle the thyisidine monophosphate is then converted to thymidine triphosphate (nucleotide) and in this form enters the DMA, I t can be seen therefore that i f thymidylciU* synthetase is inhibited* endogenous thymidine is not produced and in the absence of thymidine DMA cannot replicate, Thiss of com sc» prevents ni to?»i s > as has been observed experimentally (ToHvet tuiu Sitnon 1967), Addition of v \ternu1 thymidine can reverse the effects of THUdK because DMA replication ( requiring thymidine) can again take place.

. The actui.l reaction taking place during the formation of dTMP from dUMP is shown below and in Figure 1.43 at (1), TIk methyl group in the reaction below is transferred from r. 10 methylene tetra- hydrofoltite (CH.JF) to the 5 position of the pyrimidine ring of dUMP forming cITMF-' + dihydrofolato.

dUMP + CH.JF " • • x dTf# + DHF£ ---- - * ■ ■

tet” -ihyc!ro - dihydrofolatefolate.

The reaction is shown i*; Its context in the Metabol ic; scheme inFigure L43 below.

The actual bloi*f;<vj*< of th*1 activity of the enyynKi thyialdyloto

synthetase .occurs as foil owe. ;

dllMP normally binds the en/ynie thymidylatc synlhrtaso fK'ape) (1

mole cult, of dUHP to one snolocuH of thymidyluto synthetase), The

■inhibitor 5-4'tMR bind*, with this enzyme in the ratio two molecules

of 5-Ftk!R to one t.iolocuie of thyridylate r.ynthetsso (Jteldelbenjfir

1965), Tins is fhown in Fiouro 1,44 bo low.

TSasn + 1 dUM P-^ T fn iS ' v (d !., : r ) 1

TSase + 2FdUMr TSase - {rd lfr iP i *. ■ \ ' ” ■ ■ e.

Figure 3 .44 *.v.,v■*,' *r.V /•,. j o n i!n,:.iL'?ulaU

Once the enzy*-? has bean Huarf by dttT (normal reaction) It is used

to convert diKP to dl'IP by faeiM tal i tig the transfer of a mothvl

9**‘owl) 1 ro;»i 5» 10 f;’fct.hylc*:;o totraby dr**?r<lqte to the 6 position cf the

pyrinridine r1»K: of dUflF (K< rUran cud l!cidalboru?r 15G1). Methylene

tefcrabydrofo<»;£« is mj-MIr^d for the? inhibition of this reaction by

5-rUtid, {in the forr,! FdUItf*) a s s In ils alnerce, FdU»MP binds poorly to

tho The transfer of the Cb’5 uroup from tho mu thy low*

tetrahyrirofelate to the ddMP is shown in Figure 1.45 on page 44

K,:

d i'h y d r o f o i a t eV

o

"Clf.

5, iO-CK,«if< Foiatc/y ' methylene

teUfihydiofQlatii

Methyl Gioap0

.■A. IsShould ir. ;>), * •

this

0J p.-0-CIf , 0 {s' u

OH

t'.u,'.:?

HN ,j ^ cr^K-;.^

■Cit»<5

/ s-r-u*;:th^m idy tote*

"01F ~ < 0 ~ C H ^ O

b loct; a '*>* ” t hvt.»;•1*

ViOJ

..„Y'

0 dTMP■V

O'

Ai'~o-c;i. ^0V

7m ft It 0 s 'his

OHF-dUMJ!

Finure 1.45 :">vo;.vr,->> a/’ CH.. from to dVMP,

A further schemn of the3 5-ftldR block is shown below with 5-FUdR

entering tho cycle at (1) and forming FdUMP Instead of thymine being

able to form dTMP at (2). The FUdR enters the cycle as 5-FJdR

and is motabnl iscni to FHg as shown in Figure 1.46 on pago 45

n

eyele* 2'ke inhibition €»ic in inav1:cd I.

In conclusion* 5-FUdR inhibits the enzyme thyniidylate synthetase by

competing for its binding site and in so doing prevents the

formation of thymidine, thus preventing DNA replication. This causes

an inhibition of mitosis,

1.8.4. S-Bromodooxyuridine

5-Bromodeoxyuridine differs from 5-FUdR i,i that a nromine atom

rather than a fluorine atom is found at position 5 (see Finn re 1.41)

os shown in Figure 1.47 below,

5-BUdR replaces thymidine in the DNAS making the DNA heavier than

normal DuA. riguro 1,48 below shows the mode of entry of the 5-BUdR

into the DNA at the position marked 2.

NdR

Figure 1,47 Stmwluvc of &-l>t>m->deaxyuvid-inv (5-BUdR)

For comparison the position marked 1 again shows the 5-FUdR

block. DNA containing 5-BUdR prevents differentiation in cells in

culture although the cells g n w normally. This will be further

discussed in section *.3.5. below.

B U d K P P P

tlB U d R P P

B l i d R P

A

B U d R

Ii

BU

Bl oc ! ; in T h y m l d y l a t e S y n t h e s i s

F i g u r e _ L 48 E n t r y o f S -B U dR i n t o DNA . ( s r y b a l s k i 1 9 6 2 )

1,8.5. Effects of 5-BUdR and 5-FUdR on.cells in culture

Although cells vn ■ h 5-BUdR in their DNA are fully functional i.e.

they continue growing and dividing, the production of "luxury

proteins (proteins for the specialised functioning of the cell e.g.

myosin in muscle) is prevented. This can be reversed, as when the

5-BUdR is removed the cells proceed to differentiate normally.

Likewise the effects may be reversed if thymidine is added to

replace the shortage of endogenous thymidine. Stockdale et al (1964)

showed that myogenic cells (prospective muscle cells) in the

presence of 5-BUdR failed to fuse and form multinucleated myotubes

prior to becoming muscle.

Simon (1963) reported that HeLa cells in culture with both 5-BUdR

and 5-FUdR divided only once and incorporated slightly more 5~BUdR

than did cells in 5-BUdR alone. It could be suggested that this is

due to the lack of thymidine caused by 5-FUdR in the DN'A, (which the

cells replace with 5-BUdR). The one cell division is the final one

in the presence of 5-FUdR.

1.8.5.1. The effects of 5-FUdR on cells and organisms

when administered alone, with special

reference to concentration

Owing to the cost of 5-FUdR, prudent use was made in the administration

of this drug. As a first approximation to set a working concentration

of 5-FUdR, it was decided to follow the dosage concentrations for

Chinese Hamster cells which were worked out by Conrad and Ruddle

(1972). They showed that when Chinese Hamster cells were grown in

concentrations of 0,00005 f-ig/ml 5-FUdR, there was no reduction in

the rate of mitosis and hence no reduction in thymidylate activity.

In concentrations of 0,0001 pg/mT 5-FUdR, the rate of mitosis

initially slowed down, stabilised and then returned to normal,

showing an initial reduction in thymidylate activity. When these

workers used a concentration of 0,001 pg/ml 5-FUdR, the rate of cell

division was slowed down permanently and did not return to normal.

Thymidylate synthetase activity was reduced to 20% of its original

level, ,

Another worker (Ferguson 1978) who worked on the teratological

effects of 5-FUdR in Wister rats, injected 0,1 pg 5-FUdR per mg. of ’

body weight. ,

In the present study, tadpoles were maintained continuously in

water, and concentrations ranging from 0,2 - 20 yg/ml of 5-FUdR were

added to the water. These concentrations were 2000 times greater

than the concentrations of 0,001 jjg/ml that were effective in tissue

culture and 200 times greater than the concentrations injected into

the Wister rats. Furthermore, while the rate of uptake of the 5-FUdR

may have been relatively lower, this was compensated for by the

continuous application of 5-FUdR.

1.8.5.2. Effacts of 5-BUdR on rills and organisms

when administered alone, with special

reference to concentration

In the present project a concentration of 10 ng/ml 5-BUdR was ucid.

This was higher than that used in most tissue culture.experiments as

the tadpoles 'fere swum in the solution and it was assumed that the

chemical could be less toxic with this method than Jn tissue culture

as the tadpoles were only getting a fresh solution of 5-BUdR once

weekly.

While 5-BUdR affects differentiation, it does not affect cell

division and growth as shown by Bishoff and Holtzer (1970). The fact

that thymidine competes with 5-BUdR to enter the DMA was shown by

Stockdale et al (1964) where excess thymidine added to the culture

medium prevented the uptake of 5-BUdR, -

Abbott and Holtzer (1968) examined the effects of 5~BUdR on

chondrocytes taken from chick vertebral cartilage. The concentra­

tion used was 20 ng/nil. The resulting chondrocytes had bizarre

shapes and the clones consisted of widely scattered fibroblastic

cells i.e. true condensations required for cartilage formation were

not seen. The mucopolysaccharide required for the matrix was

destroyed.

U s h e r and Cahn (1969) in their studies of cartilage cells

found that a concentration of 10~4M was toxic to the cells, while

10 M was not toxic, but prevented differentiation.

Agnish and Kochhar (1976) showed a relationship between the

developmental stagt- of mice embryos and their sensitivity to 5-BUdR.

From each embryo they removed the limbs and grew them in cell

culture, one on normal medium and one on 5-BUdR. They found the

following:

1. At a concentration of 2 ug/ml 5-BUdP, stage 26 to 29 embryos

(eleven day) showed a complete suppression of chondrogenesis.

2. Mid-eleventh day embryos needed 10 to 25 qg/ml 5-BUdR to

achieve the same effect.

3. If twelve and thirteen day embryos were used the effects were

milder no matter how high the concentration of 5-BUdR was.

The effectiveness of the drug coincides with the various stages

of cartilage differentiation, as the first signs of differentiation

•were apparent only in the late eleventh day embryo and after

differentiation has taken place, the drug has no more effect.

Abbott and Holtzer (1968), Coleman et al (1970), (1958), Lasher

and Cahn (1969) found that 5-FUdR had a proximo-distal effect. The

scapula, first to differentiate7became increasingly resistant to the

drug, while the more distal radius and ulna retained sensitivity

throughout. This is in keeping with Tschumi (1957) who shows that

differentiation proceeds in a proximo-distal direction.

