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A silver-staining study o f the nucleolus organizer regions of the chromosomes of domestic sheep (Ovis aries )
A thesis presented in partial fulfilment o f the requirements for the degree of Doctor of Philosophy in Genetics
at Massey University
Leigh Marian Henderson March 1 9 7 9
ABSTRACT
A study was made of various aspects o f the nucleolus organizer regions (NORs ) on sheep mitotic chromosomes , using the Ag-AS and Ag-I techniques . The sheep used in this s t udy were carriers o f
the t 1 , t2 and t 3 Robertsonian translocat ions in various hetero
zygous and homozygous combinations . The lo cations o f the NORs wer e determined to be at the telomeres on the metacentric chromosomes lp , 2q and 3q and on the acrocentric chromosomes 4 and 25 . The identity o f the NO-chromosome was confirmed by a duplicate G-banding and Ag-1 technique . The terminal location o f the NORs indicates that they do no t have a. causat ive role in acrocentric association or Robertsonian translocation in domestic sheep . They also do no t coincide with the regions in which secondary constrictions are somet imes seen .
Individual animals were found to have a characteristic
silver-staining pattern . The chromosomal distribution o f the Ag
s taining NORs (Ag-NORs ) , the frequency and size of the Ag-NORs were fairly constant in different cells o f an individual and in r eplicate cult ures of an animal . Dif ferences between cells
and replicates in Ag-staining involved variation in the frequency o f staining o f Ag-NORs with small Ag-depo sits . Overall , the meta
centrics had the highest frequency of Ag-NORs , followed by chromosome 25 and then chromosome 4 .
The association frequency o f individual NO-chromosomes was found to b e p o s i t ively correlated with the frequency o f Ag-NORs and the size o f the Ag-deposit . The frequency of each pairwise combination of associating chromosomes was determined solely by the frequency of Ag-NORs of the component chromosomes . No evidence was found for a non-randJm fusion o f NORs . These obser?ations p rovide an explanat ion of the reports of non-random partic ipat ion o f
acro centric chromosomes in satellite association i n man . The increased association o f NO-chromosomes with large depo sits could b e due to an increased chance of fusion of larger nucleol i or to differences in the disintegrat i on rate o f different sized nucleo l i .
The presence of a NOR on the chromosome 25 / t3 polymorphism was utilized in inheritance studies of Ag-stainability . Five pedigree group s in which this chromosome was segregating were s tudied and in all cases the size o f the Ag-deposits and
f requency of s taining was consistent between consecutive generations . The value o f Ag-staining in genetic mapp ing s tudies is discus sed .
In 3 animals , the Ag-staining patterns of transformed lympho
cytes and fibroblasts were compared and found to be similar .
A comparat ive study was made o f 5 species o f the Bovidae:
domestic sheep (Ovis aries), domestic goa t (Capra hircus), aoudad (Ammetra�us lervia),bharal (Pseudo is nayaur) and cattle
(Bos taurus) . Five NO-chromosome pairs were found in sheep , goat , aoudad and cattle and these chromosomes have homologous b anding pat terns . The bharal has a t least four NO-chromo some pairs homologous to sheep . These results indicate a conservation of the NORs during evo lut ion in this family .
Based on the results of the f requency o f Ag-staining
o f NORs , the heritability of Ag-s tainab ility and the cons tancy o f Ag-staining pat terns found in the t issue s tudies , the nature of the Ag-staining is discus sed and a model on the basis of the Ag
s tainability o f individual chromosomes is presented .
iii
ACKNOWLEDGEMENTS
I would l ike to thank s incerely Professor A . N . Bru�re for his supervision of this proj ec t and his encouragement and help throughout its durat ion . I am grateful to Dr Helen Chapman for her useful discussions during the early part of this proj ec t . I would also l ike to thank Dr A. Wilkins for his very helpful d is cussions of parts of this thes is .
