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A silver-staining s t udy o f the nucleolus organizer regions
of the chromo somes of dome s t i c sheep (Ovis a ries )
A thesis presented in partial fulfilment o f the requirements
for the degree of Doctor of Philo sophy in Genetics
at Massey University
Le igh Marian Henderson
March 1 9 7 9
ABSTRACT
A s tudy was made of various aspects o f the nucleolus o rganizer
regions (NORs ) on sheep mi totic chromosomes , using the Ag-AS and
Ag-I techniques .
The sheep used in this s t udy were carriers o f
the t , t and t Rober t sonian translocat ions in various hetero ­
3
1
2
zygous and homozygous comb inations . The lo cat ions o f the NORs
wer e determined to be a t the telomeres on the metacentric
chromo somes l p , 2q and 3q and on the a c ro c entric chromosomes 4
and 25 .
The iden t i ty o f the NO-chromosome was confirmed by a
duplicate G-banding and Ag-1 technique .
The terminal loca t ion
o f the NORs ind icates that they do no t have a causat ive role
.
in acrocentric associat ion or Robertsonian translocat ion in
domestic sheep .
They also do no t co inc ide with the regions in
whi ch secondary const rictions are some t imes s een .
Individual animals were found to have a characteristic
s i lver-s taining p a t tern .
The chromo somal distribut ion o f the Ag­
s ta ining NORs (Ag-NORs ) , the frequency and size of the Ag-NORs
were fairly cons tant in different cells o f an individual and in
r eplicate cul t ures of an animal .
Dif ferences between cells
and replicates in Ag-staining involved variat ion in the frequency
o f staining o f Ag-NORs with small Ag-depo sits .
Overall , the me ta­
centrics had the highest frequency of Ag-NORs , fo llowed by
chromosome 25 and then chromo some 4 .
The asso ciat ion frequency o f individual NO-chromosomes was
found to b e p o s i t ively correlat ed with the frequency o f Ag-NORs
and the s i z e o f the Ag-deposit .
The frequency of each pairwise
combinat ion of associating chromosomes was determined solely by the
frequency of Ag-NORs of the component chromo somes .
found for a non-randJm fusion o f NORs .
No evidence was
These obser?a tions p rovide
an explanat ion of the reports of non-random partic ipat ion o f
a c ro centric chromosomes in satellite association i n man .
The
increased association o f NO-chromo some s with large depo s i t s could
b e due to an increased chance o f fusion of larger nucleo l i o r to
differences in the disintegra t i on rate o f dif ferent s i z ed nucleo l i .
iii
The presence of a NOR on the chromo some 25 / t
polymorphism
3
was utilized in inher itance studies of Ag-stainability . Five
pedigree group s in which this chromo some was segregat ing were
s tudied and in all cas es the s ize o f the Ag-deposits and
f requency of s taining was cons istent be tween consecutive generations .
The value o f Ag-staining in genetic mapp ing s tudies is discus sed .
In 3 anima l s , the Ag-staining pat te rns of trans formed lympho­
cytes and fib rob lasts were compared and found to be similar .
A comparat ive study was made o f 5 species o f the Bovidae:
domes t ic sheep ( Ovis aries ) , dome s t i c goa t ( Capra hircus ) ,
aoudad (Amme tra�us lervia ) , bharal (Pseudo is nayaur ) and cattle
(Bos taurus ) .
Five NO-chromo some pairs were found in sheep ,
goat , aoudad and cattle and these chromo s omes have homologo us
b anding pat terns .
The bharal has a t lea s t four NO-chromo some
pairs homologo us to sheep .
These results indicate a conserva t ion
of the NORs dur ing evo lut ion in this family .
Based on the resul t s of the f requency o f Ag-staining
o f NORs , the heritability of Ag-s tainab il ity and the cons tancy
o f Ag-s taining pat terns found in the t is sue s tudies , the nature
of the Ag-s t a ining is discus sed and a mo del on the basis of the Ag­
s ta inability o f individual chromo somes i s presented .
iv
ACKNOWLEDGEMENTS
I would l ike t o t hank s incerely Professor A . N . Bru�re for
his supervis ion of this proj ec t and his encouragement and help
throughout its durat ion .