Sal a and Rizotti (1975) used stage 40 or 47 (NF stage) tadpoles

(Xenopus laevis) and exposed them to 5-BUdR by injecting it into the

coelomic cavity (2ifl of 5 x 10'2M ) . The most severe effects were

noticed on stage 40 tadpoles reduction in pigmentation,

swelling in the anterior portion of the head, curvature of the tail

and accelerated cardiac rhythm. The effects were less severe in the

stage 47 tadpoles and at stage 51 no external modifications 'were

seen. Histologically, however, all the animals had suffered

abnormalities to some degree.

In conclusion 5-BUdR is found to affect the differentiation of

cells in culture and the severity of the effect is related to

concentration.

1 *9 Analysis of DNA by density gradient

aflalytical ultracentrifugation , '

In the present study DNA was made heavy by swimming tadpoles in a

solution of 5-BUdR. This heavy DNA was analysed by Density Gradient

Ultracentrifugati on. This technique is a means of measuring the

amount of 5-BUdR that has entereu the DNA due to the fact that the

5-BUdR makes the DNA heavy. Meselson and Stahl (1958) performed the

classic experiment using this technique on DNA labelled with 15N

heavy hydrogen. If several species of DNA of different density are

placed in an ultracentrifuge cell in a solution of caesium chloride

and spun for twelve hours or more at 20,000 to 50,000 revolutions

per second. As the DNA absorbs ultraviolet light at 260um ,

Ultraviolet photographs will display these various bands.

Hanawalt ^1968) has shown that the tjoyant density, of DNA containing

5~BUdR increases by 0,9 gem while W^i 1 et al (1935) have shown

that in the presence of 5~FUcH even more s-iiUdR is taken up as chown

by the fact tha'; the buoyant density of the DNA increases,. Further,

by this method one can distinguish between very heavy DNA which has

two strands substituted with 5-BUdR and hybrid DNA, which is

lighter as only one strand has been substituted with 5-BUdR.

The hybrid bands of DNA are found between the normal DNA and the

heavy DNA on the photographs. Vi nograd (1963) has postulated the use

of marker DNA bands using DNA of known density to determine the

density of the various bands of DNA. Each DNA species forms a band

at the position where the CsCl (caesium chloride) density equals the

buoyant density of that species in the ultracentrifuge cell. The

CsCl spins down forming a varied density solution throughout the

cell, the densest solution being at the base of the cell. The

various solutions of DNA stabilise in position in the cell when

their density is counterbalanced by the density of the CsCl

at a particular point in the cell. This takes up to 12 hours to

achieve. At this point bands can be seen on the ultraviolet

photographs.

Figure 1.49 below shows a cell with the various densities of

CsCl.

I n s i d e of c e l l

w i t h C s C l s o l u t i o n

D N A

A i r

M e n i s c u s Lowe r

d e n s i t y

L o w e r d e n s i t y H I g h e r d e n s i t y

Figure 1.49 Co Cl in the u ttmcantvifuge cell

XH i g h e r

density

Figure *].50dh p,5l shows-a typical result of an ultracentrifuge

ultraviolet photograph.

yDMA band D N A b a n d DMA bemd

Figure 1.50 Sketch of ultraviolet photograph showing

various DNA hands

' *

1.10 Objectives of this study .

The Hindiimb of Xenopus laevis appears to develop in a proximo-

distal direction (Tschumi 1957} under the influence of the apical

ectodermal ridge. The zone of polarising activity (ZPA) appears to

influence the antero-posterior patterning of the limb during its

development (Saunders anti Gasseling 1968}. Of the three limb axes,

proximo-distal, antero-posterior and dorso-ventralythe dorso-ventral

axis is established first, followed by the antero-posterior axis and

lastly the proximo-distal axis is determined.

The differentiation of the hind]imb tissues, muscle, cartilage

and connective tissue^appears to be determined by the cell's

position which the cell determines according to a gradient of a

particular chemical or it could determine its position according to

the amount of time it has spent in the “progress zone” (Wolpert

1981}.

The present study used 5-BUdR and 5-FUdR to study the above

processes further. As 5-BUdR prevents differentiation and 5-FUdR

prevents cell division, the hind!imb development was likely to be

affected in various ways. These effects could increase our

knowledge of the processes of cell differentiation and cell

patterning In the Xenopus laevis hind!imb.

52

2. METHODS AND MATERIALS

2.1 Experiments investigating the effects of 5-BUdR and 5-FUdR

on the shape and patterning of the Xenopus laevis hindlinth

2.■ 1.X. Tadpole breeding and rearing

For the experiments in which the tadpoles were swum in 5-BUdR and 5-

FUdR a reliable, regular supply.of tadpoles was required. Xenopus laevis

responds well to life in captivity, in that it breeds all year round

when kept at warm temperatures and treated with a regular dose of

breeding hormone (Pregnyl). The tadpoles respond equally well by

growing and metamorphosing as long as the temperature is kept

constantly warm. For these reasons Xenopus laevis was chosen as the

amphibian for this study.

Xenopus laevis tadpoles were bred from adults supplied by

Jonkershoek Island Fish Hatchery, Stellenbosch. Females and males

were isolated and fed on chopped beef liver twice weekly in summer.

When breeding became more difficult, in winter, they were fed daily.

The water was changed after feeding and the frogs were kept at a

temperature of 25°C->v/hich is conducive to breeding. Breeding was

induced by Pregnyl (Organon). A solution was made up of 4500 units

in 9m 1 water, about 500 units/ml. The male was given 2 doses of

0,3ml, about 300 units in total. As breeding became more difficult

to induce during the winter, it was necessary to prime the males

daily for two weeks with the above dosage until the black nuptial

pads appeared. The females did not require increased doses. The

hormone was injected through the dorsal lymph sac (Gurdon 1967). The

frogs were then placed in a laying tank in shallow water on a mesh.

The eggs dropped through the mesh and were collected the following

morning and placed in shallow trays at 25°C, At this temperature

they developed to stage 43 in three days (Nieuwkoop and Faber 1967).

At about this stage they started feeding and were fed on Liquifry

No. 2 (Liquifry Company Ltd, Dorking), about ten drops daily to

sixty tadpoles in two litres of water, The tadpoles were thinned out

constantly to prevent growth inhibition by overcrowding. The water

was changed regularly.

2.1.2. In vivo experiments ii. which Xertopus laevis

tadp^les were swum in 5-BUrlR and 5-FUdR and

analysed for growth deformities_ •

The following experiments were sot up to test the effects of 5~pJdR

and 5-FUdR on Xenopus laevis tadpole growth, A weekly analysis was

carried out under the dissecting microscope and hindlimb deformities

were noted. (See section 3.3 for pictures.) The hi'rJlimb was

therefore focussed on for further experimentation.

2.1.2.1. Pilot experiments „

The following initial experiments were set up :

a) Experiment started at stage 43 (NF stage)

Solution Volume (ml) Number of tadpoles

(2 dishes, 6 tadpoles

per dish)____________

5-BUdR 10 ug/ml +

5-FUdr 0,2 ug/ml 200 2 x 6

5-FUdR 0,2 ug/ml 200 2 x 6

5-BUdR 100 ug/ml 200 2 x 6

5-BUdR 10 ug/ml 200 2 x 6

5-0UdR 1 ug/ml 200 2 X 6

5-BUdR 0,1 ug/ml 200 2 x 6

Water 200 2 x 6

b) Experiment started at stage 46 (NF stage)

Solution

5-BUdR 10 ug/ml

"5-BUdR 10 M,g/ml ■

J5~FUdR 0,2 ug/ml

5-FUdR 20 qg/ml

Water

Volume (ml)

200

200

200

200

Number of tadpoles

(2 dishes, 6 tadpoles

per dish) ___

2 x 6

2 x 6

2 x 6

2 x 6

54

2.1.2.2. Further1 experiments using Xenopus

laevis tadpoles swum in 5-FUclR

On the basis of the results of the pilot experiments in 2.1,2,1.

further experiments were set up in which the tadpoles were swum only

in 5-FUdR as 5-BUdR was noted not to have had any externally visible

deforming effects on the hindlimb.

The following experiments were set up :

Stage Solution Cons, entrati on Volume Number of tadpoles

(Expenment tig/ml ml (2 dishes, 6 tadpoles

begun) ^ per dish)

0 FUdR 10 200 6 x 2

46 FUdR 20 200 6 x 1

46 FUdR 10 200 6 x 1

46 FUdR Id 200 6 x 2

47 FUdt* 20 200 6 x 2

4B FUdR 20 200 5 x 1

48 FUdR 10 to o o 6 x 1

49 FUuR 20 200 6 x 1

49 FUdR 10 200 6 x 1 .SO FUdR 20 200 6 x 2

52 FUdR 20 200 6 x 2

43 Water 200 6 x 4

(The tadpole stages used can be seen in Appendix A.)

In order to study the deformed limbs in further detail, and

for the purposes of clearer photography, the tadpoles were fixed and

the nmbs stained, removed and mounted for photography (whole

The tadpoles were fixed in Karnovsky's fixative (Kernovsky

1S6£>) as this was found to be less damaging than the formaldehyde

mentioned in the same article.

Kri rnov s ky1 s FI xat i ve .4g of parufmmalriwhyde are dissolved in 46ml water, The temperature

is raised to 60oC in u water bath and 1M NaOH is added dropwise

until thi* solution clears,

Al'-1 60 1:11 i w C «nd W m glutaraldeliydu. Titrate ,t

toofti t f . i n rr-s to pH 7>2 ** 7j4 usinrj IN HC'l

f

55

Buffer C -- 41,5ml soln A + 8j5m1 soln B.

Soln A « Nr.HgPO^ HgO (mouosodium phosphate) 2?26%

Soln D = -NaOH (sodium hydroxide) ?.,52$.

Washing solution 50^ buffer + 50!' glutaraldehyde solution.

The tadpoles were fixed for 24 hours and then washed for 24 hours.