Priscilla Ell is provided competent technical ass istance with this work, for which I am very appreciat ive . I would like to thank Mr T . Law for his help with photography . Dr R. Harris provided assis tance with the statis t ical analyses in this thes is . Karen Armitage aided in the final preparation of the photographs in this thes is .
I am very grateful to the farm staff of the Massey No 2 Sheep Unit for their work in the maintenance of the experimental flock and their assis tance in blood collection . I would like to thank all the s taff of the Veterinary Clinical Sciences Department who have been so obl iging in assisting me in collecting blood, ob taining samples from surgery and general assis tance in other aspec ts of this work .
I am grateful to Angela Lowe and Mrs J. Pearce for typing drafts of this manuscript . I apprec iate very much the time and ef fort put into typing the final manuscrip t by Val Darroch .
This work was funded by a demons t ratorship in the Department of Microbiology and Genetic s and I am grateful to Professor D . Bacon
for making this pos icion available to me .
Finally , my thanks go to Neville who has provided so much encouragement and help throughout the course of this work .
iv
Chapter 1 : Chapter 2 :
TABLE OF CONTENTS
Acknowledgements Table o f Contents List of Tables Lis t of figures
Introduc tion
S t ructure and Identif icat ion of Nucleolus Organizer Regions in sheep
Page iv
V
xii
� xiv
1
4
2 . 1 . Introduct ion 4
2 . 1 . 1 The nucleolus 4
2_. 1 . 2 The formation of the nucleolus on specific chromosome regions
2 . 1 . 3 Secondary cons trict ions
5 6 2 . 1 . 4 Structure o f the nucleolus organizer regions 6 2 . 1 . 4 . 1 Definit ion o f the term ' nucleolus organizer
region ' 6
2 . 1 . 4 . 2 Location o f 18S and 28S 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 in maize 10 2 . 1 . 4 . 5 Structure of the NOR in the sciarid fly 1 2 2 . 1 . 4 . 6 Structure o f the NOR in salamander 1 2 2 . 1 . 4 . 7 Clarificat ion o f t erminology 1 2 2 . 1 . 4 . 8 Other sites involved in nucleolus organ-
2 .L5 2 . 1 . 6
2 . 1 . 7 2 . 1 . 8
izat ion
Nucleolus-associated heterochromatin Staining Techniques in the identificat ion
of NORs
Duplicate banding
Robertsonian translocations and NORs in sheep 1 3 1 3
14 16 1 7
V
2 . 2 . Mat erials and Methods 18 1 8 1 8 1 9 2 1 2 1 2 . 2 . 1 Blood cul ture and harves t ing techniques
2 . 2 . 2 Animals
2 . 2 . 3 Ag-AS technique 2 . 2 . 4 Ag-I technique
2 . 2 . 5 Duplicate G-banding and Ag-l staining
2 . 3 . Result s 2 1
2 1 30 3 1 3 1 34 2 . 3 . 1
2 . 3 . 2 2 . 3 . 3 2 . 3 . 4 2 . 3 . 5 2 . 3 . 6
Location of nucleolus organizer regions Ag-AS technique
Ag-l technique Duplicate banding An anomalous cell
Ag-staining in interphase , prophase and
metaphase cells 34
2 . 4 . Discus s ion 34
2 . 4 . 1 Structure of sheep nucleolus organizer regions 34 2 . 4 . 2 Possible roles of the secondary cons trict ions
on sheep chromosomes 35
2 . 4 . 3 Association of nucl eolus organizer regions 38 2 . 4 . 4 Applicat ions of s ilver-staining 39 2 . 4 . 5 Anomalous silver-staining region 39
Chapter 3 : The Frequency o f Ag-NORs and Regulation o f
NOR act ivity 4 1
3 . 1 . Intro duction 4 1
4 1 4 1 4 1
3 . 1 . 3
3 . 1 . 1 Evidence that s ilver s tains active NORs 3 . 1 . 2 Regulation of NORs
3 . 1 . 2 . 1 Variat ion in the level of rDNA 3 . 1 . 2 . 2 NOR regulation in interspeci2s
hybrids !
