Molecular Genetic Characterization of HSP90
gene in Deoni (Bos indicus) cattle.
Thesis submitted to the
National Dairy Research Institute, Karnal
(Deemed University)
in partial fulfilment of the requirements
for the award of the degree of
MASTER OF VETERINARY SCIENCE
in
ANIMAL GENETICS AND BREEDING
By
SHAHID AHMAD SHERGOJRY
B.V.Sc. & A.H
DAIRY PRODUCTION SECTION
SOUTHERN CAMPUS
NATIONAL DAIRY RESEARCH INSTITUTE (I.C.A.R.)
ADUGODI, BANGALORE- 560030, INDIA
2011
Regn. No. 2060910
Molecular Genetic Characterization of HSP90
gene in Deoni (Bos indicus) cattle.
by
Dr. SHAHID AHMAD SHERGOJRY
Thesis submitted to the
National Dairy Research Institute
(Deemed University)
in partial fulfilment of the requirement
for the degree of
MASTER OF VETERINARY SCIENCE
ANIMAL GENETICS AND BREEDING
Approved by:
(EXTERNAL EXAMINER)
(K. P. RAMESHA)
Major Advisor & Chairman
Members of Advisory Committee
1.
2.
3.
4.
Dr. D.N. Das
___________________
Dr. A. Obi Reddy
___________________
Dr. M.A. Kataktalware
___________________
Dr. Bandla Srinivas
___________________
Senior Scientist, Dairy production section
Principal Scientist, Dairy production section
Scientist, Dairy production section
Senior Scientist, Dairy production section
DAIRY PRODUCTION SECTION
SOUTHERN CAMPUS
NATIONAL DAIRY RESEARCH INSTITUTE
ADUGODI, BANGALORE- 560030, INDIA
Dr. K. P. Ramesha
Principal Scientist
CERTIFICATE
This is to certify that the thesis entitled, “Molecular Genetic
Characterization of HSP90 gene in Deoni (Bos indicus) cattle"
submitted by Dr. SHAHID AHMAD SHERGOJRY towards the partial
fulfilment of the award of the degree of MASTER OF VETERINARY
SCIENCE ANIMAL GENETICS AND BREEDING of the NATIONAL
DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY), Karnal
(Haryana), India, is a bonafide research work carried out by him under
my supervision, and no part of the thesis has been submitted for any
other degree or diploma.
Dated: 13th June, 2011
(K. P. Ramesha)
Major Advisor & Chairman
DEDICATED
To my beloved parents,
who taught me to dream and set goals.
To my teachers,
who gave me the tools to achieve those goals
To my friends,
who taught me how to live and love.
Acknowledgement
IN THE NAME OF ALLAH, THE MOST GRACIOUS, THE MOST MERCIFUL.
All praise and thanks to Allah, the lord of the Alamin I am ever grateful to God,
(Alhamdullilah-i-rabbil aalamin) who gave me good health and peace of mind, so that I could
put in my best while attempting this study, thank you making everything that is wonderful. It is
with great delight that I acknowledge my debts to those who have contributed immensely to the
success of this thesis. First and foremost I would like to express my deep and sincere gratitude to my
guide, Dr .K. P. Ramesha, Principal Scientist, (Animal Genetics and Breeding), Southern campus
of NDRI Bangalore for his inspiring guidance, sustained encouragement, close supervision, keen
interest, critical appraisal, judicious planning of the project. His wholehearted cooperation,
patience, and care during the entire study have left certain experiences, which are worthy of
remembrance throughout life.
I wish to extend my grateful acknowledgement to the members of the advisory committee,
Dr. A.Obi Reddy, Principal Scientist and Incharge, Dairy Production section, Southern campus
of NDRI, Dr. D.N.Das, Senior Scientist, (AGB), Dr. M.A.Kataktalware, Scientist, (LPM) and
Dr. Bandla Srinivas (Animal Nutrition) Senior Scientist, Dairy production section of NDRI
Southern campus, Adugodi, Bangalore for useful advice and suggestions during my research work.
I convey special acknowledgement to Dr. A.K.Srivastava, my teacher and Director,
NDRI, Karnal and Dr.S. Kulkarni, Head, NDRI Southern Campus, Bangalore for providing
necessary facilities for carrying out this study and financial assistance in the form of Institutional
Fellowship during my Master’s programme.
I express my gratitude to Dr. Surendranath, Principal Scientist and Incharge, Education
Section, Mrs.Thivija kumari and all other staff for their help during the progress of the study. I
wish to express my thanks to Dr. K.P. Suresh and Dr. S. Jaya Kumar, for helping me in statistical
analysis and sequence data analysis.
I am also very much thankful to all the office staff members of the section for their timely
help, support and love and particularly to Sh. L. Krishnamurthy, Technical officer,
Dr. D. N. Hatkar, SRF and Ms. Chethana, SRF, Molecular Genetics Lab of AB & AI Section
for helping me in lab works, efficiency and programming skills to get my analysis completed in
time. I would also acknowledge Mr. Sri Hari, SRF for his advice and his willingness to share his
bright thoughts with me, which were very fruitful for shaping up my ideas and research. Hari sir
encouraged, motivated, empathized and simply beautified all my endeavours at NDRI,
Bangalore.
I am also grateful to express my wholehearted thanks to Mr. Nagaraj, Mr. Ramulu,
Mr. Ramalingam Mr. Kempraju, and Mr. Rangappa for their help in collection of cattle yard
data and for their assistance during blood collection.
I wish to express my heartfelt appreciation and thanks to my senior, Dr. Rengarajan
Kaliaperumal, Ph.D Scholar (AGB), Dr. R. Raja and Dr. G. Kumar, Ph.D Scholars (LPM),
NDRI Southern Campus, Dr. Indrasen Chauhan, Dr. Ritwik Hazra and Dr.Mandeep, Dr.
Nawale, Dr. Magar, Dr. Yathish, Dr. Tak, Dr. Sumit, Dr. Vijay, for their immense help,
invaluable guidance and constant encouragement at every stage of my research and dissertation.
I express my sincere love and affection to my classmates and friends with whom I shared
unforgettable moments. I thank Basir Ahmad, Fayaz Ahmad, Anil, Pankaj, Barat, Somya,
Manju, Archana, Ragu, Chandu Patil, Mushtaq, Rizwan, Shahid, Alok, Lohit, Samrat,
Zaheer, Manish, Sagar, Reddy, Raj, Tanu, Jaya and Kiran for the company, help, care and
affection, as they have been my morale booster and counselor throughout my work and for being
with me at every moment of my life at Karnal and Bangalore.
I would like to pay my heartiest regards to my beloved friends Dr. Bashir Ahmad,
Dr. Muzaffer, Dr. Awad Abdurrahman, Dr. Gulzar and Dr. Aadil for their unconditional
support when I needed the most. Words fail me to express my appreciation to shazu whose
dedication, love and persistent confidence in me, has taken the load off my shoulders. I owe her for
being unselfish in letting her intelligence, passions, and ambitions collide with mine.
My parents deserve special mention for their inseparable support, encouragement and
prayers throughout the period of my life. My Mother, Gasha Bano, in the first place is the person
who put the foundation of my learning character, showing me the joy of intellectual pursuit ever
since I was a child. My Father, Mr. Mohd Ahsan, is the one who sincerely raised me with his
caring and gentle love. Finally, I would like to thank everybody who was important to the
successful realization of thesis, as well as expressing my apology that I could not mention personally
one by one.
Dated:
(SHAHID AHMAD)
Molecular Genetic Characterization of Heat Shock Protein 90
gene in Deoni (Bos indicus) cattle
Abstract
PCR-SSCP analysis of HSP90AA1 gene was carried out in Deoni breed
of cattle to characterize HSP90AA1 gene and to evaluate the association
between genetic variants with reproductive performances. All the ten exons
of HSP90AA1 gene were amplified by PCR using a total of six sets of
overlapping primers. The genetic variants were determined by Single-Strand
Conformation Polymorphism (SSCP) analysis of amplified PCR products.
The exons 1 to 7 of HSP90AA1 gene showed monomorphism with
similar pattern in all the 72 animals studied. Three unique SSCP patterns
with a genotypic frequency of 0.250, 0.639 and 0.111, respectively were
observed in fragment 4 comprising Exon 8. Two SSCP patterns with a
genotypic frequency of 0.153 and 0.847 were observed in fragment 5
comprising Exon 9 of HSP90AA1 gene. The PCR-SSCP analysis of fragment 6
of HSP90AA1 gene comprising Exon 10 revealed two unique SSCP patterns
with a genotypic frequency of 0.236 and 0.764, respectively.