They were then transferred to acid alcohol for 24 hours (1% HOT in

70% ethyl alcohol) (Mahoney 1973), They were then stained for one

week in 1% Victoria Blue in acid alcohol, They were then differen­

tiated in several chances of acid alcohol until the desired colour

was obtained. When differentiation was complete they were transferred

to 90ri alcohol for twelve hours. They were then dehydrated thoroughly

in several changes of absolute alcohol for a few hours. When

completely dehydrated they v/sre cleared in methyl benzoate and

stored in fresh methyl benzoate, inis method stained the cartilage

blue and mode the limb transparent so that the cartilage could be

clearly seen, The limbs wort1 no»» whole mounted and photographed

having been severed iroa the animal. The preliminary experiment?

were photographed on the whole aniv.al (see 3,1). These whole mounts

were analysed by r,*oa?ts of a deformity index (see section 3.2),

2.2 Radioactive thymidine uptake* in the presence of

S-FUdK by _X;f;na;*us laevis tadpole hind!imbs

As 6-FUdR is known to depress the uptake of thymidine (by decreasing

DNA replicatioi which reduces the requirement for thymidine), it was

decided to test this by si/mming Xenqpus laevis tadpoles in

radioactive thynidinc* (^IMiiynridina). The experiments were set up as

follows ;

Control

One dish of 200»tl capacity with six stage 49 tadpoles which were

swum in the solution of 20 ug/ml 5-FUdR for one week. They ware

unfed for throe days then fed on liquifry No. 2 for four days.

Experiment

One dish of 200;al capacity with six stage 49 tadpoles which were

swum in tirj solution of 20 j j/ml 5-FUdR + 5 fjCi/ml thymidine for one

week, They v.vru unfad for three days, then fed on liquifry No. 2 for

fOur d;iyF..

The limbs of the tadpoles were removed and weighed as were the

tails. They were than placed singly into clean glass scintillation

vials containing 1ml of soluene which was used to solubilise the

tissue. The vials were immediately capped and incubated at 45°C in a

water bath for eight hours to assist the solubilisation process.

10ml of a specially prepared Toluene based mixture was added to each

vial. The vials were recapped and thoroughly shaken. The vials were

then placed in the dark in a refrigerator at approximately 6°C for

twelve hours.

The vials were then counted in a Packard Tri-Carb Scintill­

ation Counter (Series 3*000) for fifty minutes each. Counts of

radioactivity were registered on all three channels., each channel

having a window setting of 50 - 1000 and a gain setting of 53% for

■tritium. The couits per fifty minutes were then reduced to counts/

min/mg. (Price 1973.) The results are analysed in section 3.3.

2.3 Buoyant density gradient analysis of 5-BUdR DNA

It was decided to analyse the amount of 5-BUdR entering the DNA

using buoyant density gradient analysis and to compare this to the

uptake of 5-BUdR by the DNA in the presence of 5-FUdR. In the

presence of 5~BUdR DNA should become heavy and in the presence of 5-

BUdR and 5-FUdR DNA should become heavier still as the lack of

thymidine should cause the uptake of more 5-BUdR. The tadpoles were

swum in the solutions as follows :

a) 100 stage 41 (NF stage) Xenopus laevis tadpoles in 200ml water

for three days at 21°C. These tadpoles were killed at stage 43,

b) 100 stage 43 (NF stage) Xenopus laevis tadpoles in 200ml of 0,1

mg/ml 5-BUdR for five hours at 21°C.

c) 100 stage 43 (NF stage) Xenopus laevis tadpoles in 200ml

0,1 mg/ml 5-BUdR and 0,0002 mg/ml 5-FUdR (0,2 ng/ml as 0,001 mg

= 1 ug) for five hours at 21°C.

The DNA was then extracted from the tadpoles and analysed by buoyant

density ultracentrifugation. '

57

2.3,1. DNA extraction

DNA extraction was done essentially according to the method of

Marmur (1961) and Grossman, Goldring and Marmur (1969),

a) 100 tadpoles were killed in 15 ml of ice cold EDTA solution

(Ethylene Diamine Tetra-acetic Acid* Disodium Salt, Merck

Analytical Reagent). EDTA solution : 0,1M EDTA + Q J M NaCl. The

solution was adjusted to pH 8. The tadpoles were homogenised in

this sol u Li on by nujans of an ultra-turrax homogeniser, During

this process, the test tube with the solution was placed in a

beaker of ice (Ledoux and Huart 1967).

b) The cells were lysed in Sarkosyl NL 30, Sarkosyl NL 97 or

Sarkosyl 0 (samples by kind donation of Ciba-Geigy), The

Sarkosyl was used at a concentration of 1,3^. The mixture was

placed in a 50WC water bath for ten minutes and then allowed

to cool to room temperature (Marmur 1961), Sarkosyl is a new

detergent used by Grossman, Goldring and Marmur (1%9) instead

of SDS (sodiws dodecyl sulphate) used by most earlier workers.

SDS is usually used to dissociate the protein from the nucleic

acid but the ar.nds derivative, sodium dodecyl sarcosinate, is

now used due to its high solubility in concentrated CsCl.

Probably the hydrocarbon chain of sodium dodecyl sulphate

competes f*r hydrophobic bonds. The anionic detergent appears

to inhibit nucleases and the negative charge prevents inter­

action with nucleic acids (Noll and Stutz 1968). On comparing

DNA yields for tho different detergents, Sarkosyl was found to

give a higher yield than SDS. Of the three Sarkosyl NL 30 was

used instead of the Sarkosyl NL 97 used by both of the above

groups of research workers, as it gave a better DNA yield.

Sarkosyl surfactants are high molecular weight carboxylic acids

or their own sodium salts of the form : OIL (CiL) CON (CH )

CH2 ~ COCH (or COOKii). 3

They are modified fatty acids in which the hydrocarbon chain is

interrupted by an ami dome thy] group (-CONCIIg-). They are the

amide derivatives of SDS (also known as sodium lauryl

sulphate). This modification improves the solubility and

crystal!inity of the molecule, enhances the acidity of the

carboxylic acid group and increases the absorption characteristics.

58

They are soluble in most organic solvents and are appreciably

soluble at mildly acidic pH's. They foam less than SDS

(Handbook - Geigy Industrial Chemicals),

c) Sodium perchlorate (pure reagent) was added at a high concen­

tration to separate the protein from the nucleic acid (Harmur

1961). 1*838 g were added to the lysate thus forming a 5M

solution which was then adjusted to pH 5.

d) The solution was deprote'?niscd by shaking in 30ml chloroform r

isoatuyl alcohol 24 ; 1 (isoamyl alcohol 3 methyl butanol

CgHijOH pure reagt.it, Merck), The solution was shaken gently

for thirty minutes in a stoppered flask.

The chloroform causes surface denaturation of the protein,

while isoa.r.j 1 alcohol reduces foaming, aids the separation and

maintains the stability of the layers of the centrifuged

deproteinised solution {tfarmur 1961).

b) The emulsion was separated into three layers by a ten minute

centrifugation at 2000 rp:n, The upper aqueous layer containing

the nucleic acids was removed with a Pasteur pipette. The

protein interface and chloroform layer were discarded.

f) Further deprotelnisation was accomplished with pronase

griseus, Wiles-Seravac (Grossman, Goluring and

Harmur lOC'J)),

It was made up as follows :

300ag dissolved in 300«1 water and placed in a water bath at

37 C for forty-five minutes. The solution was then adjusted to

pH 5 with concentrated MCI and left a t t e m p e r a t u r e ^

forty-five minutes. Solid Tris Buffer (Seravac) was th, ,ed

to adjust the pH to 7 and the enzyme was used at a final

concentration of 1 mg/ml and allowed to act for three hours at

37 C (Stern 19G8). As well as digesting protein, pronase partly

digests itself (McCarthy 1968),

g) The lysate was dialysed against SSC for three hours at room

temperature and then overnight at 4°C (Grossman, Goldring and

Ha ray r 1963), SSC (standard saline citrate) - 0,1514 NaCl +

0,015M Trisodium Citrate (?~Hydrate) (Analytical Reagent,

llorck). 5

h) The lysate was treated with Ribonuclease (RNase) (Bovine

Ribonuclease Grade II 5 x crystallised Mi'les-Seravac). The

enzyme was used at a final concentration of 50 u,g/ml and

allowed to act at 37°C for three hours (Marmur 1961). RNase

also partly digests itself (McCarthy 1968). The RNase was made

up as follows (Kalf and Greee 1968) :

At a concentration of 1 mg/ml in acetate buffer pH 4,5 and

incubated at 37°C for thirty minutes. Acetate buffer - 0,001M

sodium acetate (trihydrate) (Analytical Reagent BDH) + 0,001M

EDTA. pH was adjusted with concentrated glacial acetic acid. The

solution was heated at 80°C for ten minutes to inactivate any

DNase.

i) The lysate was dialysed at room temperature for four hours and

then overnight at 4°C (Marmur 1961). It was dialysed against

0,2% SSC + Q,15M NaCl at a pH of 5.

The dialysis tubing was handled only with rubber gloves, as

enzymes on the hands could denature the DNA. Dialysis removes

the detergent, enzymes and the prodi • if enzyme degradation

(Kalf and Grece 1968).

j) The nucleic acids were precipated by addition of absolute ethyl

alcohol (Kalf and Grece 1968). The DNA was then centrifuged

down and dried in a vacuum for two hours and redissolved in 3ml

SSC. A few drops of chloroform were added and it was stored at

4°C. •

2.3,2. Buoyant analytical density

gradient ultracentrifugation "

The extracted DNA was analysed by buoyant density ultracentrifuqation.