I am grateful t o Dr Helen Chapman for
her useful discus s ions during the early part of this proj ec t .
I woul d also l ike to thank Dr A . Wilkins for his very helpful
d is cussions of parts of this thes is .
Pris c illa Ell is provided competent technical as s is t ance wi th
this work, for which I am very appreciat ive .
I woul d like to
thank Mr T . Law for his help with photography .
Dr R. Harris
p r ovided assis tance with the s ta t is t ical analyses in this thes is .
Karen Armitage aided in the final preparat ion of the photographs
in this thes is .
I am very grateful to the farm s t a f f of the Mas s ey No 2 Sheep
Unit for their work in the ma intenance of the experimental flock
I would like to thank
and their a s s is tance in blood colle c tion .
all the s ta f f of the Veterinary Cl inical Sc iences Department who
have been so obl iging in as s i s t ing me in collecting blood, ob taining
samples from surgery and general a s s is tance in other aspec ts of this
work .
I am gra te ful to Angela Lowe and Mrs
of this manuscrip t .
J.
Pearce for typing drafts
I apprec ia te very much the t ime and ef fort put
into typ ing the final manuscrip t b y Val Darroch .
This work was funded by a demons t ratorship in the Department of
Microb iology and Genet ic s and I am grateful to Professor D . Bacon
for making this p os icion available t o me .
Finally , my thanks go to Neville who has provided so much
encouragement and help throughout the course of this work .
V
TABLE
OF
CONTENTS
Page
iv
Acknowledgements
V
Tabl e o f Contents
xii
Lis t of Tables
Lis t of figures
Chapter 1 :
Introduc tion
Chap ter 2 :
S t ructure and Identif icat ion of Nucleolus
�
Organizer Regions in sheep
2.1.
x iv
1
4
Int roduct ion
4
2.1.1
The nucleolus
4
2_. 1 . 2
The fo rma tion o f the nuc leolus on specific
chromosome regions
5
2. 1.3
Secondary cons trict ions
6
2 . 1 .4
S t ructure o f the nucleo lus organizer regions
6
2 . 1 .4 . 1
Definit ion o f the term ' nucleolus organizer
region '
2 . 1 .4 . 2
6
Locat ion o f 18S and 2 8S rRNA genes in
the NOR
9
2 . 1 .4 . 3
Struc ture o f the NORs in man
10
2.1.4.4
Struc ture o f the NOR i n ma ize
10
2 . 1 .4 . 5
Structure of the NOR in the sciarid fly
12
2.1.4.6
Struc ture o f the NOR in salamander
12
2.1.4.7
Clarificat ion o f t erminology
12
2 . 1 .4.8
Other s ites involved in nucleolus organizat ion
2 .L5
Nucleolus-associated heterochromatin
2 . 1 .6
S taining Techniques in the ident ificat ion
of
NORs
13
13
14
2.1.7
Dup l icate banding
16
2 . 1 .8
Robertsonian trans locat ions and NORs in sheep
17
vi
2.2.
2.3.
Mat erials and Me thods
2.2.1
B lood cul ture and harves t ing techniques
18
18
2.2.2
An imals
18
2.2.3
Ag-AS technique
19
2.2.4
Ag-I t echnique
21
2.2.5
Duplicate G-banding and Ag- l staining
21
Result s
21
2.3. 1
Location of nucleolus organizer regions
21
2.3.2
Ag-AS t echnique
30
2.3.3
Ag-l technique
31
2.3.4
Duplicate banding
31
2 .3.5
An anomalous cell
34
2.3.6
Ag-s taining in interphas e , prophase and
metaphase cells
2.4.
34
34
Dis cus s ion
2.4.1
Structure of sheep nucleolus or ganizer regions 34
2 .4 .2
Pos s ible roles of the s econdary cons trict ions
35
on sheep chromosomes
2.4.3
As sociation of nucl eo lus organizer regions
38
2.4.4
Appl icat ions of s ilver-staining
39
2 .4 . 5
Anomalous s ilver-s t a ining region
39
Chap ter 3 :
The Frequency o f Ag-NORs and Regulat ion o f
NOR act ivity
3. 1 .