Based on the differences in the SSCP patterns, allelic variants were
selected and corresponding PCR products were sequenced to confirm
polymorphisms. Our sequences were compared to sequence of HSP90 AA1
gene in GenBank accession number NC-007319 for cattle by CLUSTAL-W
multiple sequence analysis. The analysis of fragment 4 comprising Exon 8
revealed T
G transversion at position 3650 of HSPAA1 gene. The observed
polymorphism (T
G) at position 3650 results in substitution of an amino
acid from Phenylalanine to Leucine. The cows having pattern III of fragment
4 which comprises Exon 8 had significantly higher age at first calving
as
compared to cows with pattern I and pattern II (P≤0.01). There was no
difference in calving interval in cows with different SSCP patterns in Exon 8.
The detected polymorphisms at position 4111 (C
position 4578 (A
G) in Exon 9 and at
G) in Exon 10 were silent mutations in the coding region
and had no association with reproductive performances in Deoni cattle. The
present study indicated the usefulness of genetic characterization of HSP 90
gene in association studies between reproductive traits and SSCP patterns.
CONTENTS
Sl.
No.
Title
Page
No.
1
INTRODUCTION
1-4
2
REVIEW OF LITERATURE
5-30
2.1
Heat stress
5
2.2
Effect of heat stress on productive and reproductive
performances in dairy cattle
6
2.3
Heat Shock Proteins (HSPs)
10
2.4
Function of HSP during stress
12
2.5
Genes involved in the bovine heat stress response
13
2.6
Structure and common features of HSP90
15
2.7
Mechanism of action of HSP 90
16
2.8
HSP90 and Glucocorticoid Receptors
18
2.9
Role of HSP90 in cancerous cells
19
2.10 Clinical significance of HSP90
2.11
HSP90 gene polymorphism and its role in thermotolerance
20
20
2.12 DNA Isolation
21
2.13 Polymerase Chain Reaction (PCR)
22
2.14 Single Strand Conformational Polymorphism (SSCP)
25
2.15 Deoni breed of cattle and its reproductive performance
28
2.16
Polymorphism in HSP genes and their association with
performance traits
29
3
MATERIALS AND METHODS
31-43
3.1
Experimental animals
31
3.2
Collection of blood
31
3.3
DNA Extraction by High Salt method
31
3.4
Primer Designing
34
3.5
Polymerase Chain Reaction – Single Strand
Conformational Polymorphism (PCR-SSCP)
34
3.6
Agarose gel electrophoresis
36
3.7
Custom Sequencing
42
3.8
Reproductive performance data of Deoni Cows
43
3.9
Statistical Analysis
43
4
RESULTS AND DISCUSSION
44-58
4.1
Yield and purity of DNA
44
4.2
PCR amplification
44
4.3
Single Strand Conformation Polymorphism (PCR-SSCP)
analysis
45
4.4
Association of SSCP patterns with reproductive
performance in Deoni cattle.
50
4. 5
Sequencing and analysis
55
5
SUMMARY AND CONCLUSIONS
59-62
BIBLIOGRAPHY
i-xix
APPENDIX
i-v
LIST OF TABLES
S.
No
Title
Page
No
3.1
Description of primers used for PCR amplifications of
HSP90 AA1 gene.
35
3.2
Composition of reaction mixture for PCR
36
3.3
Conditions of electrophoresis for SSCP analysis
38
4.1
Annealing Temperature and product size of different
fragments
45
4.2
Optimized Conditions for SSCP analysis
46
4.3
Genotypic frequencies and genetic diversity in fragment4 of HSP90 gene in Deoni cattle.
48
4.4
Genotypic frequencies and genetic diversity in fragment5 of HSP90 gene in Deoni cattle.
48
4.5.
Genotypic frequencies and genetic diversity in fragment6 of HSP90 gene in Deoni cattle.
49
4.6
Reproductive performances in Deoni breed of cattle
53
4.7
Effect of SSCP patterns of fragment-4 of HSP90 gene on
reproductive performances in Deoni cattle
53
4.8
ANOVA of SSCP patterns of fragment-4 of HSP90 gene
on reproductive performances in Deoni cattle.
54
4.9
Effect of SSCP patterns of fragment-5 of HSP90 gene on
reproductive performances in Deoni cattle
54
4.10
ANOVA of SSCP patterns of fragment-5 of HSP90 gene
on reproductive performances in Deoni cattle.
54
4.11
Effect of SSCP patterns of fragment-6 of HSP90 gene on
reproductive performances in Deoni cattle
55
4.12
ANOVA of SSCP patterns of fragment-6 of HSP90 gene
on reproductive performances in Deoni cattle.
55
4.13
Single Nucleotide Polymorphisms observed in HSP90
Gene in Deoni cattle
58
LIST OF FIGURES
Figure
No.
Title
After
Page
No.
2.1
A schematic description of the possible mechanisms
for the effect of heat stress on reproduction in the
lactating dairy cow
8
2.2
Domain structure of HSP 90
16
2.3
HSP90 chaperone system
18
2.4
Schematic diagram of the translocation of the GR
from the cytoplasm into the nucleus assisted by
HSP90.
18
2.5
Single-strand conformation polymorphism analysis
27
3.1
Deoni cow maintained at Cattle Yard, NDRI Southern
Campus
32
3.2
Deoni herd maintained at Cattle Yard, NDRI Southern
Campus
32
4.1
Quality checking of DNA on 0.8% agarose gel
45
4.2
Resolution of PCR amplified product of fragment -1 on
1.5% Agarose gel
45
4.3
Resolution of PCR amplified product of fragment -2 on
1.5% Agarose gel
45
4.4
Resolution of PCR amplified product of fragment -3 on
1.5% Agarose gel
45
4.5
Resolution of PCR amplified product of fragment -4 on
1.5% Agarose gel
45
4.6
Resolution of PCR amplified product of fragment -5 on
1.5% Agarose gel
45
4.7
Resolution of PCR amplified product of fragment -6 on
1.5% Agarose gel
45
Title
After
Page
No.
4.8
PCR-SSCP patterns of HSP90 Gene fragment -1 in
Deoni cattle
49
4.9
PCR-SSCP patterns of HSP90 Gene fragment -2 in
Deoni cattle
49
4.10
PCR-SSCP patterns of HSP90 Gene fragment -3 in
Deoni cattle
49
4.11
PCR-SSCP patterns of HSP90 Gene fragment -4 in
Deoni cattle
49
4.12
PCR-SSCP patterns of HSP90 Gene fragment -5 in
Deoni cattle
49
4.13
PCR-SSCP patterns of HSP90 Gene fragment -6 in
Deoni cattle
49
Figure
No.
ABBREVIATIONS
A
:
Adenine
AFC
:
Age at first calving.
A260
:
Absorbance at 260 nm
APS
:
Ammonium Persulphate
A280
:
Absorbance at 280 nm
ATG
:
Start codon
BLAST
:
Basic Local Alignment Search Tool
bp
:
Base pair
CI
:
Calving interval
C
:
Cytosine
DDH2O
:
Double Distilled Water
DNA
:
Deoxyribonucleic Acid
dNTPs
:
Deoxyribonucleotide Triphosphate
dATP
:
Deoxy Adenosine Triphosphate
dCTP
:
Deoxy Cytidine Triphosphate
dGTP
:
Deoxy Guanidine Triphosphate
dTTP
:
Deoxy Thiamine Triphosphate
EDTA
:
Ethidium Diamine Tetra Acetic Acid
EtBr
:
Ethidium Bromide
FSH
:
Follicle-Stimulating Hormone
G
:
Guanine
GR
:
Glucocorticoid receptor
GLM
:
General Linear Model
HBD
:
Hormone-Binding Domain
HS
:
Heat stress
HSP
:
Heat Shock Protein
Kb
:
Kilobase
Kg
:
Kilogram
LH
:
Luteinizing Hormone
Lt.