3 . 1 . 2 . 3 rRNA gene activation during
44
development 4 7
3 . 1 . 2 . 4 The effect o f NOR dosage on
NOR activity 48
3 . 1 . 2 . 5 The effect o f NOR dosage on Ag-NOR
numbers 50
3 . 1 . 2 . 6 Summary o f evidence en
the control o f rRNA transcrip t ion Variation in frequency o f Ag-NORs
50 52
vi
3 . 2 . Materials and Methods
3 . 2 . 1 Mater ials
3 . 2.2 S coring o f size polymorphisms
3 . 3 . Result s
3 . 3 . 1 Average frequency and range o f Ag-NORs per metaphase
5 2
52 5 2 54
5 4 3 . 3�2 Individual variat ion in number of Ag-NORs 55 3 . 3 . 3 Variation between repl icates in the number
of Ag-NORs
3 . 3 . 4 Chromosome dis tribut ion o f Ag-NORs
58 58
3 . 3 . 5 3 . 3 . 6
3 . 3 . 4 . 1 Pooled data for all individuals 58 3 . 3 . 4 . 2 Frequency of Ag-NORs on C25 and t3. 59 3 . 3 . 4 . 3 Ind ividual variat ion in distribut ion
of Ag-NORs 60
Differences in depos it size between individuals 60 Relat ionship of deposit size to Ag-NOR
frequency 65
3 . 3 . 7 Relationship o f deposit size to between-cul ture
differences 65
3 . 4 . Discussion
3 . 4 . 1 Var iat ion in the number o f Ag-NORs between
animals 6 7
3 . 4 . 2 Chromosomal distribut ion o f Ag-NORs 68 3 . 4 . 3 Polymorphism in s ize o f Ag-NORs 69 3 . 4 . 4 Relation of rDNA content to Ag-staining 69 3 . 4 . 5 Ag-stainability and NOR regulation 7 1 Chapter 4 : Assoc iation o f nucleolus organizer regions 7 2
4 . 1 . Introduct ion 72
4 . 1 . 1 Satel lite associations and nucleolar fus ions 72 4 . 1 . 2 Studies on NOR-associations in spec ies other
than man 7 3
4 . 1 . 3 Participation o f individual chromosomes in
satellite as sociation 7 4
4 . 1 . 4 Relationship of satellite association t o NOR
morphology 75
vii
4 . 1 . 5 Relationship o f satellite association to rDNA content
4 . 1 . 6 Relationship of satel lite association to staining polymorphisms
4 . 1 . 7 As sociation of NORs in sheep
7 6
7 7 8 3
4 . 2 . Materials and Methods 4 . 2 . 1 Materials
83 8 3 83 4 . 3 .
4 . 4 .
4 . 2 . 2 Method o f scoring associations
Result s
4 . 3 . 1 Observat ions on associat ion complexes of NO- chromosomes
4 . 3 . 2 Frequency of association
4 . 3 . 3 Relationship of asso c ia t ion frequency to the frequency o f express ion
4 . 3 . 4 Relat ionship of the ass ociation frequency the deposit size
4 . 3 . 5 Combinations of associating chromosomes
Discussion
4 . 4 . 1 Frequency of assoc iation
4 . 4 . 2 Factors affecting the associat ion index o f individual chromosomes
4 . 4 . 3 Comb inations of chromosomes in association complexes
to 83
83 84 84
9 1 9 1
9 5 95
97
99
Chapter 5 : Inheritance o f Ag-s tainab ility 1 0 1
5 . 1 . Introduction
5 . 2 . Mat erials and �1ethods
1 0 1
1 0 1
5.2 . 1 Mat ing records 101
5 . 2 . 2 Establishment of mat ing groups 1 04 5.2 . 3 Determinat ion o f karyotypes o f lambs 104 5.2 . 4 Culture s taining and analysis techniques 105
5 . 2 . 5 Notation 105
Vl.l.l.