41
Int ro duct ion
41
3.1.1
Evidence that s ilver s t ains active NORs
41
3.1.2
Regulat ion of NORs
41
3.1.2.1
Variat ion in the level of rDNA
41
3 . 1 . 2 .2
NOR regulat ion in interspeci2s
hybrids
3.1.2.3
!
rRNA gene activation during
development
3.1.2.4
The effect o f NOR dosage on
NOR a c t ivity
3 . 1 .2.5
The effect o f NOR dosage on Ag-NOR
numbers
3.1 .2.6
47
48
50
Summary o f evidence en
the control o f rRNA transcrip t ion
3.1.3
44
Variation in frequency o f Ag-NORs
50
52
vi i
3.2.
3 . 3.
Ma terials and Methods
52
3.2 . 1
Mater ial s
52
3 . 2.2
S coring o f size po lymorphisms
52
Resul t s
3.3.1
54
Average frequency and range o f Ag-NORs per
metaphase
54
3 . 3�2
Individual variat ion in number of Ag-NORs
3.3.3
Variation between repl icates in the numb er
55
of Ag-NORs
3.3.4
58
Chromosome dis tribut ion o f Ag-NORs
58
3.3.4.1
Poo led data for all individuals
58
3.3.4.2
Frequency of Ag-NORs on C25 and t .
3
59
3.3.4 . 3
Ind ividual variat ion in distribut ion
of Ag-NORs
60
3.3.5
Dif f erences in depo s it size between individuals 60
3.3.6
Re lat ionship of depo s it size to Ag-NOR
frequency
3.3.7
Relat ionship o f depos i t size to between-cul ture
differences
3.4.
65
Discussion
3.4. 1
Var iat ion in the numb er o f Ag-NORs between
animals
67
3.4.2
Chromo somal distribut ion o f Ag-NORs
68
3.4.3
Polymorphism in s ize o f Ag-NORs
69
3.4.4
Rel a t ion of rDNA content t o Ag-staining
69
3.4.5
Ag-stainability and NOR regulation
71
Chapt e r 4 :
4.1.
65
As soc iat ion o f nuc leolus organizer regions
72
Int roduc t ion
72
4.1.1
Satel l ite as s o c ia t ions and nucleolar fus ions
72
4.1.2
S t ud ies on NOR-a s s o c iat ions in spec ies o ther
than man
4.1.3
Participat ion o f individual chromosomes in
s atell ite as s o c ia t ion
4.1.4
73
Relationship of satellite asso c iation t o NOR
morphology
74
75
Vl.l.l.
4 . 1 .5
Relationship o f satellite association
to rDNA content
4 . 1 .6
Relationsh ip of satel l i t e association to
s taining polymo rphisms
4.1.7
4.2.
4.3.
As so c iation of NORs in sheep
77
83
Materials and Methods
83
4.2. 1
Materials
83
4 .2.2
Me thod o f scoring associations
83
83
Resul t s
4.3. 1
Observat ions on as sociat ion complexes of NOchromo somes
4.3.2
Frequency of as sociation
4.3.3
Relat ionship of asso c ia t ion frequency to the
frequency o f express ion
4.3.4
4.3.5
83
84
84
Relat ionship of the as s ociat ion frequency to
the depo si t s ize
4.4.
76
Comb ina tions of a s sociating chromosomes
91
91
Discu s s ion
95
4 .4 . 1
Frequency of assoc iation
95
4.4.2
Fac tors a ffecting the associat ion index o f
individual chromo somes
4 .4 . 3
Comb ina t ions of chromo somes in association
complexes
Chapter 5 :
97
Inheritance o f Ag-s tainab ility
99
101
5.1.
Introduc t ion
101
5.2.
Mat erials and �1ethods
101
5.2 . 1
Mat ing records
101
5.2.2
Estab l ishment o f mat ing groups
1 04
5.2 . 3
Determinat ion o f karyo typ e s o f lambs
1 04
5.2 . 4
Culture s ta ining and analy sis techniques
105
5 .2.5
No tat ion
1 05
I
ix
5.3.