2.3.2.1. Theory of analytical density

• ■' dient ul tracentri fugati on

As 5-BUdR has been found to enter the DNA, and as bromine has a

higher molecular weight than the methyl group which it replaces, the

buoyant density of the substituted DNA has been found to increase

and this principle has been used by Meselson, Stahl and Vinograd '

(1957) to study molecular weights and partial specific volumes of

macromolecules.

h) The lysate was treated with Ribonuclease (RNase) (Bovine

Ribonuclease Grade II 5 x crystallised Miles-Seravac). The

enzyme was used at a final concentration of 50 i^g/ml and

allowed to act at 37°C for three hours (Marmur 1961). RNase

also partly digests itself (McCarthy 1968). The RNase was made

up as follows (Kalf and Grece 1968) :

At a concentration of 1 mg/ml in acetate buffer pH 4,5 and

incubated at 37°C for thirty minutes. Acetate buffer - Q,001M

sodium acetate (trihydrate) (Analytical Reagent BDH) + 0,001M

EDTA, pH was adjusted with concentrated glacial acetic acid. The

solution was heated at 80°C for ten minutes to inactivate any

DNase.

i) The lysate was dialysed at room temperature for four hours and

then overnight at 4°C (Marmur 1961). It was dialysed against

0,2% SSC + 0,15M NaCl at a pH of 5. ,

The dialysis tubing was handled only with rubber gloves, as

enzymes on the hands could denature the DNA. Dialysis removes

the detergent, enzymes and the products of enzyme degradation

(Kalf and Grece 1968).

j) The nucleic acids were precipated by addition of absolute ethyl

alcohol (Kalf and Grece 1968). The DNA was then centrifuged

down and dried in a vacuum for two hours and redissolved in 3ml

SSC. A few drops of chloroform were added and it was stored at

4°C. •

2 -3>2‘ Buoyant analytical density

gradTsnt ultracentn'fugation *

The extracted DNA was analysed by buoyant density ultracentrifugation.

2.3.2.1. Theory of analytical density

, gradient ultracentrifuoation

As 5-BUdR has been found to enter the DNA4 and as bromine has a

higher molecular weight than the methyl group which it replaces, the

buoyant density of the substituted DNA has been found to increase

and this principle has been used by Meselson, Stahl and Vinograd '

(1957) to study molecular weights and partial specific volumes of

macromolecules.

The method involves an observation of the equilibrium

distribution of the macromolecu! ar material in a. density gradient,

itself at equilibrium. The density gradient is established by the

sedimentation of a low molecular weight solute in a solution subject

to a constant centrifugal field. A solution of low molecular weight

is centrifuged until equilibrium is closely approached. The

opposing tendencies of sedimentation and diffusion have then

produced a stable concentration gradient of Lhc low molccular wpight

solute. The initial concentration of low molecular weight solute,

the eentrifug field strength and the length cf the liquid column

are chosen so that the range of density at equilibrium encompasses

the effective density of the macromolecu!nr mate. ial. At equilib­

rium, the reacromolecules reach positions at which the density of the

surrounding liquid is equal to their own, thus m a c m o l e c u l e s of

different easil y band at different positions in the ultra centrifuge

cell (Meselson, Stahl and Vi nograd 1557, and Heckman Review).

As the molecules sediment towards this bottom of the cell a trans­

parent solvent solution region is created and the image of this

portion of the cell becomes darter. Where the ultraviolet light is

absorbed by the DNA light bands will be seen. By means of a

densitometer the image on the film can be converted into a plot of

optical density versus distance from the axis of rotation (Meselson

and Stahl 1958 Beckman 1962).

The advantage of this optical system is that concentrations of

0,0012 of D M can be detected which is of the order of maqnitude

likely to be found in the experimental samples (Beckman Instruction

Manual E~ 114-3).

2.3.2.?,. ^ethod Jised

One of the most effective low molecular weight substances found for

DNA is CsCl, which combines with it to form the cncsiu... salt.

Meselson, Stahl and VinogrM (1957) chose it due to its high

solubility, high molecular weight and high density which allows the

obtaining of steep gradients, 1,25 to 1,90 g/ml. It also has a low

viscosity suitable (or rapid sedimentation, is non-toxic to DNA and

is soluble in water (Dirk::) and salt solutions (Szyhalski 1968).

The average buoyant density of UNA in CsCl is +/- 1,7 g/ml

and thus a caesium chloride solution was <„ade up at average density

1 ,699 g/ml at 25°C, t’e temperature used the ultracentrifuge

runs (Szybalski 1968).

'rhe experimental work was done: on a Beckman Model E Analytical

Ultrac^ntrifuge, A 30mm charcoal-filled Epon centrepiece was used as

CsCl is corrosive to aluminium (Vinograd and Hearst 1962). The

longer centrepiece was chosen, as low volumes of DNA of a certain

density were more likely to be detected. 0,2 to 0,5 ug enn be used

(Vinograd and Bruner 1966), A counter-balance of 30 mm v/as used.

The optical density of the sample v/as read on a Beckman

Ultraviolet Spectrophotometer at 260

.The sample was diluted to an optical density of approximately

0,65 with SSC. 2,85 g of CsCl (Optical Grade - Suprapur Merck) were

added and the sample made up to a weight of 5,09 g with SSC, The

refractive index of the solution was read off art Abbe Refractometer

and the density read off from t ties (Chervcnka 1969).

The upper plain window of the cell was replaced with a 2°

negative wedge window to correct for the light scatter caused by the

density of the solution (Vinograd and Hearst 1962).

The AN.E Rotor was used and the machine was run at 44 770 rpm

in order to allow the sedimentation of the macromolecules in the

dense gradient (Meselson, Stahl and Vinograd 1957, Grossman,

Goldring and Marmur 1969).

The duration of the run was eighteen hours and photographs were

taken at two hourly intervals with an exposure of two minutes.

The photographs were analysed in a Beckman RB Analytrol with a

Film Densitometer attachment.

The following three runs were performed :

Optical Refractive Density

Density Index CsCl

DNA CsCl

M m l )

(9/ml)

Volume

used

M L1. Water 3 hrs. 0,58 1,3991

2. 5-BUdR 5 hrs. 0,52 1,4000

3. 5-BUdR h* 5-FUdR 0,46 1,4005

5 hrs.

The photographs and densitometer traces are shown in section 3.4,

1.699

1.699

1,720

1.3

1,8

1.3

2.4 Investigation into the growth of tadpoles

‘ under different methods of feeding

It was decided .to investigate the -optimum growth conditions for the

tadpoles, as far as feeding was concerned, in both large trays (2

litre capacity normally used) and small dishes (200 ml capacity used

in this study due to scarcity of 5-FUdR).

Tadpoles were taken at stage 47 (NF stage) and placed into

trays of 2 litre capacity and large surface area. Sixty tadpoles

were placed in two litres of water as this was the concentration of

tadpoles used in the small (200 ml) dishes.

Experiment A

Food Pose Number of

Tadpoles

Commercial

baby food

(liver and

vegets

Complan

Yeast

Liquifry

1 9/day for 2 weeks

2 g/'de.y for rest

60

60

60

CO

Volume of

Water (litres)

2

22

Experiment B

Commercial

baby food

(liver and

vegctaMos)

Fish fond

(Tetram In)

Yeast

Liquifry

Lettuce

Dose

1 g/day for 2 weeks

2 9/day for rest

Number of

Tadpoles

60

60

60

60

60

Volume of

Water (litres)

2

2

2

2

2

63

Experiment C

Food

Commercial

baby food

(liver and

v e g e t

Yeast

Fish Food

(T c t r a m in )

Lettuce

Liquifry

Dose Number of

Tadpoles

0,16 g/day - V weeks

0 S32 g/day for rest

6

6

6

Volume of

Water (mis)

2C0

200

200

200

200

The tadpole'" were analysed for rate of growth once weekly by

counting thv, nuober of arrivals at any one stage (NF stage). The

results are shown in section 3.5.

64

£.5 Details of the chemicals used

and source -11st of suppliers

1. BLIdR, 5-Brcmo 2‘deoxyuridin?, 5 1gma Chemical Corporation,

2. 5-FUdR, 5-fluoro 2'deoxyuricHne kindly donated by Dr, Raw of

Roche Products (Pty) Limited,

3. Victoria Blue, 4R Merck, Art 8539.

4. ■■Tetramin, Lopis* Johannesburg, containing fish meat, fish roe,

fish liver, crayfish, water plants, mussel meat* brine shrimp*

wheat germ, cod liver* insect larvae, kelp,

5. Liver and vegetables, Licence Gerber Prod. Company, USA,

Purity Ricket and Column SA Ltd, containing modified maize

starch, liver, soya protein, tomatoes, yeast, onion extract,

caramel, iodised salt.

6. -Yeast* Anchor Yeast (Pty) Limited, Vlatt Street, Industrie,

OohnnnesburQ, .

7. Comp!an, Glaxo Allenbury's $A (Pty) Limited, Manchester Road,

K'adeville, containing protein 3lg» fat 16g, carbohydrate 44gs

calcium 850,'Jig, phosphorus 7S0isig, sodium 400mg, chloride 750sng,

potassium llOGawt, iron 7,7nig> iodine 44ug, Vit A 1100 units,

Vit Bj i,0mg.

8. CsCl, Art 2041 Merck 2525019 extrapur tm, 168,36,

9. Soluene 350 (Packard) is an extremely efficient solubiliser of

animal tissue. Since most of the Biological samples containing

radioactivity measured by liquid scintillation counting are not

readily soluble in the aromatic hydrocarbon based scintillation

solution (Toluene) special solubilisers are required to obtain

a hort'ogenous system for reproducible measurement of radioactivity.

10. Toluene scintillant is composed, of 100ml Permafluor III

(Packard) in 1 litre of folueno (Packard), The resulting

solution contains 5 ,0y PPO and 0,bg bi s~MSB per litre, PPO and

bis-MSB are highly efficient scintillators which will not form

quenching adducts with tissue solubilisers, aminos and acids,

11. Toluene mixture. The solubilising mixture was made up as

follows :

Porniafluor 111 Packard : 70 •>

Triton X 100 Packard : 3d.: ,

65

Perntafluor is a concentrated solution of stable efficient

scintillators (PPO and bis-MSB), It does not form quenching

adducts with tissue solubiliser, amines and acids, Triton X

100, especially purified for liquid scintillation counting,

is an efficient tissue solubiliser* so that the mixture of

Permafluor and Triton is an extremely efficient scintillant.

This mixture modified from Thornley 1971» was found to be the

most efficfc»;t method of detecting radioactivity in aqueous

biological samples. Samples were* incubated in the dark to allow

them to equilibrate and to reduce cherniluminescence to a

minimum (Packard handbook), .