I
5 . 3 . Results
5 . 3 . 1 Pedigrees t aken from mat ing records 5 . 3 . 1 . 1 Pedigree 1
5.3 . 1 . 2 Pedigree 2
5. 3 . 2 Result s o f mating experiments
5.3 . 2.1 Lamb ing result s o f Pedigree 5 . 3 . 2 . 2 Lambing results of Pedigree 5 . 3 . 2 . 3 Lambing results o f Pedigree 5 . 3 . 3 Recomb ination of Ag-NORs
5 . 3 . 4 Inheritance o f frequency o f Ag-NORs s ize polymorphism
5 . 4 . Discussion
5 . 4 . 1 Inheritance of Ag-stainab ility
5". t. .l
Recombina tion of Ag-NORs
3 4 5 and
Chapter 6 : Comparison o f silver-staining pat terns in f ibroblas t s and lymphocytes
6 . 1 . Introduct ion
6 . 2 . Materials and Methods 6 . 2 . 1 Tissue Source
6 . 3
6. 2 . 2 Fibroblas t culture technique 6 . 2 . 3 Sub culturing
6 . 2 . 4 Harves t ing of fibroblas t cul tures 6 . 2 . 5 Ag-staining
Results
105 105 105 106 106 106 1 09 1 1 5 1 15 1 1 7
1 1 8 1 1 8 1 24
1 2 6
1 2 6
1 2 7 1 2 7 1 2 7 1 2 8 1 2 8 1 2 8
1 2 9 6 . 3 . 1 Frequency o f Ag-NORs in dif ferent tissues 1 2 9
6 . 4
6 . 3.2 S ize polymorphism of Ag-NORs in different tissues
Discuss ion
Chapter 7 : Homo logy of NORs in the s�eep , goat , aoudad , bharal and cattle
1 32
1 3 2
1 3 7
ix
7 . 1 . Introduction
7 . 1 . 1 Chromosomal evolut ion in the Bovidae 7 . 1 . 2 S tudies on the evolution o f NORs 7 . 1 . 3 NOR studies in the Bovidae
7 . 2 . Materials and Methods 7 . 2 . 1 Animals
7 . 2 . 2 Karyotyping and comparative s tudies 7 . 3 . Resul t s
1 3 7 1 3 7 1 39 1 4 1
144 1 44 1 44
146
7 . 3 . 1 Location of NORs 146
7 . 3 . 2 Variation in number and s ize o f Ag-NORs 1 52
7 . 4 . Discussion 152
7 . 4 . 1 Assignment of NORs to specific chromosomes 152 7 . 4 . 2 Nomenclature and presumpt ive homologies o f
the chromosomes o f the Bovidae 7 . 4 . 3 The ring chromosome of the bharal 7 . 4 . 4 Frequency of Ag-NORs
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
Chapter 8 : Conclus ions
8 . 1 . Relation of Ag-staining to NOR act ivity 8 . 2 . The nature o f the Ag-staining polymorphisms
8 . 3 . A model of NOR regulat ion and Ag-staining in
diploid organisms 8 . 4 . The s tabilit� o f NORs
8 . 5 . S ummary of advances flade in this s tud}:
1 55 1 5 7 1 5 7 158 1 60 1 6 3
1 6 3 1 65
1 6 6 1 69 1 7 1
X
Appendices 1 7 3
I Personal communicat ions 1 73
II Leuco cyte culture technique 1 74
Ill Giemsa s tain 1 75
IV Fibroblast cul ture materials 1 75
V G-banding technique 1 7 6
VI Washing of laboratory glassware 1 7 7
VII Photography techniques 1 7 7
VIII His tory and management of experimental animals 1 7 7
IX Dis tribution o f counts o f number of Ag-NORs per metaphase
X Two-way ANOVA for replicat e cul tures
XI Example of s ize polymorphism data s core
XII Data on Ag-NOR frequency in inheritance study
XIII Copy of the p ubl ication "Association of nucleolus organizer chromosomes in domestic sheep (Ovis aries ) shown by s ilver staining"
XIV Copy of the p·..1blication "Conservat ion o f nucleolus organizer regions during evolut ion in sheep , goat , cattle and aoudad"
XV Abs tract o f paper presented t o New Zealand Genet ical Society Conference , 1 9 7 8
Bibl iography following page
1 7 9
1 80
1 8 1 182
184
185
1 86