Resul t s
105
5 . 3. 1
Pedigrees t aken from mat ing records
5. 3 . 2
5.3. 1.1
Pedigree 1
105
5.3 . 1 . 2
Pedigree 2
106
Resul t s o f mat ing experimen t s
106
5.3 . 2 . 1
Lamb ing resul t s o f Pedigree 3
106
5.3.2.2
Lamb ing results of Pedigree 4
1 09
5.3.2.3
Lamb ing results o f Pedigree 5
1 15
5.3.3
Recomb inat ion of Ag-NORs
5.3.4
Inheritance o f frequency o f Ag-NORs and
s iz e polymorphism
5.4.
105
1 15
1 17
Discussion
1 18
5 .4. 1
1 18
Inher itance of Ag-stainab i l i ty
5". t. .l
Recomb ina t ion of Ag-NORs
Chapter 6 :
1 24
Comparison o f silver-staining pat terns in
f ib roblas t s and lymphocyt es
126
6.1.
Introduct ion
126
6.2.
Ma t erials and Methods
127
6.2 .1
Tis sue Source
127
6. 2 . 2
Fib roblas t culture technique
127
6.2 . 3
Sub culturing
128
6.2.4
Harves t ing of f ibroblas t cul tures
128
6.2 .5
Ag-s tain ing
128
6.3
129
Results
6. 3 . 1
Frequency o f Ag-NORs in dif ferent t issues
6 . 3.2
S ize polymo rphism of Ag-NORs in different
tissue s
6.4
Discus s ion
Chapter 7 :
129
1 32
132
Homo logy of NORs in the s�eep , go at , aoudad ,
bharal and cattle
137
X
7. 1.
7 .2.
7.3.
7.4.
Int roduc t ion
137
7.1.1
Chromosomal evo lut ion in the Bovidae
137
7. 1 .2
S tudies on the evolut ion o f NORs
1 39
7 . 1 .3
NOR studies in the Bovidae
141
Materials and Methods
144
7.2. 1
Animals
1 44
7 . 2 .2
Karyotyping and comparative s tudies
1 44
146
Re sul t s
7.3.1
Locat ion o f NORs
146
7.3.2
Varia t ion in number and s ize o f Ag-NORs
1 52
Dis cus s ion
152
7 .4 . 1
Assignment of NORs t o specific chromosomes
152
7 .4.2
Nomenclature and presumpt ive homologies o f
the chromosomes o f the Bovidae
1 55
7.4.3
The ring chromosome of the bharal
157
7 .4 . 4
Frequency of Ag-NORs
157
7 .4 . 5
Homology o f NORs in sheep , goat , cattle ,
aoudad and bharal
7.4.6
Relat ionship of NORs to Robertsonian
translocations in the Bovidae
Chap ter 8 :
158
Conclus ions
1 60
163
8.1.
Rel a t ion of Ag-staining to NOR act ivity
163
8.2.
The na ture o f the Ag-st aining po lymorphisms
1 65
8.3.
A mo del of NOR regulat ion and Ag-staining in
d ip l o id organisms
166
8.4.
The s tabilit � o f NORs
1 69
8.5.
S ummary of advanc es flade in this s tud}:
171
Appendices
173
I
Personal communicat ions
1 73
II
Leuco cyte cul ture t echnique
1 74
Ill
Giemsa s tain
1 75
IV
Fibroblast cul ture materia l s
1 75
V
G-banding technique
176
VI
Washing of laboratory glas sware
177
VII
Pho to graphy techniques
177
VIII
His tory and management o f experimental animals
177
IX
Dis tr ibut ion o f count s o f numb er o f Ag-NORs pe r
179
metaphase
X
Two-way ANOVA for rep l icat e cul tures
1 80
XI
Exampl e o f s ize polymo rphism data s core
181
XI I
Dat a on Ag-NOR frequency in inheritance study
1 82
XIII
Copy of the p ubl icat ion "As s o c iat ion of nucleolus
organizer chromosomes in dome s t ic sheep (Ovis aries )
1 84
shown by s ilver staining"
XIV
Copy o f the p·..1blication "Conservat ion o f nucleolus
organizer regions dur ing evolut ion in sheep , goa t ,
1 85
cat t l e and aoudad"
XV
Abs tract o f p aper presented t o New Zealand
Genet ical Soc iety Con ferenc e , 1 9 7 8
Bibl iography
following page
1 86
1 86
Xl.l.