:
Litre
MgCl2
:
Magnesium Chloride
Min
:
Minute
mM
:
Milimolar
NaOH
:
Sodium Hydroxide
NCBI
:
National Centre for Biotechnology Information
nm
:
Nanometer
ORF
:
Open Reading Frame
OD
:
Optical Density
PAGE
:
Polyacrylamide Gel Electrophoresis
PCR
:
Polymerase Chain Reaction
pH
:
Negative logarithm of hydrogen ion concentration
RBC
:
Red Blood Cells
rpm
:
Revolutions per minute
Pmol
:
Pico mole
QTL
:
Quantitative Traits Loci
SSCP
:
Single-Strand Conformation Polymorphism
ssDNA
:
Single stranded DNA
SDS
:
Sodium Dodecyl Sulphate
SDP
:
Selective DNA Pooling
SNPs
:
Single Nucleotide Polymorphisms
T
:
Thymine
Taq
:
Thermus aquaticus
TBE
:
Tris Borate EDTA
IU
:
International Unit
UTR
:
Un-Translated Region
UV
:
Ultraviolet
V
:
Volt
WBC
:
White Blood Corpuscles
µl
:
Microlitre
µM
:
Micromolar
CHAPTER - 1
───────────────
Introduction
───────────────
1. INTRODUCTION
Most part of India lies in tropical region with temperature even
going up to 46°C during summer. These high temperatures are usually
associated with high humidity and availability of poor quality forage in
most parts of the country. Global warming has resulted in extensive
climatic changes in tropical regions, resulting in increased heat stress
in dairy animals of the region. The excessive heat stress during
summer season has led to observable reduction in milk production,
decreased reproductive performance and fitness in cattle.
Indigenous (Bos indicus) cattle survive and perform better under
heat stress as compared to temperate breeds or their crossbreds
(Collier et al., 2008). Deoni breed is one of the important dual purpose
breed of the Southern region of India having specific qualities like
disease resistance, heat tolerance, ability to survive and reproduce
under stress, low feed input and potential for improvement in dairy
traits (Das et al., 2011).
In India, crossbreeding of cattle has been practised to improve
milk production. We have to respond by propagating thermo adaptable
crossbred cattle in future to achieve optimum profits under climate
change scenario. Although India produced about 110 million tonnes of
milk by the year 2010, it is losing nearly two percent of the total milk
production among cattle and buffaloes due to rise in heat stress and
global warming. The loss of milk production due to heat stress in
monetary terms amounts to a whopping Rs 2,661.62 crore per year
(Upadhyay, 2010). An increase of about 0.9°F in a cow’s body
1
Introduction
temperature has been estimated to cause 12.8 per cent decline in
conception rate in cattle (Pete, 2006).
Genetic differences in thermo tolerance at the physiological and
cellular levels are documented by a number of studies on Bos indicus
and Bos taurus cattle breeds (Paula-Lopes et al. 2003; Hansen., 2004;
Lacetera et al., 2006). One possible approach for reducing the impact
of heat stress on cattle productivity is to select and breed animals with
thermo tolerance. The above scenario emphasizes the importance of
the study of genetic mechanisms that play a role in providing heat
tolerance in cattle, so as to implement proper breeding program for
developing cattle having better thermo tolerance. This requires
simultaneous identification and selection of cattle for improved heat
dissipation mechanisms.
It has been reported that about a 20% drop in conception rates
(Lucy., 2002) or decrease in 90-day non-return rate to the first service
could occur in summer among lactating dairy cows (Al-Katanani et al.,
2002). Heat stress can alter follicular growth (Roth et al., 2000),
steroid secretion (Wolfenson et al., 2000; Ozawa et al., 2005) and
gene expression (Argov et al., 2005). Heat shock protein expression
has been detected in gametes and early-stage embryos (Edwards and
Hansen., 1996; Sagirkaya et al., 2006; Collier et al., 2008; Wilkerson
and Sarge, 2009). These reports suggest that HSP gene expression is
related to embryonic survival and overall pregnancy success, thereby
affecting reproductive performance.
Cellular tolerance to heat stress is mediated by a family of
proteins named Heat Shock Proteins (HSP). Among members of the
HSP family, HSP70 and 90 are the most abundant proteins in
eukaryotic cells. These HSPs protect cells from toxic effects of heat
2
Introduction
and other stresses. The thresholds for expression of HSPs are
correlated with levels of stress naturally undergone by the animals.
The protective function of HSPs relies on their chaperone activity which
consists of assisting the non-covalent assembly and or disassembly of
other macromolecular structures (Ellis, 2006). The chaperone known
as HSP90 (90-kDa) is one of the most abundant proteins in eukaryotic
cells, comprising 1-2% of cellular proteins under non-stress conditions.
There are two major isoforms of HSP90 which have arisen by gene
duplication,
(constitutive
HSP90AA1
form).
(alpha)
HSP90
(inducible
proteins
have
form)
and
key
roles
HSP90
in
β
signal
transduction, protein folding, protein degradation, and morphological
evolution.
At the cellular level, genetic adaptations to resist deleterious
effects of elevated temperature results in successful preimplantation of
embryos in zebu breeds during embryo development compared to
embryos from taurine breeds. Few studies confirm a genetic linkage
between species, breed, and individual differences to heat tolerance at
the cellular level (Collier et al., 2008) The zebu genotype has been
utilized in crossbreeding systems to develop cattle for beef and dairy
production systems in hot climates but success has been limited by
other
unfavourable
genetic
characteristics
of
these
cattle.
An
alternative scheme is to incorporate specific thermo tolerance genes
from zebu cattle into European breeds while avoiding undesirable
genes.
Once specific genes responsible for thermo- tolerance in zebu
cattle have been identified or mapped, breeding strategies such as
Marker Assisted Selection (MAS) and transgenics can be applied to
further the utilization of the zebu genotype for cattle production
3
Introduction
systems. Therefore, information about variation in specific genes, such
as those affecting resistance to stressful conditions can constitute a
valuable tool for ecological genetic studies, so that we can propagate
thermo adaptable cattle as future generation to achieve optimum
profits under global warming scenario. HSP gene family have been
widely discussed as candidate genes for heat resistance (Hoffmann et
al., 2003) and few studies have shown association between Single
Nucleotide Polymorphisms (SNPs) at HSP genes and stress resistance
in different species (Reddacliff et al., 2005; Sun et al., 2007;
Li et al.,
2009).
Very few studies have been made in cattle for identifying genetic
variation in HSPs heat tolerance. Determining genetic variation in HSP
90 gene in Bos indicus cattle (Deoni breed) and its association with
reproductive performance will aid in selecting cattle for thermotolerance. Hence the present study was undertaken with the following
objectives:
i.
Molecular characterization of HSP90 gene in Deoni breed of cattle.
ii. To study the association of HSP90 gene haplotype/variation with
reproductive performances.
4
Introduction
CHAPTER - 2
─────────────────────────
Review of Literature
─────────────────────────
2. REVIEW OF LITERATURE
Homeothermic animals require relatively constant core body
temperature for their vital and productive processes. All animals have
a comfort range of ambient environmental temperatures termed as the
thermo-neutral zone. The range of temperature that is conducive to
health and performance varies from 15 to 25°C for crossbred cattle
and buffaloes and 15-28°C for indigenous cattle (Singh and Upadhyay,
2009). Diurnal variation of body temperature doesn’t exceed 1°C, if
the animal is exposed to a natural environment. The upper critical
temperature is the point at which heat stress begins to affect the
animal’s performance.
2.1
Heat stress
Heat shock or stress response is a cellular adaptive response,
which helps to maintain cellular homeostasis under stress. Stress
denotes the magnitude of external forces which tend to displace the
bodily system from its resting or ground state (Yousef, 1985). Stress
may be climatic, such as severe cold or heat; nutritional, due to feed
or water deprivation; or internal, due to some physiological disorder,
pathogens or toxins (Stott, 1981). Heat stress occurs when any
combination of environmental factors cause the effective temperature
of the environment to be higher than the animal’s thermoneutral zone
(Armstrong, 1994).
The effects of heat in the organisms are related to hyperthermia
and consequent impairment of tissue and organ functions through
reduction in blood flow (Kregel, 2002). These effects can be observed
in
several
tissues
and
systems,
5
including
reproductive
system
Review of literature
compromising spermatogenesis and viability of oocytes and embryos
(Rivera & Hansen, 2001; Krininger et al., 2003; Roth & Hansen,
2004). Thermo-tolerance describes the phenomenon whereby prior
exposure of cells / tissues to a mild heat stress (e.g. 1 hr at 42°C for
mammalian cells) confers resistence to a subsequent exposure to an
otherwise lethal stress (eg. 1 hr at 45-48°C) (Landry et al.,1994).
2.2 Effect of heat stress on productive and reproductive
performances in dairy animals
Heat stress is one of the major factors that have a negative
impact on milk production and reproduction in dairy cattle. Stress from
the thermal environment is a major factor negatively affecting
productive and reproductive performances of dairy cattle. Because of
the major economic importance of this problem, heat stress in dairy
cattle has been extensively discussed by many researchers (Yousef,
1985; Shearer and Beede, 1990a and b; Nienaber et al., 2003; StPierre et al., 2003; West, 2003).