1 86
List of Tab les
2-1 . Examples of varying usages o f the term ' nucleolus organizer region ' and its derivatives
3- 1 . Summary o f evidence on the control o f rRNA t ranscript ion
3-2 . The dis tribut ion o f average number o f Ag-NORs per metaphase in 33 individuals
3-3 . Average frequency and dis tribut ion o f Ag-NORs in
Page
8
5 1
54
individual sheep 56
3-4 . Analysis of variat ion due t o replicate differences
in between-animal diffe rences 5 7
3-5 . Dat a o n the chromosomal distribut ion o f Ag-NORs 58
3-6 . Chromo some distribution o f Ag-NORs in individuals o ther than those involved in pedigree study
3-7 . Distribut ion of the number of cell s in which a chromosome has a given size deposit
3-8 . Relationship o f deposit s ize t o between-culture
59
63
differences 66
4-1 . Comparison of associat ion frequency with expected
values based on frequency o f Ag-NORs 86
4-2 . Relationship between deposit s ize and assoc�at ion
frequency o f C4 , C25 and t 3 89
4�3 . Relat ive participation in nucleolar organization of NO-chromosomes in 5 animals used in
comb inat ional analys is
4-4 . Observed and expected values o f pair-wise comb inat ions of N07chromosomes
92
94
Xl.l.
4-5 . Reported frequencies of satellite assoc iat ion in man 9 6 5-1 . Lamb ing and Ag-staining results of pedigree 3
5-2 . Lamb ing and Ag-staining results of pedigree 4 5-3 . Lambing and Ag-staining results of pedigree 5 .
5-4 . Inheritance o f stainability in sheep heterozygous for Ag-stainability
5-5 . Size o f Ag-NORs in 5 animals and their progeny
6-1 . The frequency o f Ag-NORs in lymphocyte and f ib roblast cul tures from 3 animals
6-2 . Size polymorphism of Ag-NORs in each tissue from
107 109 116 -
117 119
129
3 animals 133
7 -1 . Comparison o f location and number o f NORs in man ,
chimpanzee , orangutan and gorilla 140
7-2 . G-banding homology between sheep , aoudad , cattle , goat and bharal autosomes
7-3 . Modal values and range o f number o f Ag-NORs per
145
metaphase in sheep , aoudad , cattle and goat 153
7-4 . Involvement of chromo somes in cent ric fusion
during karyo typic evo lut ion and within species 161
Xiii
List of figures
2-1 . Drawing showing the morphology of the NOR in man ( t ext figure)
2-2 . Drawing showing the morphology of the NO-chromosome in maize ( text figure)
Page
7
1 1
2-3 . Flow-diagram o f Ag-AS and Ag-I staining techniques 20
2-4 . Karyo type showing locat ion o f Ag-NORs on chromsomes
1 , 2 , 3 , 4 and 25 22
2-5 . Karyotype showing Ag-NOR on t 3 chromosome 2 3
2-6 . Ag-I stained metaphase showing absence of Ag
staining on a secondary cons tric t ion
2-7 . Absence of silver�staining on secondary constrict
ions in 3 metaphases stained by Giemsa , destained 2 4
.and Ag-stained 25
2-8. Met aphase stained by Ag-AS technique (colour plate) 26 2-9 . Metaphase s ta ined by Ag-I technique (colour plate) 27
2-10 . Met aphase treated by Ag-I technique and then G
b anded (colour plate)
2-1 1. Karyotype o f NO-chromosomes ident ified by dup l icate G-banding and Ag-I stain ing
2-1 2. Karyo type o f a cell with Ag-deposits on both telomeres of a chromosome 1 .