12. Methyl 3H Ihytr.idino specific activity 18 Ci/mmol labels DNA.,3

13. 5 }6 H Uridine (methyl) specific activity 4U,0 Ci/mmol label A,

14. Liquifry tlo, 2 (liquifry Company Limited, Dorking),

15. Pregnyl (Orgr.non), This is a mammalian gonadotropin which in

the male fro:; stimulates the growth of the testes* sexual

maturityand release of the sperm9 as well as the formation of

dark nuptial pads on the insides of the fore-limbs. The

formation of the nuptial pads indicates that the males are in

mating condition,

16. EDTA, Ethyl one Diamine Tctra - acetic acid, di sodium salt,

Merck analytical reagent,

17. Sarkosyl tit. 30* sarkosyl NL97* sarkosyl 0, by kind donation of

Ci ha Go i

18. Isoamyl alcc-hol - 3 methyl butanol CgH^OH pure reagent Merck.

19. Pronase, Sjjhscus Miles.Soravac.

20. ftibomicler.., t Bovine Ribonucleaso Grade II 5X crystal! H o d

Miles Seravac.

21* Sodium Acetate, Analytical Reagent BOH.

3. RESULTS

The hind!itnb buds of the tadpoles swum in 5-FUdR were found to be

deforr J (see section 2.1) either in shape or lacking the full

number of digits or both. A method was devised to approximate the

degree of deformity of each limb, In the limb each defect was given

equal weighting as described on page 73. For convenience, the limbs

were divided into three types: adult limbs (56 - 65) were given a

count of 9.1 when normal, medium mature limbs (54 - 5b) were given a

count of 32 when normal and immature limbs were given a count of 24

when normal. From these normal counts points were removed depending

on the type of deformity and the result is referred to as the

deformity index for each limb.

3.1 Results of pilot experiments in which Xenopus laevis

tadpoles were swun in solutions of 5-FUdR and 5-BUdR

A series of initial exp r ments revealed the hindlimb deformities on

observation of the tadpoles under the dissecting microscope. These

tadpoles were subsequently photographed to show the deformities. The

photographs of these tadpoles are shown in the next few pagt#*. In

section 2.1 the limbs were further analysed by staining the

cartilage and taking magnified photographs of the result. Below, in

Figure 3.1, is a picture of a tadpole (NF stag-' <f0) with three toes

on the left hindlimb and two toes on the right hindlimb.

Figure 3.1 Dorsal view of stage 60 tadpole swim in O^uglmi

S-FUdR. A claw is visible on the third toe oj

the left foot.

In Figure 3.2 below are sketches of the hindlimbs of the above

tadpole compared to the sketch of the normal tadpole hindlimb for

that stage.

Fiqure 3.2 Sketch of the hindlimbs of the tadpole in Figuve----r.------------ - & '3.1 conrpaved to that of tthe normal stage GO hindlimb.

The tadpole in Fi g u r e 3.1 can be compared to the normal stage 60

tadpole shown in Figure 3.3 below.

Figure 3.3 Photograph of normal stage 60 tadpole.

This type of result was found consistently throughout the various

preliminary experiments as will be seen in the following photo­

graphs (Figures 3.4 to. 3.11),. A stage 53 tadpole was seen to have a

bifurcated hindiimb bud instead of the normal division into five

toes at this stage. See Figure 3.4 below.

Figure 3.4 L a t e r a l v iew o f phage 58 tad p o le swum i n ,. lOfcg/ml

5-BudR + %^{j/mi s-nm.

The tadpole in Figure 3.1 can be compared to the normal stage 60

tadpole shown in Figure 3.3 below.

1 1

*; 1 J ! «jf' -

Figure 3.3 Photograph of normal stage 60 tadpole.

This type of result was found consistently throughout the various

preliminary experiments as will be seen in the following photo­

graphs (Figures 3.4 to 3.31). A stage 53 tadpole was seen to have a

bifurcated hindiimb bud instead of the normal division into five

toes at this stag*. See Figure 3,4 below.

ti

Figure 3.4 Lateral view of stage 53 tadpole miun in .. 10/-<g/ml

5-BUdR •/- lf^g/ml 5-Fl

The same tadpole was-enlarged to show only the abdominal region so

that the hindlimb was clearly visible (see Figure 3.5 below).

Figure 3.5 Abdominal region of stage 53 tadpole enlarged to

show detail of bifurcated hindlimb.

Figure 3.6 Sketch of the above hindlimb with a normal

limb for ooinparison.

70

A photograph of the normal stage 53 tadpole is shown below

comparison to the photograph in Figure 3.4.

Figure 3.7 Photograph of normal- stage S3 tadpole

In Figure 3.8 below is a photograph of a stage 57 tadpole with

a hindlimb having three toes.

Figure 3.8 Lateral view of stage 5? tadpole swum in lOojJg/ml

5~BUdR + , OjJjHg/ml 5-FUdR. Only three toe $ c m

be seen.

71

The photograph in Figure 3.8 has been enlarged to show the

hindlimb more clearly.

Figure 3.9 Enlargement of the hindlimb region of the tadpole

in Figure 5.8 on page 70. Three toes are clearly seen.

A sketch is shown in Figure 3,10 below comparing the three-toed

stage 57 hindlimb to the normal stage 57 hindlimb.

Figure 3.10 Sketch showing the hindlimb from Figure 3.9 above

* compared to normal tadpole hindlimb.

In Figure 3.11 below an enlarged hindiimb of a normal stage 57

tadpole hindiimb'is seen for comparison to Figure 3.9.

Figure 3.11 Enlarged "hindiimb region of stage 5? tadpole

to show normal hindiimb

These preliminary results provided the basis for further exhaustive

experimentation. The hind/limbs were studied in greater detail by the

more refined method of cartilage staining (see section 2.1.2.2.) and

the limbs were analysed in detail in section 3.2.

3.2 Effect of 5-BUdR and 5-FUdR on tadpole

hindiimb development - a detailed analysis

The tadpoles were immersed in solutions of 5-BUdR and 5-FUdR* which

were renewed weekly (see section 2.1). The tadpoles were staged and

studied each week under the dissecting microscope, in order to

detect any deformities in the development of the hindiimb. The

progress of the deformity was traced until no further deformity

appeared. Subsequently the tadpoles were killed and the Hindiimbs

stained to make the cartilage clearly visible (see section 2.1)

These stained hindlimbs were photographed. The photographs were

analysed in a semi-quantitative manner, according to the degree of

the deformity. A penalty number was given which reflected the degree

of this deformity.

In order to attain a norv-weighted figure (overall) a measure of

the degree of deformity with regard to the temporal aspect of the

formation of the deformity was adopted. In this approach a score was

allocated which reflected the absence or magnitude of the deformity.

For convenience the developing hindlimb was classifed into three

stages :

1. The most mature limb (adult limb) in which the digits are

clearly visible and demarcated and all the cartilaginous

elements as would be seen in the adult limb are present, Stages

56 to 65. (See Figures 3.IE and 3.15.)

2. The medium mature limb in which the cartilage elements are not

clearly seen but demarcation of toes was visible. Stages 55 to

56. (See Figures 3.13 and 3.17.)

3. Immature limbs in which only a paddle was visible. Only

deformities in shape could be detected in these limbs. Stages

54.and younger. (See Figures 3.14 and 3.18.)

In this approach each element was awarded a point of 4 and from this

number a penalty of 2 v/as imposed on a deformed element, while a

penalty of 4 was imposed on an absent element. (Toescould be visible)

3.2,1. Adult hindlimb -

In this limb according to the point system above a total score of 91

was accumulated in the normal adult limb as follows :

Femur 4 .

Ti bio-fibula 4

Tibi ale fibiale 4

Metatarsals 20 for 5 elements = 20

Phalanges 56 2(2x4) + 2(3x4) -I- 4(4) * 16 + 24 + 16 = 56

Claws 3 (1 per claw)

Total 91

In a typical deformed adult limb one could calculate a penalty

index as follows :

bent (4-2)

bent (4-2)

mildly bent (4-1)

two absent (20-2(4))

digits 4 and 5 absent (56-4(4)-4(3)

Femur

Tibi0-fibula

Tibiale fibulare

Metatarsals

Phalanges

Total

22

3

12

28

47

This indicates approximately 50$ deformity. '

To illustrate these analyses more clearly a series of graphs

has been drawn alongside each limb photograph, next to the penalty

index analysis. In Figure 3.12 below a normal adult limb is shown on

this graph and below it a typical deformed limb as described above

with a penalty index of 47. ______

Deformity Indor.

= 91.

Kj:Y;

Pe FemurTF 'Tiblo F ibu laToFe Tibicile F ibu la reMi M e ta ta rsa l sP|t .Phalong-ecCl .Claw*' .

K IT T p e p o r m IT YPro lent

„'‘j Abiftnl — .4.*-.- M.svv/ B ad ly B tn t

fracas M11 <1! y - & '■<'' '

T V AW S •o PI ei crvl

A b ie n I

j_p .!IE-Wtmslnormi! rtotnui

i

• ; ’1 „ , i ■

J ? 1 'X -C.|....HoiVrM

’ • 1•

I !1

3 JX-. m l t o

N o r m a 1.1 imb

j D c ( o r m11 y Index i » 91 -11 :' = 69 '

noffnfll >,;"»•>* rnr/m 5 ^|{!iVlV| fei)«Lv Pm s

................................................. W ,

f 1bent?/ ;—.

1 teswS!

,4„j - 6 1-2 — V -

— r ! : ■ t \»wim■■»«» | Mtwu

....... i !, - + *- M * f t

sinpj,

JL__

4

Ulyii No

D e f o r m e d l i mb

Figure 3.12 Gmphiaal roproBoniaiion of novml adult

Hindiimb and a typical defonned hindiimb

3.2.2. Medium mature hindlimb

In this stage of limb development, although the metatarsals and

phalanges were present they were not clearly demarcated and could

not easily be counted. For this reason penalties in the distal

region of the hindlimb were only given for the presence or absence

of toes.