2.2.1 Decreased milk production
Reduced milk yield under heat stress is caused by associated
effects on thermal regulation, energy balance and endocrine changes
(Yousef, 1985; Ominski et al., 2002) and reported a decrease of 4.8 %
in milk production when cows were exposed to heat stress compared
to their milk production in the thermo neutral zone. Bouraoui et al., 2002
reported that for every degree above temperature humidity index of 69
in dairy cattle a 0.4 kg decrease of milk production. Berman (2005)
estimated
that
effective
environmental
heat
loads
above
35°C
activated the stress response system in lactating dairy cows.
6
Review of literature
The
reduction
of
productive
performance
in
cattle
with
devastating economic consequences to the global dairy industry due to
warm environment has been documented by researchers (St-Pierre et
al., 2003; Bernabucci et al., 2010). They have estimated a total
economic loss incurred by the US livestock sector due to Heat stress at
between 1.69 and 2.36 billion US dollars. About 58% of this loss
occurs in the dairy industry, 20% in the beef industry, 15% in pigs and
the remaining 7% in the poultry industry (Bernabucci et al., 2010).
There is normally a decrease in milk production in a cattle and
buffaloes under heat stress. This decrease can be either transitory or
long term, depending upon the length severity of heat stress. The
decrease in milk production ranges from 10-25% (Upadhyay et al.,
2009a).
2.2.2 Decreased reproductive performance
Heat stress dramatically lowers conception rates, influences
estrus behavior, modifies endocrine function, alters the oviduct and
uterine
environments,
and
delays
or
interrupts
early
embryo
development of dairy cattle. Heat stress does not prevent the
occurrence of normal estrus cycles. It does, however, amplify the
problem of heat detection by reducing the length of the estrus period,
from 18 hours down to about 10 hours, and lowering the intensity of
estrus behavior (Shearer and Beede, 1990a). Heat stress compromises
oocyte growth in cows by altering Progesterone, Luteinizing hormone
and Follicle-stimulating hormone secretions during the oestrus cycle
(Ronchi et al.,2001), as well as impairing embryo development and
increasing embryo mortality (Wolfenson et al., 2000). Heat stress
reduces the fertility of dairy cows in summer by poor expression of
oestrus due to reduced estradiol secretion from dominant follicle
7
Review of literature
developed in a low luteinizing hormone environment (De Rensis and
Scaramuzzi, 2003).
Lucy (2002) reported 20% drop in conception rates among dairy
cows in summer while Al-Katanani et al. (2002) reported decrease in
90-day nonreturn rate to the first service in lactating dairy cows due to
heat stress in summer. Heat stress can alter follicular growth steroid
secretion (Wolfenson et al., 2000; Roth et al., 2000; Ozawa et al.,
2005) and gene expression (Argov et al., 2005). In goats, heat stress
reduced plasma concentrations of oestradiol and lowered follicular
oestradiol concentration, aromatase activity and LH receptor level, and
delayed ovulation (Ozawa et al., 2005). Previous studies conducted on
rats have demonstrated that heat stress reduced the levels of
gonadotropin receptors and aromatase activity of granulosa cells and
the follicular fluid concentrations of oestradiol (Shimizu et al., 2005).
Heat stress could lead to increase in calving interval, lower birth rate
and a reduced farm milk yield per year (Roth et al., 2000). The
principal site of action of heat stress on the reproductive axis appears
to be hypothalamic region. However, additional sites of action in the
ovary and the uterus also appear to be important (Figure 2. 1).
There appear to be two distinct and largely independent
pathways by which heat stress leads to infertility. The first is a direct
effect of hyperthermia on the reproductive axis. Hyperthermia leads to
increased lethargy and a compromised uterine environment, both of
which can lead to worsening infertility through poor estrus detection
and embryo loss.The second is an indirect effect related to the effects
of heat stress on appetite and dry matter intake, both of which are
reduced by heat stress. The consequence is worsening energy balance
and since the postpartum dairy cow tends to be in negative balance,
8
Review of literature
Figure 2.1: A schematic description of the possible mechanisms for
the effect of heat stress on reproduction in the lactating
dairy cow adopted from Ahmad and Tariq (2010).
the consequences of heat stress on fertility are more likely to be
severe.
Negative energy balance produces lower blood concentrations of
insulin and insulin like growth factor (IGF-I) and higher blood
concentrations of growth hormone (GH) and non essential fatty acid
(NEFA) and this altered metabolic profile acting via the hypothalamopituitary system reduces GnRH and LH secretion, leading to reduced
estradiol secretion by the dominant follicle. The consequences of
reduced estradiol secretion from the dominant follicle are poor estrus
detection, compromised oocyte quality, and in extreme situations,
ovulatory failure. The role of progesterone in summer infertility, if any,
remains uncertain. There is a reduction in LH secretion leading to
reduced estrogen secretion, impaired detection of estrus, reduced
oocyte quality, implantation failure and infertility. Heat stress will also
change
the
secretion
adrenocorticotrophic
of
(cortisol)
thyrotrophic
and
(thyroxine)
adrenomedullary
and
hormones
(adrenaline), these potentially impair fertility (Ahmad and Tariq 2010).
There are a number of changes in reproductive performance that
have been reported (Upadhyay et al., 2009b). These include:
¾ Decreased length and intensity of the oestrus period
¾ Decreased conception (fertility rate)
¾ Decreased growth, size and development of ovarian follicles
¾ Decreased foetal growth and calf size
¾ Increased risk of early embryonic deaths
¾ Increased number of AI per conception
¾ Increased incidence of silent heat especially in buffaloes.
9
Review of literature
2.3 Heat Shock Proteins (HSPs)
The heat shock response is one of the most ancient and
conserved cellular stress responses, which is characterized by the
transcriptional activation and accumulation of the Heat Shock or Stress
Proteins (HSPs) (Schlesinger, 1990). They are classified into 5 families
according to their molecular weight, HSP100, HSP90, HSP70, HSP60
and the small HSPs. The heat shock genes are highly preserved and
show
low
variation
between
species,
suggesting
evolutionary
importance of cell protection during and after stress (Chen et al.,
2005). The chaperone known as HSP90, 90-kDa heat shock protein, is
one of the most abundant proteins in eukaryotic cells, comprising 1–
2% of cellular proteins under non-stress conditions.
There are two major cytoplasm isoforms of HSP90 which have
arisen by gene duplication, HSP90α (inducible form) and HSP90β
(constitutive form). The contribution cytosolic HSP90 to various
cellular processes including signal transduction, protein folding, protein
degradation, cell survival, and morphological evolution has been
extensively studied (Chen et al., 2006). Additionally, a quantitative
trait loci study performed on Drosophila melanogaster (Morgan and
Mackay, 2006) revealed a genomic region on chromosome 3 with an
important effect on heat stress resistance. That region contained
several positional candidate genes, including HSP83, which is the
homolog of the mammalian HSP90 family in Drosophila.
Heat shock proteins, as a class, are among the most highly
expressed cellular proteins across all species. As name implies, heat
shock proteins protect cells when stressed by elevated temperatures.
However when cells are heated, the fraction of heat shock proteins
increases to 4–6% of cellular proteins (Crevel et al., 2001). Many
10
Review of literature
members of these HSP families are present constitutively (heat shock
cognates) in cells while some are expressed only after stress. Heat
shock proteins function as molecular chaperones that help animals to
cope with stress. They can be induced by environmental factors or
genetic stress (inbreeding). HSPs are primarily involved in protein
quality system, they fold proteins and prevent aggregation of
misfolded proteins (Sorensen et al., 2003). HSP expression is fine
tuned (not being only an on-off mechanism) and are also continuously
expressed after a mild chronic stress exposure (Lindquist and Craig
1986; Hoffmann et al., 2003).
The induced and the constitutively expressed members of HSP
families are well known as molecular chaperones which i) help in
normal folding of various polypeptides, ii) assist mis-folded proteins to
attain or regain their native states, iii) regulate protein degradation
and/or
iv) help in translocation of proteins to different cellular
compartments (reviewed in Hartl and Hayer-Hartl, 2002; Kregel 2002;
Soti et al., 2005).The above functions imply that the HSPs interact
with a very large variety of cellular proteins and thus are important
components of cellular networks (Csermely, 1998). This is also
reflected by their roles, especially of HSP90, in evolvability (Craig et
al., 1993; Rutherford and Lindquist, 1998).