List of Tab les
2-1 .
Examples of varying usages o f the t erm ' nucleolus
organizer region ' and i t s derivatives
3- 1 .
51
The dis t ribut ion o f average number o f Ag-NORs per
metaphase in 3 3 individuals
3-3 .
56
Analysis of varia t ion due t o replicate differences
in b etween-animal diffe rences
3-5 .
Dat a o n the chromosomal distribut ion o f Ag-NORs
3-6 .
Chromo some distribut ion o f Ag-NORs in individuals
o ther than those involved in ped igree study
3- 7 .
63
Comparison of associat ion frequency with expected
86
Relat ionship b etween dep o s i t s ize and as soc�at ion
frequency o f C4 , C25 and t
4�3 .
59
66
values based on frequency o f Ag-NORs
4-2 .
58
Rel a t ionship o f depo sit s ize t o be tween-culture
d i f ferences
4-1 .
57
Dis t r ib ut ion of the numb er of cell s in which a
chromosome has a given size d eposit
3-8 .
54
Average frequency and dis tribut ion o f Ag-NORs in
ind ividual sheep
3-4 .
8
Summary o f evidence on the control o f rRNA
t rans c ript ion
3-2 .
Page
3
89
Relat ive part icipation in nucleolar o rganizat ion
of NO-chromo somes in 5 animals used in
c omb inat ional
4-4 .
analys is
92
Ob served and expected values o f pair-wise
comb inat ions of N07chromo somes
94
Xiii
4 -5 .
Reported frequencies of satellite assoc iat ion in man 9 6
5-1 .
Lamb ing and Ag-staining resul t s of pedigree 3
107
5-2 .
Lamb ing and Ag-s taining result s of pedigree 4
109
5-3 .
Lamb ing and Ag-s taining resu l t s of pedigree 5 .
116 -
5 -4 .
Inheritance o f stainab ility in sheep het erozygous
for Ag-stainab ility
5-5 .
Size o f Ag-NORs in 5 animals and their pro geny
6-1 .
The frequency o f Ag-NORs in lymphocyte and
f ib roblast cul tures from 3 animals
6-2 .
133
140
G-banding homology between sheep , aoudad , cat t l e ,
goat and bharal autosomes
7-3 .
129
Comparison o f location and number o f NORs in man ,
chimpanzee , orangutan and go rilla
7-2 .
119
S i ze polymorphism of Ag-NORs in each tis sue from
3 animals
7 -1 .
117
145
Modal values and range o f numb er o f Ag-NORs per
metaphase in sheep , aoudad , cattle and goat
153
7-4 . Involvemen t of chromo somes in cent ric fusion
during karyo typ ic evo lut ion and within species
161
x�v
Page
List of f igures
2-1 .
Drawing showing the morphology of the NOR in
7
man ( t ext f igure)
2-2 .
Drawing showing the morpho logy of the NO-chromo some
11
in maize ( text figure )
2-3 .
Flow-diagram o f Ag-AS and Ag- I staining techniques
2-4 .
Karyo type showing locat ion o f Ag-NORs on chromsomes
22
1 , 2 , 3 , 4 and 25
2-5 .
Karyotype showing Ag-NOR on t
2-6 .
Ag- I s t a ined me taphase showing absence of Ag­
3
chromo some
staining on a secondary cons t ric t ion
2- 7 .
20
23
24
Absence of s i lver � staining on secondary constrict­
ions in 3 met aphases s tained by Giemsa , des tained
.and Ag-sta ined
25
2-8.
Met apha se s ta ined by Ag-AS t echnique (colour plat e )
26
2-9 .
Met aphase s ta ined b y Ag-I t echnique ( colour plate)
27
2-10 . Met aphase t reated by Ag-I te chnique and then G­
b anded (co lour plate)
28
2-1 1. Karyo type o f NO-chromo some s ident ified by dup l icate
G-banding and Ag-I s t a in ing
29
2- 1 2. Karyo type o f a cell wi th Ag-deposits on both
telomeres of a chromosome 1 .