According to the point system a total score of 32 was

accumulated for the normal medium mature limb as follows :

Femur 4

Tibio-fibula 4

Tibiale fibulare 4

Toes 20 (5x4)

Total 32 •

In a typical deformed medium mature limb one could calculate a

deformity index as follows :

Femur 4

Tibio-fibula 4

Tibiale fibulare 2 slightly bent

T°es 12 (3x4) 2 toes missing

Total 22

This indicates approximately 33% deformity.

To illustrate these analyses more clearly, a series of graphs

has been drawn alongside each limb photographs next to the deformity

analysis. In Figure 3.13 below is a graphical representation of a

normal medium mature limb and below it the typical deformity

analysed above.

.Deform Ity liulox .32 7~ 0.= 3 2 .. - .

,52 - J= ?.5.

k e y : 1 - •

Te Femur TF ’ T 1 b I o F ibula Tefe Y lb la le Flbulcire Mt M e ta ta r s a l s

. '

K E Y j D E r O t U A l T Y

. P r o i o n t • • » '

............. / i b s o n I . r r . ' t — • -

I W i t H y B o u t — *

v m t ' M i i J ! y - C c n l !

C L A W S i -

o i* r c j o n I — - •

A b i o n 1 -------

N o r m a l I i m b

D e f o r m e d l i m b

Figure 3.13 Graphical representation of the normal medium

mature hindlini) and a typical deformed kindlimb

3,2.3. Immature hindlimb

In this stage of limb development deformities in early limb shape (

were noticed, which often went on to form the typically reduced

number of digits in the adult limb. Penalties were given only for

shape and possible absence of toes judged by indentations on the

paddle at stage 53 only. According to the point system a total of 24

was accumulated for the normal immature limb as follows :

Shape 4 ,

Toes 20 (5x4)

Total 24 -

In a typical deformed immature limb one could calculate a deformity

index as follows :

Shape 1 1imb very bent

Toes 16 (4x4) stage 53 smaller paddle in width i,e*

one toe less

Total 17

A 28% deformity. '

To illustr&te these analyses more clearly a series of g/aphs^

has been drawn alongside each limb photograph, next to the deformity

analysis. In Figure 3.14 below is a sketch of a normal immature limb

and below it the'deformity analysed above.

D o fo rm i!y In d o *. . . . ..2 4 0 „

„S . Sh«pe

T,..ToesST

Wtsiing | 3

s -2

T - 8

Figure 3.14 Graphical representation of the normal immature

hindlimb mid a typical deformed hindlimb

This detailed analysis is summarised as follows :

There are three classes of limb development for this analysis,

The earlier two classes were analysed to show the on-going progress

of the deformity while the adult class was a detailed analysis of

the deformities themselves. This semi-quantitative approach gave

interesting results even though the deformities were not found to be

consistent either with stage or concentration.

The detailed analysis of the limb deformities is carried out in

3.2.5. for adult limbs, 3.2.6. for medium mature limbs and 3.2.7.

for immature limbs.

The results show limbs with one or more digits missing. In the

thirty cases of adult limbs analysed in detail :

Digit 1 missing

Digit 2 missing

Digit 3 missing

Digit 4 missing

Digit 5 missing

23

13

6

16

16

30 cases

30 cases

30 cases

30 cases

30 cases.

3.2.4. Analysis of normal hindlimbs

Figure 3.15 Normal hindlimbs

79i

1 s j iopa

I 1 O 0 s’. 'I,— '

Normal S tage 50

. ■

■t-w

iN.ormal Stage 51

Nqi m. o I Slag o 5 2

T t o o sf-

Normcii S tage . 50

|N.ormal Stage 51

AKtU.iU.flJ S ta g e 5 2

'^psr

k e y :— -------------- j

1C C Y :pEFORMl T YFe Femur _______ P K M e n i

TF ’Tlbio F ibu la ____ "j A b t o n l - t . 4 .

Te Fe Tibia lo F lb u la r e , f B o o ’ l y Bonl 2

Mt M e ta ta r s a l s r r e r w t M i l d l y - C e n t 1

Morm oi. Stoge 54

n Q f n t a l

XLoof mnlX?£a J U _ L £ i

normal«normal

, i1 i 1

ii- 23 \A

>Dl9t( Ho

.Normal S to g g 55

Norm a] Stage 57

JE—

igrm.ii noiin.ilT£_ Ic.Lfi

no f Hint normal,normalJJ.U.norma)

J U j X

r s o / m a j

” p h >t 1 c i .

n o r m a l * n o , r m a 1 ;

r > . - ............................. K !0 .

1 '* *

1 ■ 3 <

n i • !i 4

'Digit No|

KE'FaI F

Fern ii r'T ib !® F i b u l a KEY:DEF0RIAITY

TeF© T lb la lc r-lbulare t Z T * P'* * * "1Mt M e ta l arsa Is jPh .Phctlnngas C l , Cl aw 4 JESSrf

Absent

Dotlly Ml |d ly . n*n< "" 1

B e n t 1

N.ojipg} Stag^ _S8.

_UL_ Jste- P h J I I Jill-2 J- ti'- S- normal norma! normaj

. . P h 4 _

j l f l o V m a i normal

Ifcior ma I,..Stags 5 9

a —Jncitmal

T P Tor-f t (4 I __r t o r n ' s l ■ y o f r n . i l normal

i i

EhJ—normal normal

P h Jnormal

rr>- I*

_PJiAnormal norma)

X " DTgjt^o

3.2.5. Detailed analysis of adult hindlimbs

Figure 3.16 Adult hindlimbs

k e y : - • KEYsFa Pom u r ■

1 t r- •T lb lo f lbulo ......; Te l:o Tlb lo le r*lbu are W>V’w

Wt M e to la rs a l s ?;*kzl, Ph ph a la n g es • -; c i . C1 a w ^ •

P I o I c n I •

Alii nn I

B a d l y ' n o " 1— 7

C t AW-SP | 0 I B 1 *

A In o n I .t : '■

1 ADULT LIMBS 83*

S ta r t vlctgo 4 7 0 01 , m t) /in | C-llUdft 20 jta /mI 5-FUdR

Lt—nQimal

EU—i 4Hannnl •ijilnl' 7­

2 J —:T4 ( —IB —16 1 • * J*...... |»* w J.

4 t . , *■«***«* . L... J— ’»'*** i,|' . ; i _ . ’

ill' 2.i» i s smn

A

P.Ji.3..riissii'O

3

PM_.unssii’g

'12

Leu

X ! ______issir.n 3 ____‘

■* 3

i . \

' I

6 6

...... v-

t

0(

. A - .

JLDigit Not

D e fo rm ity indexS tti r I Hugo <1 7 Oj.01 mg / in I 5- BUdt?

l o ]\9 /ml 5-FUdR Right

normalJL jr-

aatmai'i.o I" e I„.M t

t-z'U l4

i----«:

ELb.Ji___ EJiA

' 5-16

mssino 5

_PJl_3_iuS5(H<i

3

- uy-r- j—

wwVWW**,

li___'U---I]___

_ - 1-6 i — ■ 0

-o

-12

I’Mmissing

Imssinn.

3

I 2

o ♦ 3 t, J 4

I --Digit No

D o I o r m I ly IruJo x — .9.1 " 8 1

=z 4

S ta rt stage 49 7,0^9 /ml 5-FUdR

Loft

E z n r i i ^ i U uj HQ fm ilts5• nT.'»s« n«,sSin.

-20

ELhJ—*ru$fl!/x

-2 0

mmi s S»f*P

5

L.M,tit 5 smg

3

J P J l J —missing

X

-20— 0

-12

OISS1I13,3 I

I

U

j. I**

9 ■ V - oi;--- i"

Iu .«*«♦*»»y *Vtw ‘ Digit No

}

D o ( o r m 11 y I n cf o % 7.1 t * 7 .

Stci rt sU tgo <1?20/^cj /m l 5-FUdR

\

1 Z I E P 3 5 S

r i= T - ( ;

Highl

j 5u Z u u J I k £ wp jt j L ;»ni,ssin» •nus'M <<\l S si'll' if)1 s.sidy miosit'o

4 i 5 5 3“ l r ~” : .1 6 "*•*20* — 2 0 - 1 T

♦ _ — — O ' . i

pa*M jI— — 4“" ^I___ L--- • -U-r

Is z : ■r r-?“

o

lyssiry ; 3 i

“ 3_ j _■ J L ._ L _ l ' t.

--01 git No

\

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k_p y ; . .

Fe Femur T!: *T|blo N b u la TePe Tib la Id f l bulcire Mi M o I a I a r s a 1 s 4Ph /Miolcmges C l. .C la w i •

l( E V : D E |! 0 R M I T Y ___P io i on i . ...

AbtenI .rr.'fv/vWr'’ fiotlly Ban I *“2

jssass M11 <J | y. I iU —* i

Deformity I ndox 91 “ 73

' ~ 18

S,t a r t . s I o g 0 4 £ I0 i|cj/m l (>•* PU ilR

X L___________ Top.nl M i__

^—«<»»’ sjiAg

*2 i-16

Left

Elut.tnissiia

4'16

I:

---ll_ . . >11----J.

Deformity Index .9 1 " 7 3

: 18

>«i s 5in'j5

i ’J 'A .ttlssing

3

- M J . .ttifS isis/1

1, c . l _rn ss> rig ,

2 i“ 20 -12 — 4 — ST , *■

/*: - » 1WW***. $ • - ■ ■ - j. ?

■ I . J L LI . . . j 4I

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j 15

f

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Sl a f |. stage 4,5 j ,0p « / m l 5 ~ f U ciR I

x Z J l ^ i X M j J ^ Z L C u i -lnQiniag’c'{idt!r’l>“i'iV” *B«nly2'H,5S"ltn1'Sam9

Wght-Cl .hissing

s i V. ,

,1 1: 2

3 t,...4 .