Many studies have shown that the heat shock proteins play
critical roles in modulating the apoptotic cascades (Samali and
Orrenius, 1998; Kim et al., 2009). Cultured bovine, ovine, equine and
chicken lymphocytes have all been shown to respond to heat stress by
increasing the expression of both HSP70 and HSP90 (Hansen et al.,
1991; Rutherford and Lindquist., 1998). Control of heat shock protein
synthesis has been studied extensively (Collier et al., 2008). Heat
11
Review of literature
shock protein expression has been detected in gametes and earlystage embryos (Edwards and Hansen, 1996; Sagirkaya et al., 2006;
Wilkerson and Sarge, 2009). Earlier reports suggest that heat shock
protein gene expression was related to embryonic survival and overall
pregnancy success (Wilkerson and Sarge, 2009).
2.4 Function of HSP during stress
During stress, heat shock proteins associate with denatured,
damaged proteins, thereby preventing them from interacting with
other proteins and cellular constituents. Following the stress period,
some HSPs function as molecular chaperones to catalyze the refolding
of denatured proteins (Parsell and Lindquist, 1994). The protective
function of HSPs against multiple stressors is manifested in part by
their ability to confer thermotolerance to cells in which their expression
is elevated. The protective effects of a pre-heat stress have been
shown to increase survival rates to subsequent heat stress in a
number of organisms, including yeast, cultured mammalian cells and
Drosophila (Landry and Gierach, 1994).
Fluctuation of the environmental temperature can lead to
induction of cellular stress responses, including the HSP family in
mussels (Hofmann et al., 2003). Sudden changes in temperature are
most ubiquitous stress that cells had to cope with to preserve their
structural and enzymatic integrity (Nadeau and Landry, 2006). The
variation in the environmental temperature will generally impose
stress upon the organism, which may result in the evolution of
adaptative genetic mechanisms to cope with temperature extremes in
nature (Hoffmann and Parsons, 1991).
12
Review of literature
2.5 Genes involved in the bovine heat stress response
Genetic differences in thermo tolerance at the physiological and
cellular levels are documented by a series of studies on Bos indicus
and Bos taurus cattle (Paula-Lopes et al., 2003; Hansen 2004;
Lacetera et al., 2006). A genetic linkage between species, breed, and
individual differences to heat tolerance at the cellular level has been
reported by various researchers (Collier et al., 2008). Selection of
animals for thermo tolerance is one possible approach for reducing the
impact of heat stress on cattle productivity.
Since genetic variation for heat tolerance exists in dairy cattle,
there is possibility of specific genes controlling heat tolerance could be
introduced into the gene pool of the population. Olson et al., (2006)
identified a single gene in cattle affecting hair coat density and hair
length. This slick hair gene was also associated with increased
Evaporatory Heat Loss (EVHL) in homozygous cattle under heat stress
conditions (Olson et al., 2006). Slick hair gene originally described in
Senepol cattle, subsequently identified in Carora cattle, and introduced
into Holsteins by crossbreeding (Olson et al., 2003). Slick hair gene
has been mapped to chromosome 20 (Mariasegaram et al., 2007). The
other genes influencing heat tolerance are group of genes referred to
as the Heat Shock Protein (HSP) gene family.
2.5.1 Heat shock gene families
A group of proteins are synthesized during heat stress called
Heat Shock Proteins (HSPs),they are a family of proteins from
pleiotropic genes that protect cells from toxic effects of heat and other
stresses. The thresholds for expression of HSPs are correlated with
levels of stress naturally undergone by the animals (Feder and
Hofmann, 1999).
13
Review of literature
Members of the HSP60 family are called chaperonins. HSP60
protein expression increases in response to heat shock and other
stressors. The mammalian HSP60 proteins are ring-shaped molecular
chaperones that are synthesized in the cytoplasm and are then
translocated to the mitochondria where they use the energy of ATP
hydrolysis for protein folding (Gething and Sambrook, 1992; Welsh,
1992).
Among members of the HSP family, HSP70 (namely, HSP70.1
and HSP70.2) is the most abundant and temperature sensitive
(Dokladny et al., 2006) HSP70 transcription is increased by heat shock
as
well
as
other
stress
stimuli
such
as
oxidative
stress,
ischemia,inflammation, or aging and can be an indicator of stress in
cells (Favatier et al., 1997; Sonna et al., 2002; Saibil, 2008)
2.5.2 The HSP 90 family
HSP90 (Heat Shock Protein 90) is a molecular chaperone and is
one of the most abundant proteins expressed in cells (Csermely et al.,
1998). These cells were stressed by heating, dehydrating or by other
means, all of which caused the cell’s proteins to begin to denature
(Prodromou and Pearl, 2003). It is a member of the heat shock protein
family which is upregulated in response to stress. HSP90 is found in
bacteria and all branches of eukaryotes, but it is apparently absent in
archaea (Chen, 2007). Cytoplasmic HSP90 is essential for viability
under all conditions in eukaryotes. (Thomas and Baneyx, 1998). Like
all heat shock proteins, members of the HSP90 family maintain a high
degree of sequence conservation across diverse species (Chen et al.,
2006). The function of HSP90 includes assisting in protein folding, cell
signaling, and tumor repression. This protein was first isolated by
extracting proteins from stressed cells.
14
Review of literature
2.5.3 HSP90 genes and isoforms
HSP90 is highly conserved and expressed in a variety of different
organisms from bacteria to mammals. (Chen et al., 2006) There are
two major cytoplasm isoforms of HSP90 which have arisen by gene
duplication, HSP90α (inducible form) and HSP90β (constitutive form)
2.6 Structure and Common features of HSP90
The overall structure of HSP90 is similar to other proteins (Pearl
and Prodromou, 2001). It contains all of the common secondary
structural elements (i.e., alpha helixes, beta pleated sheets and
random coils) HSP90 consists of four structural domains
¾ a highly conserved N-terminal (NTD) domain of ~25 kDa,
¾ a "charged linker" region, that connects the N-terminus with the
middle domain,
¾ a middle domain (MD) of ~40 kDa, and
¾ a C-terminal domain (CTD) of ~12 kDa (Pearl and Prodromou,
2000; Prodromou et al.,2000).
HSP90 forms homodimers where the contact sites are localized
within the C-terminus in the open conformation of the dimer. The Ntermini also come in contact in the closed conformation of the dimer
(Sato et al., 2000). Crystal structures are available for the N-terminal
domain of yeast and human HSP90, for complexes of the N-terminus
with inhibitors and nucleotides, and for the middle domain of yeast
HSP90 (Prodromou et al., 1997).
2.6.1 Domain structure of HSP 90
Domain
structure
of
HSP
90
as
reported
by
Pearl
and
Prodromou, 2001 is shown in (Figure 2.2).
15
Review of literature
N-terminal
domain:
The
N-terminal
domain
shows
high
homology not only amongst members of the HSP90 chaperone family,
but also to members of the ATPase/kinase GHKL (Gyrase, HSP90,
Histidine Kinase, MutL) superfamily
(Pearl
and Prodromou., 2001).
Amino acids that are directly involved in the interaction with ATP are
Leu34, Asn37, Asp79, Asn92, Lys98, Gly121, and Phe124.
Middle domain: The middle domain is divided into three
regions: a 3-layer α-β-α sandwich, a 3-turn α-helix and irregular loops,
and a 6-turn α-helix (Pearl and Prodromou,2001).
C-terminal domain: The C-terminal domain possesses an
alternative ATP-binding site, which becomes accessible when the Nterminal Bergerat pocket is occupied (Soti et al., 2002).
2.7 Mechanism of action of HSP 90
The HSP90 protein contains three functional domains, the ATP
binding, protein binding domain, each of which play a crucial role in
the function of the protein.
ATP binding: The ATPase binding region of HSP90 is currently
under intense study, because it is the principal binding site of drugs
targeting this protein. Antitumor drugs targeting this section of HSP90
include the antibiotics geldanamycin, herbimycin, radicicol, deguelin,
derrubone, and macbecin (Grenert et al., 1997).
Protein binding: The protein binding region of HSP90 is located
towards the C-terminus of the amino sequence. The ability of HSP90 to
clamp onto proteins allows it to perform several functions including
assisting folding, preventing aggregation, and facilitating transport.
(Buchner, 1999).The full functional activity of HSP90 is gained in
16
Review of literature
Figure 2.2: Domain structure of HSP 90
concert with other co-chaperones, playing an important role in the
folding of newly synthesized proteins and stabilization and refolding of
denatured proteins after stress. Apart from its co-chaperones, HSP90
binds to an array of client proteins, where the co-chaperone
requirement varies and depends on the actual client. There is a
growing list of HSP90 client proteins, now including several hundred
proteins. Interestingly, most of the clients include molecules involved
in signal transduction (Ali et al., 2006).