2-13 . Ag-stained cells showing differences in amount o f Ag-staining material a t different stages o f the
28
2 9
32
c ell cycle 33
x�v
3-1 . Diagram o f Nucleolar dominanc e in F1 hybrids o f D . mulleri and D . arizonens is
3-2 . Two metaphase cells showing Ag-deposits o f different s izes
3-3 . Histograms illustrating the average number of Ag-NORs on the 3 chromosome types
3-4 . Histograms o f size of Ag-NORs in 8 animals 3-5 . Graph showing co rrelation o f deposit size and
46
53
6 1 6 2
frequency o f express ion o n chromosome 2 5 and t3 64
4-1 . Metaphase cell showing chromosom�in association 78
4-2 . Metaphase cell showing chromosomes lying adj acent but not associated
4-3 . Mataphase cell with chromosomes associated by one chromat id and by both chromatids
4-4 . Two metaphases with associated chromosomes j oined by a long connecting s t rand
4-5 . Metaphase cell showing an associated chromosome with no detectable Ag-stain
4-6 . Met aphase cell showing four chromosomes in one as sociation complex
4-7 . Metaphase cell with seven chromosomes involved in association complexes
4-8 . Scattergram showing correlation o f association index with frequency of Ag-NORs
4-9 . Graph showing correlat ion between Ag-de?os i t size and association index
78
79
80
8 1
82
82
88
90
XV
5-1 . Pedigree chart o f pedigree 1 5-2 . Pedigree chart o f pedigree 2
5-3 . Ag-s tained karyotype o f one o f the sires o f
102 103
pedigree 3 ( 1 2 1 / 5 ) 108
5-4 . Karyo type o f NO-chromos omes of two progeny of
pedigree 3 108
5-5 . Ag-stained karyotype o f s ire o f pedigree 4 (60/5) 1 10 5-6 . G-banded karyotype o f lamb 12 3/8 1 1 1
5-7 . Karyotype of NO-chromo somes o f four p rogeny of
p edigree 4 1 1 2
5-8 . Karyo type o f sire o f pedigree 5 ( 1 7 6 / 6 ) 1 1 3
5-9 . Karyotype o f NO-chromosomes o f s ix progeny of
pedigree 5 1 14
5-10 . Scatt ergram showing correlation between Ag-NOR frequency in parent and progeny
5 -1 1 . Scattergram showing relationship of Ag-NOR size polymorphism in parent and progeny
6-1 . Histograms of average number of Ag-NORs per metaphase in fib roblast s and lymphocytes in
three animals
6-2 . Histograms o f size po lymorphism o f Ag-NORs in lymphocyte cultures
6-3 . Karyotyp e of NO-chromosomes from three tissues o f 155/6
120
1 2 1
1 30
1 3 1
1 34
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 . Metaphase cells from 1 /29 bull showing Ag-NORs on 2 small acrocentric chromosomes
7-4 . Karyotype of Ag-s tained metaphase of a goat showing location of NORs
7-5 . Karyotype of Ag-stained metaphase o f an aoudad showing locat ion of NORs
7-6 . Part ial metaphase from an aoudad showing an Ag-NOR on chromosome 1
7-7 . Metaphase c.�ll from bharal
7-8 . Karyotype of an Ag-stained metaphase cell from a bharal showing the location o f NORs
7-9 . Two metaphases from a bharal showing ' ring ' chromo
somes
7-:- 10 . Idiogram of the NO-chromosomes in the five bovid species showing homologies
xvii
143
147
147
148
149
149 150
1 50
1 5 1
156
.I