32
2 - 1 3 . Ag-st ained cells showing d i fferences in amount o f
Ag-staining material a t dif ferent s tages o f the
c ell cycl e
33
XV
3-1 .
Diagram o f Nucleo lar dominanc e in F1 hyb rids o f
46
D . mulleri and D . arizonens is
3-2 .
Two metaphas e cells showing Ag-dep o s i t s o f different
53
s izes
3-3 .
Histograms illustrating the average number of Ag-NORs
61
on the 3 chromosome types
3-4 .
Histo grams o f s ize of Ag-NORs in 8 animals
3-5 .
Graph showing co rrelation o f depo s i t s ize and
frequency o f express ion o n chromosome 2 5 and t
62
3
4-1 .
Metaphase cell showing chromos om�in association
4-2 .
Metaphas e c el l showing chromosomes lying adj a cent
but not associated
4-3 .
82
82
Scattergram showing c orrelation o f association
index with frequency of Ag-NORs
4-9 .
81
Metaphas e cell with seven chromosomes involved in
associa tion complexes
4-8 .
80
Met aphase cell showing four chromosomes in one
as sociation complex
4- 7 .
79
Met apha se cell showing an assoc iated chromos ome
with no detec table Ag-stain
4-6 .
78
Two met aphases wi th associa ted chromosomes j o ined
by a long connecting s t rand
4-5 .
78
Mataphase c el l with chromos omes as s o c iated by one
chromat id and by both chromatids
4-4 .
64
88
Graph showing correlat ion between Ag-de?os i t
s ize and associat ion index
90
5-1 .
Pedigree char t
o f pedigree 1
102
5-2 .
Pedigree chart
o f pedigree 2
103
5-3 .
Ag-s ta ined karyotype o f one o f the sires o f
pedigree 3 ( 1 2 1 / 5 )
5-4 .
108
Karyo type o f NO-chromos omes of two progeny of
pedigree 3
1 08
5-5 .
Ag-s tained karyo type o f s ire o f pedigree 4 ( 6 0 / 5 )
1 10
5-6 .
G-banded karyotype o f lamb 12 3 / 8
111
5-7 .
Karyo type of NO-chromo somes o f four p rogeny of
p edigree 4
5-8 .
Karyo type o f s ire o f pedigree 5 ( 1 7 6 / 6 )
5-9 .
Karyo type o f NO-chromo somes o f s ix progeny of
pedigree 5
112
113
1 14
5 - 1 0 . Scat t ergram showing correlat ion between Ag-NOR
frequency in parent and progeny
1 20
5 -1 1 . Scatt ergram showing relat ionship of Ag-NOR size
polymorphism in parent and p ro geny
6-1 .
121
Histograms of average number of Ag-NORs per
metaphas e in fib rob las t s and lymphocytes in
three animals
6-2 .
His tograms o f s i z e po lymorphism o f Ag-NORs in
lymphocyte cultures
6-3 .
1 30
131
Karyo typ e of NO-chromosomes from three tis sues o f
155/6
1 34
xvi i
7-1 .
Taxonomy of bovid species used in this study
7-2 .
Karyo type o f Ag-stained metaphase from cattle
showing location of NORs
7-3 .
1 49
Par t ial me taphase from an aoudad showing an Ag-NOR
on ch romosome 1
7-7 .
Metaphase c.�ll from bharal
7-8 .
Karyo type of an Ag-stained metaphase cell from a
bharal showing the location o f NORs
7-9 .
148
Karyo type of Ag-s tained metaphase o f an aoudad
showing lo cat ion of NORs
7-6 .
147
Karyo type of Ag-s t a ined metaphase of a goat showing
loca tion of NORs
7-5 .
147
Metaphase cells from 1 / 29 bull showing Ag-NORs on
2 small acrocentric chromosomes
7-4 .
143
149
150
1 50
Two metaphases from a bharal showing ' ring ' chromo­
somes
15 1
7-:- 1 0 . Id io gram of the NO-chromosomes in the f ive bovid
spec ies showing homo logies
156
.I