Normal cells: In unstressed cells, HSP90 plays a number of
important roles, which include assisting folding, intracellular transport,
maintenance, and degradation of proteins as well as facilitating cell
signaling.
Protein
degradation:
Protein
molecules
are
continuously
synthesized and degraded in all living organisms. The concentration of
individual cellular proteins is determined by a balance between the
rates of synthesis and degradation, which in turn are controlled by a
series of regulated biochemical mechanisms. The only way that cells
can reduce the steady state level of a particular protein is by
degradation. Thus, complex and highly-regulated mechanisms have
been evolved to accomplish this degradation. Cells which are subject
to stress such as starvation, heat-shock, chemical stress or mutation
respond by increasing the rates of degradation. Selective degradation
of particular proteins may occur in response to internal and external
signals. Furthermore a constant supply of functional HSP90 needed to
maintain the tertiary structure of the proteasome. Experiments done
with heat sensitive HSP90 mutants and the 26S proteasome suggest
that HSP90 is responsible for most, if not all, of the ATPase activity of
the proteasome (Pratt and Toft, 2003).
17
Review of literature
Protein folding and role as chaperone: Heat shock proteins
are molecular chaperones that assist in protein folding and in the
prevention of cytotoxic aggregations resulting from improperly folded
proteins adhering to one another (Benjamin and McMillan, 1998). They
also help shuttle proteins from one compartment to another inside the
cell, and transport old proteins to “garbage disposals” inside the cell.
HSP90 is known to be associated with the non-native structures of
many proteins, which has led to the proposal that HSP90 is involved in
protein folding in general (Miyata and yahara,1992). Furthermore
HSP90 has been shown to suppress the aggregation of a wide range of
"client" or "substrate" proteins and hence acts as a general protective
chaperone. However HSP90 is somewhat more selective than other
chaperones (Fig. 2.3) (Imai et al., 2003; Saibil, 2008).
2.8 HSP90 and Glucocorticoid Receptors
HSP90 in association with one glucocorticoid receptor (GR) has
an important role in the hormonal response to stress. The GR-HSP90
complex consists of one molecule of steroid-binding protein associated
with two molecules of HSP90 (Mendel and Orti, 1988). It is known that
HSP90 binds to the hormone-binding domain (HBD) of the GR (Pratt et
al., 2006). HSP90 must be bound in order for the HBD to be in a high
affinity steroid-binding conformation as it was found that in the
absence of HSP90 there was no binding of steroid by intact GR
(Bresnick et at., 1989). Once steroid binds to the receptor-HSP90
heterocomplex, HSP90 dissociates from the GR and this transformation
somehow allows the receptor to dimerize, bind to DNA and begin
transcriptional activating activity.
In the cytoplasm, GR is complexed with HSP90 and the
immunophilin FKBP51 (51). Binding of hormone to GR causes a
18
Review of literature
Figure 2.3: HSP90 chaperone system - adopted from Saibil.(2008).
Figure 2.4: Schematic diagram of the translocation of the Glucocorticoid
receptor( GR) from the cytoplasm into the nucleus assisted
by HSP90 (90) adopted from Lurje et al. (2009)
conformational change in the complex which results in exchange of
FKBP51 for FKBP52 (52). FKBP52 in turn binds the dynein (dyn) motor
protein which attaches to the cytoskeleton and transports the GR
complex into the nucleus (Davies et al., 2002; Dollins et al., 2007).
Once in the nucleus, the complex disassembles releasing GR which
dimerizes and binds to DNA where it facilitates transcription of DNA
into mRNA. The glucocorticoid receptor (GR) is the most thoroughly
studied example of a steroid receptor whose function is crucially
dependent on interactions with HSP90. In the absence of the steroid
hormone cortisol, GR resides in the cytosol complexed with several
chaperone proteins including HSP90 (Figure 2.4). These chaperones
maintain the GR in a state capable of binding hormone. A second role
of HSP90 is to bind immunophilins that attach the GR complex to the
dynein protein trafficking pathway which translocates the activated
receptor from the cytoplasm into the nucleus (Davies et al., 2002).
Once in the nucleus, the GR dimerizes and binds to specific sequences
of DNA and thereby upregulates the expression of GR responsive
genes. HSP90 is also required for the proper functioning of several
other steroid receptors, including those responsible for the binding of
aldosterone, androgen,
estrogen, and progesterone (Pratt et al.,
2006; Lurje and Lenz, 2009)
2.9 Role of HSP 90 in cancerous cells
Cancerous cells over express a number of proteins, including
growth factor receptors, such as EGFR, or signal transduction proteins
such as PI3K and AKT (Inhibition of these proteins may trigger
apoptosis). HSP90 stabilizes various growth factor receptors and some
signaling molecules including PI3K and AKT proteins, hence inhibition
of HSP90 may induce apoptosis through inhibition of the PI3K/AKT
signaling pathway and growth factor signaling (Calderwood et al.,
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Review of literature
2006). Another important role of HSP90 in cancer is the stabilization of
mutant proteins such as v-Src, the fusion oncogene Bcr / Abl, and
mutant forms of p53 that appear during cell transformation. It appears
that HSP90 can act as a "protector" of less stable proteins produced by
DNA mutations (Eustace et al., 2004; Oh et al., 2007)
2.10 Clinical significance of HSP90
HSP90 plays a Janus-like role in the cell, where it is essential for
the creation, maintenance, and destruction of proteins. Its normal
function is critical to maintaining the health of cells, whereas its
dysregulation may contribute to carcinogenesis. The ability for this
chaperone to both stabilize the 26S proteasome (which enables the
cell to degrade unwanted and/or harmful proteins) and to stabilize
kinases against the same proteasome demonstrates its functional
diversity (Goetz et al., 2003).
2.11 HSP90
gene
polymorphism
and
its
role
in
thermo
tolerance
Various studies have confirmed the association between Single
Nucleotide Polymorphisms (SNPs) at certain HSP genes and stress
tolerance (Reddacliff et al., 2005; Sun et al., 2007; Li et al., 2009).
HSP90 AA1 gene is located on chromosome No.21 in Bos taurus.
HSP90 gene is composed of 10 exons with a total length of 5332 bp
(http://www.ncbi.nlm.nih.gov/BLAST). The amount of HSP90 gene
expression increases with the levels of stress undergone by the
animal. In eukaryotes HSP90 belongs to a multigene family. In yeast,
the genes coding for HSP90 (also called HSP82) and for HSP90 (Heat
shock cognate, a constitutively expressed isoform) have been cloned
(Chen et al., 2006). In S. cerevisiae the HSP90 protein (HSP82) is
essential for survival at all temperatures. Further, the amount of
20
Review of literature
HSP90 required for growth increases as the temperature increases
(Parsell and Lindquist, 1994; Chen et al., 2006), which suggests that
the HSP90 proteins have acquired additional protective functions in
eukaryotes which they do not share with prokaryotic HSP90 proteins.
Chen et al. (2006) assessed the function of HSP90 in vivo by utilizing a
mutant of S. cerevisiae, which contained a mutation in the HSP90
gene resulting in a complete loss of HSP90 protein activity at high
temperatures, and consequent inability to survive heat shock. HSP90
facilitates folding of a specific subset of proteins that would otherwise
not attain their native conformation, which suggests that in S.
cerevisiae, HSP90 functions not to prevent thermal-damage to
proteins, but rather to enhance reactivation of heat-damaged proteins.
The common view on the protective function of HSP relies on their
chaperone
activity
which
consists
of
assisting
the
noncovalent
assembly and/or disassembly of other macromolecular structures
(Ellis, 2006).
2.12 DNA isolation
Genomic DNA is the starting material for most of the molecular
biology techniques. Whole blood and semen are convenient sources of
nucleated cells for the extraction of genomic DNA from farm animals.
The primary objective of the isolation process is to recover the
maximum yield of high molecular weight DNA devoid of protein and
enzyme inhibitors (Sambrook and Russel, 1989).
Phenol-chloroform extraction method, (Andersson et al., 1986),
Guanidine hydrochloride method (Jeanpierre, 1987) and High salt
method (Miller et al., 1988) are commonly used for extraction of DNA
from blood of farm animals. Many researchers followed High salt
method for the isolation of genomic DNA with an additional chloroform
21
Review of literature
extraction method from blood samples of cattle (Kantanen et al.,
1995; Ramesha et al., 2002).