Digit No!«?w;wtpw*D e fo rm ity I it d o >c

?1 ~ 3 1 .60

S t a r t stag a 43

° ) 2 ^ g / , n l 5-PtJdR

JL JJL Jiulcl-Mlocjfftiat <roim,iI g'gjfl* Wfln.j Ju,j JihX, f* It 1

«> i 5«n*.tiissidii2 1

—8 - 4

D 0 1 o t m 11 y ! n d x . 5>,1 .“ 4 6

S 1 n r t tu ifja /IS 0)2MS/ml 5‘PUr)R

- ■. ..J L J .L - .-lJ !^ ■....... . U

X K E Y : DH l: 0 R W ! T Y

p ( C, 10 It I . *

4 .A b t c n I

iv y ;(: o " FemurTP ' Tlblo F i b u l a ^Tero Tlblcile F lbu iaro L j , Dc.diy Ben' ' ~ 7l

~ i <-i ( m rr.Cl I S ' iffflEKMl IA l i ( l ! y - l ic n l_____ . 1

C LAW 5ji" I' I C1 0 n <\V.. , A b 1 O f> l — I

k e y : ' *,Fe> F e m u rTl: ‘Tlblo F ibu la , to !:e T Ib lc i l e P f b u l a r eMl M o t a t a i ft ti I sf’l) (P Ik i tangosC l . ,CIaw

D o { o r m 11 y Index 91. — 43

= 42

K E Y : Di- FO R M IT Vf‘ / o i o n t

. . . » A 1j i o n ( c tA ..VWV*1 Hail I y Bon I “ " 2

3SKTJ M i l d l y Bcnl — 1

C LA W s ! o I' f 0 I n n l

U A b t o n I ~ I ,

86

Start s t ci y o 4 8 20 US/ml 5-FUdR

■ r X L03 f fU«< I

IAE js. J jLL- fib J.J.P.I).?..normal (iSS'iyj 8^HiL,«'lssin3

__ m I i MI«V \ 3- Il i Q . l _ J r

I MWy | --- * |• • I

t i ' Xnussirg

3 1 2 ~

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A

£ 1 _ , rjijsing.

■ A>

Hanoi | ttmnsr 0IiT

J

I

J ■L.5— .- D i t| 11 Ui>

D.atorml ty Intlcx 9.1 — 4 9 „

= 4 1 .

Stcirt s l ct g ft 4 ii 2 0 /Ag /m l 5 ‘ PU d R

____ —- . r

. ■ ! 'I . . * i * * > ,! * :

i ;= 1

:•

1

! . ■ - |

“ 11 - 1

U l - . J : ....... 1

D o l o t m l l y lnc>.‘ )- .9.1 ” 57.

"- ■T"'1 ; , 3 4

Right

i t t A ,t n !VU .1K'-"' 1 ’ I «Mllmi lljty Vt fll 'l .3____?

-12’I4 tntJSina ’

I H i x <

Ph.-t\ l Hints ftilssing

2 1~ e — 4

(Y>7rA

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t A f * .....I.■*"**. I —

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t . I

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o

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n>«ma 3__

~3

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•o 3 _ t

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& o f o r m 11 y Index5> 1 — 56

.. ~ 35 *

■ fcAw'.J'fct ^

„S la r t clugo 4<52 0 fU) /ml 5 -r-UdR

\yrn.>»ii j : „ .

di 6S r-'U5S\V(— Ip M !* . . « * M ^ U i~ .

? . . "1 2 ^ 1.2 ! ! z l 6 a;- ;■ * * I >A*Am | ft8tf5W»i | — f . JI /VVWv . laftocsK ( * I ’1 1 1 r' . 11 . Bmnvu 1 hBSB9

| w — ' ^ L _ ,— : 1—

I ...1“ '" irr*:.!— ,.i;.... J : . . :

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JLr - V

C — 111 rii nrnight |

.Ci.Oiiiii^g.,,

3 I "3

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!— ?t—

:■ A..

: ()|tiU No

>

k e y :1

r a Foin 11 rT l: •Tlblo P l b u U )

!To I:g Tlblct lo r i b u l c o o

■Ml M 0. (ct I a r s a Is

n (Phc i lc inoosc . .C l o w i ,

K E Y t PE r -OKMI TYi ,i;. p f <r t c n i .

A b ie n l --‘.'J -

nodiy 1,0,11'P5?»!) Ml NUy. tlent r

[Do (o r m 1 iy Index ? j - 2 6 ~ 65

C l AW S[■ r 11 f n I

. ,A 1)1 «i.n I

TF To Fo M t

■format piss mg )

Ph1

fni ssi nq l

Pit 2

miss int I

Ph3

nissmg 2

S I 0 r I s I a g q 4 7

0,01 mg/ml 5-BUdR 10ug/ ml 5 - f U d R

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■tissing\

Cl

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I i 2i i

I ,

D o l o r m l t y h itl«v .. 9.1 ~ ,2,6.

.:■==• 65

normalTP.

nstmalTcFe

riOftrlfilML.nvissin ■ !

5 l a r i via go »1 7O.JM.mtj/m l.ir-fludR

5 - F U d R *

‘t 3— JG Z" 1 BfgTl No

Right

EJxJL.fni53trr*

£1) J2„

\

I h.5,.r.tSRintj

i8

Ph;l..missmij1

£L

<33Ifr

•V

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D e f o r m i t y Index 7.1 ™ 26

JL

JL

■ * 65JLsssas „ . * -

r Y i L L j I l *rxjfmai fuimnt riormat

i ; ; . j : 1I ' - : . - L _ _ i

Sior l . Mage 47“ ' mi 6— ru tiR

Digit No

'Xcft ]

m 1.1 Sir. 1

i; 4

eiurniss1 miitiijg 4 1 - 5

I l i X(01l iSing iflusi«<3

7 <- a — 4

i!L M

1 i

r1 lln 0

% I.,

!

J— J—l

I ifWOHj

5 tor i Mago 47

iO^g/ml 5— ru UR

Digit No

jgr"**

87

—>»t—riV.mxn.0".

k k y : 1

V " l:0im/rr r ' TIb 1 o F ibu laTe Po Tlblcile IMbulareMl M o I a 1 d rs a 15

fJ h h ci 1 a n {1 e sCl . .Claws

KGYfPE r ORMl TY----.--P t o i on I

. _ A b i o n I •tt.'S

WAV* U tiJly B o n l“

WKEtf M l |tl )y . (lent ~

D o ( o r m I ( \> I n tl o x ...?1 - - 3 4 . . .

< = 57 _

C LAW S i .O' I’loionl . . .

w '.. A 1) i a n I ]...

S iu n Etcirjo 5220 qg /ml 5-PUdR

88

[ U f t

X X L XsJLa.ftO(rn.il

,,.L.

iI <_ I !I (___

Mj . i ..

, M j . J R l i J __ tM> 2_{H}aivi:m,.«.ra«r-miSSir«. -

I

6fifty*

8(THssir;^

£ i'A .missing

1

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14 j

I. - d

Cl__rt 1901 nQ

3~3 " J ____

ii •« foam ,

j I

D e f o r m i t y Intlox . 30 .* = 53

’ 3 i . . A

_5■Ltlcill (4oj

S (a .r .L s ttig o 52 2 0 ju$ / m 5-PUdR

nqimaiTF

rdirl&l-U‘J>

cie»f m«J

I Right

tvn s1 - .7l»ra«s(} tuannr L

H J3 L . 8

a.- 8‘

£ k L-t>, ssmg

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njtSSing_ J _■— 4 “

Iod

i •O'

' | i; ., i... •'**• ja* ■ <-»*,, ««*» >t

D e f o r m H f In t lo x „ ,9,1 - r.4 5

: = . 4 6

fltSMriJ,.;3 t

•3~~-1___ J23 t

4— , I- • -1

--Dig ,S"fo r i ,s I o g o 52 ,

2.0po /ml 5-FUdR

ri>qirua<

X L

1!i * i!

j .

mii iiijiji

- 12-' i--~l/vwv j- |/Vw !*■ /W*A* , •

I AVMVt

E X r t T J J i l a ”lic'nl't mtSMf-g

sj-

s

miiSipmisfioji

6 1-14 ‘

D eform ity Intlox . P.1 45 .

'= i 46**>«» **>»

ngtmolX L ™fiormAl

i^.llanor mrtl

f-

|V1 <11.V, is,M(>3|m i iM ^

■12 i“ ~6

V., I .

| AVVA 1.. ... .| j»-—_I tow * I*——_<

I /VVN\ I

lk*wv l*» j—* W«#

S iu r I .s ia^ o G2 20 pg /in 1 5-PUdU

Ph, i iMt iT l j ’T ?• n i H v . h n d i u 1 1

2

I*JI

:

f

k e y : I — —

Fe FemurTF ' T! b I o F1 b v 1 clToFe Tlbldl-e f:|butar-eMl M c 1 a l a i s a i sn ,P h a I a i] (j-e sCl. ,C 1 ci w si

K 1: V : DEFORM I T YI't o 11- n I , ...

___ A b i on I ~ t A ... ,

HUA^ Dudly Beni--?.wasa Milfljy. &»«< ~ l

Detor l i i l t v Index S>1 - 3 9

— 52 ...

_E— JJ ... ..UP.Uu.il floim

unsung lOisiigj ! -8 1-6

i .

2 -- 1 0 1- o... «.... rr-k.. . __i,-

C LAW 5 : © Pioicnl .

A b t o n I !.

89

S.tci r t s l a p j o_ 0 ... ID uy / ni I 5— I’U lU!

left

ii •I -

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ftQ

j : j .__rniMif'g n < svt ng.,...1 ' 3 i

“ 3,.J.__j

■ ; . __■ ; 3 v.■ . . A

j | /iiwm, | ftsw. [ ««*(\

D oto i ni I ty Index .9 1 .” .39.

• — 52 . ..

MoS.Ut.r t „ static-. 0 .

1 0 ^ ,/ m l 5 ~ TU ilR

l i _ Tr-I.uf| Ofmil

.: i.

f •lL _1?

laiJE.uZDiD7-hastyn.tvdiy iBtmi rf.htntT /;,benr J '

“ 8 I “ 6 1-10—__J ^ ... f*v_____i _____ u,

C)ii

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S,J>A. Mi •(oi**nr«5, i t l lS H '.g

? f

— 8 — 4

I i ' , i:

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• o i

t f 1 «•

Deformity Index .9.1 - 30.