2.12.1 Yield and quality of DNA
Senthil (1995) extracted DNA from blood samples of cattle by
high-salt method and obtained pure DNA in the range of 450µg to
800µg with an average of 625µg per 15ml of blood. Appannavar
(2001) isolated DNA from whole blood of Deoni cattle by High salt
method and the yield of DNA was in the range of 350µg to 575µg per
10ml of blood.
2.13 Polymerase Chain Reaction (PCR)
The PCR is an in vitro method for the enzymatic synthesis of
specific DNA sequences, using two oligonucleotide primers that
hybridize to opposite strands and flank the region of interest in the
target
DNA.
The
PCR
allows
the
amplification
of
a
specific
deoxyribonucleic acid (DNA) region that lies between two regions of
known DNA sequence.
2.13.1 Components of PCR
PCR Components are critical to a successful PCR amplification.
These Components include DNA Template, Oligonucleotide primers,
Magnesium Concentration, Deoxynucleotides, Taq DNA Polymerase
Concentration, PCR Buffers (Sambrook and Russel, 1989).
DNA
polymerase:
The
most
commonly
used
Taq
DNA
polymerase is isolated from Thermus aquaticus. A recommended
concentration of Taq DNA polymerase varies between l.0 and 2.5 units
per
high
100 l reaction mixture when other parameters are optimum. Too
concentration
results
in
the
22
accumulation
of
nonspecific
Review of literature
background products, while at too low concentration of enzyme, the
yield may be insufficient.
Deoxy
ribonucloeoside
triphosphates:
PCR
is
normally
performed with dNTP concentration around 100µM, although at lower
dNTP concentration (10-100µM) Taq DNA polymerase has a higher
fidelity. The four dNTPs should be used at equivalent concentrations
to
minimize
misincorporation
errors
and
lowest
possible
dNTP
concentration appropriate for the length and composition of the target
sequence must be used (Innis and Gelfand, 1990).
PCR primers: For most applications of PCR, the primers are
designed to be exactly complementary to template DNA.
Generally,
the primers used in PCR are between 20-30 nucleotides in length and
higher
GC
content,
which
allows
a
reasonably
high
annealing
temperature to be used. Primer concentration between 0.1 and 0.5 µM
are considered optimum (Innis and Gelfand, 1990).
PCR buffer: PCR buffer is an important component of PCR which
affect the outcome of amplification. In particular, the concentration of
MgCl2
has
a
profound
effect
on
the
specificity
and
yield
of
amplification. Innis and Gelfand (1990) recommended a PCR buffer
that contains 10-50mM Tris HCl with pH between 8.3 and 8.8 at 20°C.
Generally excess Mg2+ will result in the accumulation of nonspecific amplification products and insufficient Mg2+ will reduce the
yield (Innis and Gelfand, 1990). A magnesium concentration of 1.5–
2.0 mM is optimal for most PCR products generated with Taq DNA
Polymerase.
Optimization
normally
involves
supplementing
the
magnesium concentration in 0.5 or 1.0 mM increments (Sambrook and
Russel, 1989).
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Review of literature
DNA Template: Use of high quality, purified DNA templates
greatly enhances the success of PCR reactions. Optimum concentration
of template DNA per reaction could vary substantially from typical
conditions (100ng per reaction) depending on the primer-template
combination use (Sambrook and Russel, 1989). According to Invinson
and Taylor (1992), 100 to 500 ng of genomic DNA should be used as
target,
although
anything
between
50
to
1000
ng
produces
satisfactory amplification (Ramesha et al., 2002)
Taq DNA polymerase Concentration: Taq DNA Polymerase is
normally used at a final concentration of 25 units/ml (1.25 units/50 µl
reaction), but can range from 5–50 units/ml (0.25–2.5 units/50 µl
reaction) in specialized applications.
Cycling Parameters: PCR is performed in three steps in a
cycle of amplification, namely denaturation, annealing and extension.
In a typical reaction, the double stranded DNA is denatured by briefly
heating the sample to 90°C for 30sec or 97°C for 15sec (Innis and
Gelfand, 1990). Several workers have used template denaturation
conditions of 94°C for 30 sec (Van Eijk et al., 1992; Kantanen et al.,
1995) and 94°C for 45sec (Ramesha et al., 2002). For difficult
templates such as GC-rich sequences, a longer denaturation of 2–4
minutes at 94°C is recommended prior to PCR cycling to fully denature
the template.
The primers are allowed to anneal to their complementary
sequences by briefly cooling (Innis and Gelfand, 1990). Annealing
temperatures should be chosen to match the Tm values of the primer
pair and are typically 45–68°C. If extra bands are observed, higher
annealing temperatures should be considered. The absence of product
can indicate the need for a lower annealing temperature. Annealing
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Review of literature
times of 15–60 seconds are usually adequate (Sambrook and Russel,
1989).Extensions are normally done at 72°C. As a general rule,
extension times of one minute per kb should be used. For products
less than one kb, an extension time of 45–60 seconds should be used.
A final extension of 5 minutes at 72°C is recommended (Sambrook
and Russel, 1989).The number of cycles is usually between 25 and 35.
2.14 Single Strand Conformational Polymorphism (SSCP)
Single strand conformational polymorphism analysis is a rapid
method for detection of minor sequence changes in polymerase chain
reaction amplified DNA. Since the first reported use of SSCP in 1989
(Orita et al., 1989) this technique has been widely used to detect
mutations. SSCP have been used to detected base substitutions, small
insertions and deletions, and rearrangements. SSCP technique could
detect even a single base substitution in a PCR fragment of several
hundred nucleotides in length (Orita et al., 1989).
The technique requires relatively less labour and less expensive
than most other approaches used to detect mutations. It is based on
the assumption that subtle nucleic acid change affects the migration of
single-stranded DNA fragment, therefore results in visible mobility
shifts across a no denaturing Polyacrylamide gel. Non denaturing
Polyacrylamide gel without Urea is used for analysis of DNA fragments.
In non denaturing PAGE, the components used to synthesize
DNA separating matrix are Acrylamide monomers, N,N-methylene
bisacrylamide,
Ammonium
Per
Sulphate
(APS)
and
N,N,N’,N’-
Tetramethylenediamine (TEMED) which are dissolved in 1X TBE.
Ammonium persulphate (APS) when dissolved in water generates free
radicals, which activate acrylamide monomers inducing them to react
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Review of literature
with other acrylamide molecules forming long chains. These chains
gets cross-linked with N,N-methylene bisacrylamide to form the DNA
separation matrix. TEMED act as catalyst for gel formation because of
its ability to exist in free radical form. The casted Non denaturing PAGE
gels are run in vertical gel electrophoresis unit containing 1X TBE for
resolving DNA fragments in PCR-SSCP analysis. (Orita et al., 1989)
Single-strand Conformation Polymorphism (SSCP) analysis is one of
the most widely used technique for mutation detection due to its
simplicity and versatility, together with its comparatively high rate of
mutation detection (Cotton, 1993) make it a method of choice for
screening
DNA
fragments
in
many
research
and
diagnostic
applications.
Many modifications to the original protocol developed by (Orita
et al., 1989) include those affecting the gel matrix, e.g., percentage of
acrylamide monomer, cross-linking ratio, buffer systems, addition of
neutral compounds to the gel, and electrophoresis temperature. The
most preferred gel characteristics for successful differential separation
of single strand conformers in the range of 200–300 bp, product, are
12% acrylamide (Savov et al., 1992) and cross-linking ratios (%C)
between 1 and 3 (Glavac and Dean, 1993). Under these conditions,
long-fiber gels are formed, large-pore matrices that are sufficiently
dense for successful electrophoretic separations with better flexibility.
Protocols developed so far rely on at least two temperature standard
points for electrophoresis with the gels, between 4°C and 25°C.
Addition of neutral compounds like glycerol, 50–150 mL/L gives better
electrophoretic separation of conformers in some cases (Glavac and
Dean, 1993).
26
Review of literature
2.14.1 Identification of new Polymorphisms
A
variety
of
techniques
are
available
for
new
mutation/
polymorphism identification. One commonly used strategy for SNP
screening is to amplify genes of interest by PCR, scan the PCR
products for the presence of DNA variants by confirmation-based
mutation scanning methods, and then sequence positive PCR products.
The development of new-generation DNA sequencers also allows direct
heterozygote sequencing frequently used in SNP identification. With a
vast amount of human expressed sequence tags (ESTs) and genomic
clones in the public domain, computer-based sequence alignment and
clustering also provide a rich source for SNP identification (Makino et
al., 1992).