61

S fa.r.t . slag c. 0,.10;.ui / m l S - P U t l R

oztrnz f ncm.n l & t l f e >•»»**^ *** 91 n>t s5«r mi v s»ucJtfSv & j >nq

. 1“ 7.6' j-S 1-6 1-5>•1a

. ; . i.

D e Io r m11y Index „9 ,1 — .3 0

.-61.-

Lo't

M )£ .. .CJ_____n ssmg ,

1 ' 3 !-■4 - 3 ;>. ...... r '. . . . • 7

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: 45•■* ...J•Digit No

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i ' ‘( ’ *

R'Ciw

IL.

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6 I 5 1 ’ 5 I 4

— ' ~~_i~~~~ I l ___|._____j___ _■; i .1

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C L ._ ..

3 !:'3~ ”

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<1 ”.... ___

••• f1 i fj i! Is'n

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khy; .f'« Fem ur Tl: ' T l h l o F i b u l a

To Fe> T lb la l f t F| bular-e Mi M o l a ( a re a Is Pli JMia lcuu je ' j C !. ,Cla\vi * •

K E V i D E F O R M l T Y | C L A W 5~~— , f*( o ion I . .

Abienl “ <4____*W*Vtv 11 a <! I y Bon! 2

IlSEtSEl M t l d i f Si oil I ]

> P ( c m n l

(1™ A b i c.n I 1,,

90

D e f o r m i t y I » cl e x ?,1 “ 34

.57..

S.tci u .,.s tago 52 , <0/.^/ m l 5- FU ii K

■r Right

' 3.2.

Figure 3.

3. Detailed analysis of medium mature Hindiimbs

L7 Medium mature hindlimks

\

f

ft

2 M E D I U M M A T U R j

k u y :t:-e Femur TP 'T lblo Fibulu ToFo Tlb ia le Flbulcne Ml M e t a t a r s a l s

Dofo r tn ( ty Index.12 ~ U

J'.o. .. .

KEY: DEFORMITY ______ I’ i •> i o n I

_____ Mis cii I. 77A —

tloJly Bont-

355tifd fAi Id !y* ,u‘n* "

S ( a r i s logo 4 5 0,01 mtj/ 111)

■ ’s-nucJR

Led

.Deform 11 y Index .32 .-.12

_“ 20_. ■

lr t fnptnjisl normal no* mo-*

• { . .

S l " t l s t ago 43 O./.OI.mtj/inl &-IlUrfRt O/jljng/m.1 5"FUdR

Right

....... l

t ' T

D Cto tm t ty i title x,5 2 J 3

19 ._____ j i . J - 'nuti»o)S!c}i i.tnoK,>!l1

S t a r t Mc)t}0 4 7IO^ci / ml C—_ru cfl-f

Left

„De(o rm I t y Index i 3 2 ~ r . t4 .! = 18 .

S t a r t t i <i a o 4 1 lQ^tt/ mI 5— I U ill?

Right

J.ef «miid I y.

e,n i - - mi :• s i 3

t.J

n

2 M ED lUfA M ATUR E L I M B S92

k i3 y : 1 -

F'tt !:c mu r

TP ’TIbIo !:lljulci

To no T I! j 1 a I e l: l b u l a r e Mt Molctlcirsuls

D clo r m 11 y 1 ncto x . 1 2—12

. . 2 0 . ......

K E Y : P C i : O R M I T Y. ™ ~ , (’ t '■ i (i n 1

...... A lii on I. rr.4- ,CJ I y B o o t “ 2

SH5ST3 (A i I i! ! y ■ I1 cn1 ~ '

; S t ci r i stciga <13 o.,.01. mg/ml &-«Ur)R 0/2 / nil 5-FUdR

Loft

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nsjim al r>of m al

* *" I

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_ 3 _ ?-12

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1

,D o lo rni H y I ndex . S 2 ,~ .1 2

*i=. 20

S t a r t stag a 43 O.j.Ol.mtj/m I 5-SiUtlR) 0,'Zjt g/ml S'FUciR

Right

j :r_ . i

Deform l l y 1 ntlox .3 2 .- 1 3

— 1_9 _

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rt i -

t il!

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in « '3 (

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, 3 . , 1

4

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Loft

P e(p r m It y I ndox 3 2 ~.-.14 .

18

M o i l vt( igo 4 7 10 f.\g /_m I 5 *- )■ U tl f!

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1 .

mi** inn’'3 i*12

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1 ftKOtfUl

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B3

ii k e y :

Pci F emu r TP T lb lo i; IbulciTe Te Tibialo F ibula re sodiy non 1 — 2Ml M e t a t a r s a l s ' " ~ ‘ 'Deform 11 y I nde x

. S 2 ~ .1 3 = 19

l( E Y : DE T OR M I TV 1* I 0 * 0 !• J

..... A ti 10 ri I -~A6

M ild j y B cf> * 1

S ta r,i ,.s tog 0 52,,20 /ml 5-FUdR

Left

1 , r... TF 1taUeM-yu.ojmisl ■aJ-. rr12.

r -

J . ___X __3 (

■ .. ;_j Mo

u d o i m 11 y I ncicx , . ,52 8.,= '24

S t a r t staao 43 Ot 2 pcj/'ml 5-FUdR 0- ;0Lm y/ id! S-ftUcJR,

Left□

i-LL...

natmiii-sL-

l-*8

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JLX

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,D o(o r m 11 y t ode x .5 2 r : . 8

24

Start tuigo <15

0*2 fig/m l 5"FUttR/ml C“ BUtt«

Riflht

34

k e y : ‘ ••rft Femur TP ‘Tlblo Plbulo

;Vol;« Tlbiulo Pi bulare •M» Metutuisab

K E Y : D E F O R M I T Y„ „ P r c i o n J . .

. . . A l> i e n I --Otttily I ^

W 5SS*' M l l ' U y . Dcnl - I

D o to r m i I y I nciox .5 2 .”10 ...

.= 16

Sta rt si ago 4?2 0 /mI S-FUdR

Left

L E LTEjJe.r.eBan |V--.no/n><ll

-2t

V*rt«s ftVn

T JL

Dcto i m I ty Index .5 2 ~ 7

= 25

Sta rt s t age 49 AiS /ml 5-FUdR

Right

“ ■-saw-srDole rm I t y Index

, S 2 — .6 „= 26

s ! a r i 11 a f| o <1 y 2 0 j.(ci /ml 5-FUdR

u, Left

! - V ‘ F J I F I TeFff

,-1 r-,1 .

' I

\tk4*. i-..

I J ._ J . _ _ L 3. »

rrulitTyj j. on i,,;..j ;.lT .fo il

■porffl.il

'*1;■ it.!-1 ....... 1 . . . .

Jslfc.fftissmi ■ I > - 4

l J U .

1 A

igps*

1 J ...*i ^

L l — . .

!0TrjT?Ho •

e

D ofo t m I t y 1 iicJ dx , .5 2 “ .6 .~ 2 6 , . .

' S t a l l i.(ao o 47 '20 }tg /m 1 5-FUdR

.............

Right

K

k e y : 1I'o Femur

T F ’ Tlblo r-1 bu ln ,To|‘e Tiblcile F ibu la reM t M o t a t ci r s ci I s

K t: Y : D F. r 0 R / A1 T Y, I’ I 0 I 0 ft I

. . „ _ A ti ( o n I , T , ‘1 . ■

yWANV Bvdly I ' " I I1— 7

msms* i/ .lN ly - CH' 1 “ ’

D o l o r m i l y I ndex ..,3 2 . “ 14 .

. ~ . 18 _____

M.i.•>QC

.1 i..

S Ui r t s ( a tj o 54 , ; ! i ° | i i j/ m i 5 - r-u.iiR

712

D cto

.. .. "i

r

rm 11 y 2 -r .1

r r |met «K-S'.y *

t : * •~2 ‘

* ,J i

i: i 1l ; _ 2 ____i • 3 l

" ™

JliCLlU iQ1 ' '

S.t o x 1 s Ui g o mi 5-

- X,,,, „ .ru ciR

' '-, * 0 |_ ___ i

I -.12 r , _ _ _

lilL

WrA>(V>VrA *-.3,.(

■*01«11 Ue

° o ( o rm I l y I ltd ox >32 — 4.

J = 28

SlOri slqyo/jp20j^g /nil 5-FUtir

* ‘ " ■* * ■■

y . J r__ -Ur.c.no, my.

'.V,v i:;. j

. :v 1:::■LLL 1

.::;: I.; ■: r— j . . . . . . L _

D o(o rm I ly I ndox 32 — 4.

— 28

* "—•Olyil J\'o____

S t ci r 1 1 1 ct p u 4 0 ,20||g /ml 'i-FUdR

'Urtwir„ F __ JJF .-.

----- ----- -—------

fiyinml rwrrfltti mifiTify ; ..1 1 * 1

I, " T ~ “

■ . 1 , . ; ~ 7 | ,1... '

• ■ .. . [ w ^__ , U lL

-J. J' Digit Koj

eft

H"- 111

Hight

k e y : -Pe Femur TP 'Tlblo F ibu la To Fe Tlblcilo. F lbu la re Ml Me let tars ci IsD q I o rm 11y I ik I ox

, 3 2 ~ 4 .= 28

IC E Yi PEf-OKMI T YPromnl

A b t on l .rr .4 -

Radiy bent-"Mildly H on I ~

Star t sUig o <1 8 2 0 (./a /ml 5 -FUdR

Led

D o 1o r m 11 y I ndex . 5 2 — .4

= 28

Stor i s l ot ) o4B 2 0 ff j /r,i I 5-PUdR

r

F T F J S lE a MjL-.iQimai fO«» I rvorm,\l r\ i,j »iivj

- 4 j

■' ' '

---- - :

- ch r r . J

x

DHilt Mo,,«-v— J —

Author Christie C AName of thesis The effect of 5-bromodeoxyuridine and 5-fluorodeoxyuridine on differentiation and metamorphosis in Xenopus Laevis Tadpoles 1982

PUBLISHER:University of the Witwatersrand, Johannesburg

©2013

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