2.14.2 Conformation based mutation scanning
Single-strand conformation polymorphism analysis is one of the
most widely used methods for mutation detection. In single-strand
conformation
polymorphism,
DNA
regions
with
potential
polymorphisms are first amplified by PCR. Single-stranded DNAs are
then generated by denaturation of the PCR products and separated on
a no denaturing Polyacrylamide gel. A fragment with a single base
modification generally forms a different conformer and migrates differ
entirely when compared with wild-type DNA Single-stranded DNAs are
generated by denaturation of the PCR products and separated on a no
denaturing
Polyacrylamide
gel.
A
fragment
with
a
single-base
modification generally forms a different conformer and migrates
differently when compared with wild-type DNA as shown in Fig. 2.5
(Makino et al., 1992). The SNP is a genetic variability that appears in
approximately 1% of a population this nucleotide variability arises in a
population through mutation variability persists and is maintained in
27
Review of literature
the gene pool through genetic drift or the founder effect biodiversity
within species concerns the amount, distribution, and adaptive value of
variation within and among populations in their natural environment
(Ryder, 1986).
Figure 2.5: Single-strand conformation polymorphism analysis
adopted from Makino et al. (1992)
2.15 Deoni breed and its reproductive performance
Deoni is a medium sized dual purpose indigenous breed, is
generally found in northern part mostly in Bidar district of Karnataka
and south eastern part of Maharashtra covering Latur district. The
home tract is mainly hilly with an average altitude of 480 to 705
meters above mean sea level. The minimum ambient temperature in
the home tract ranges from 9°C to 26°C and the maximum ambient
temperature ranges from 29°C to 44°C. This breed is hardy and well
adapted for the tropical draught prone areas. The body color is usually
spotted black and white. The forehead is convex and bulging. The
horns in typical animals take a characteristic outward and backward
28
Review of literature
curve. The skin is loose and of medium thickness. Deoni animals have
soft and short hair. The cows have a fairly well-developed udder.
The average lactation milk yield was 779.27 ± 18.31 kg in Deoni
cows with 4.3 percent of fat (Das et al., 2011). The Average age at
first calving in Deoni cattle was reported by many researchers as 50
months (Deshpande and Singh, 1977a) 46 months (Singh et al., 2002)
and 38 months (Das et al., 2011). The average inter-calving period
reported in Deoni cattle was 466 days (Deshpande and Singh, 1977),
447 days (Singh et al., 2002) and 447 days (Das et al., 2011).
2.16 Polymorphism in HSP genes and their association with
performance traits
The reports with regards to specific application of SSCP for
molecular characterization and determination of genetic variants HSP
gene family in livestock are very limited. The ovine gene encoding the
inducible
form
of
the
HSP90α
(HSP90AA1)
has
been
recently
characterized and mapped to chromosome OAR18 Marcos-Carcavilla et
al. (2008) They identified, several SNPs located in the 5′ flanking
region and intron 10 and found to be associated with the response to
scrapie in sheep. Marcos-Carcavilla et al., (2010) reported that a SNP
in the HSP90AA1 gene 5′flanking region is associated with the
adaptation to differential thermal conditions in ovines. Further they
reported Single nucleotide polymorphism (SNP) at position −660 in the
HSP90AA1 5′flanking region among different breeds of ovines.
Umaporn et al., (2006) reported association of HSP90 gene
polymorphism with heat tolerance traits in crossbred dairy cattle and
Thai native cattle. They detected polymorphism of HSP90 gene in Thai
native crossbred cattle. They used PCR-RFLP-SSCP technique for the
29
Review of literature
identification of polymorphism. PCR products were cut by restriction
enzyme (Hpall) and tested for polymorphism using Single-Strand
Conformation Polymorphism (SSCP) method. They obtained a PCR
product of 600 base pairs in both native and cross bred Thai cattle.
They reported 2 SSCP patterns of HSP90 gene and their association
with heat tolerance. Single point mutation, deletion, insertion, or
single nucleotide polymorphisms (SNP) of the HSP70 gene, such as the
base change at 2,033 (G > C) of the HSP70 gene that results in an
amino acid change from glycine to alanine in the translated products
had an effect on milk yield and milk content (Lamb et al., 2007).
Charles
et
al.,
(2010)
reported
that
single
nucleotide
polymorphisms in the promoter region of the bovine Hsp70 gene (base
positions 895, 1125, and 1128) were related to calving percentages.
The deletion of cytosine at base position 895 had the greatest effect
on average calving date. Only 8% of cows homozygous with the
cytosine deletion calved, and those cows that calved had an average
calving date of 109 days which was approximately 35 days longer than
cows without the deletion.
Earlier studies on polymorphism in HSPs
indicate the possibility of identifying markers for heat tolerance by
targeting HSP family genes in cattle.
30
Review of literature
CHAPTER - 3
────────────────────────────
Materials and Methods
─────────────────────────────
3.0 MATERIALS AND METHODS
3.1 Experimental Animals
The
present
study
was
conducted
on
72
Deoni
animals
maintained at the Cattle Yard, National Dairy Research Institute
(NDRI), Southern Campus, Bangalore.
Deoni
is
a
medium
sized,
dual-purpose
breed
of
cattle
predominantly distributed in North Karnataka and South Maharashtra
(Figure 3.1). The body coat is usually spotted black and white. The
forehead is convex and bulging. The horns in typical animals take a
characteristic outward and backward curve. The skin is loose and of
medium thickness. Deoni animals have soft and short hair. The cows
have a fairly well-developed udder. The average lactation milk yield
was 779.27 ± 18.31 kg in Deoni cows with 4.3 percent of fat. The
Average age at first calving, and inter-calving period was reported to
be 38.73 ± 0.73 months and 447.22 ± 6.64 days respectively (Das et
al., 2011).
3.2 Blood Collection
About 10 ml of blood was collected aseptically from each of the
representative Deoni cows from Jugular vein in a vacutainer tube
containing 0.5 per cent EDTA. After collection, the samples were
stored at 4 C and DNA was isolated within 24 hrs.
3.3 DNA Extraction by High Salt method
DNA was isolated from the blood samples using modified High Salt
Method (Miller et al., 1988), with minor modifications. The method
involved the following steps.
31
Materials and Methods
1. Ten ml of whole blood was taken in a 50ml graduated centrifuge
tube. To this, 25 ml of ice-cold RBC lysis buffer was added. The
contents were mixed thoroughly and incubated in ice for 10 minutes
with intermittent shaking to allow complete lysis of RBCs.
2. The contents were centrifuged at 4000 rpm for 10 minutes. The
supernatant containing lysed RBCs was discarded, while the pellet
of white blood cells was retained.
3. To remove the unlysed RBCs the white cell pellet was resuspended
in the RBC lysis buffer and centrifuged at 4000 rpm for 10minutes.
This step was repeated till the WBC pellet was devoid of unlysed
RBCs.
4. The pellet was washed twice in Tris Buffered Saline (TBE) by
centrifugation at 3000 rpm for 10minutes.
5. The WBC pellet was resuspended in 9ml of Tris EDTA buffer (TE
buffer, pH 8.0) by vigorous vortexing, till the pellet was completely
resuspended in it.
6. Fifty microliter (µl) of Proteinase-K and 0.5ml of 0.5M EDTA (pH
8.0) was added and mixed thoroughly by vortexing
7. Sodium Dodecyl Sulphate (SDS) was added at the rate of 500 µl to
the above solution with gentle mixing and incubated at 50°C for
overnight in a shaker water bath.
8. After incubation, 4.3ml of saturated Sodium Chloride was added to
the Proteinase-K digested sample and shaken vigorously for 30-60
seconds.
9. Equal volume of Chloroform: Isoamylalcohol (24:1) was added and
mixed thoroughly by inversion of the tube and then centrifuged at
4000 rpm for 15minutes.
32
Materials and Methods
Figure 3.1. Deoni cow maintained at Cattle Yard, NDRI Southern
Campus
Figure 3.2. Deoni
cattle
herd
Southern Campus
maintained
at
Cattle
Yard,
NDRI
10. The upper aqueous phase was transferred to a fresh tube and the
Chloroform: Isoamyl alcohol extraction was repeated.
11. The aqueous layer containing DNA was transferred to a sterile
100ml beaker and DNA was precipitated with two volumes of 95 per
cent ethanol.
12. The precipitated DNA was spooled out on a glass rod, washed with
70 per cent ethanol, air dried and then resuspended in 400µl of TE
buffer (pH 8.0).
3.3.1 Determination of purity and yield
The purity and concentration of DNA samples were estimated
using 0.7% Agarose gel electrophoresis and Biophotometer. DNA (2 l)
was dissolved in 98 l of double distilled water and loaded into a 100 l
cuvette. Optical density (O.D.) was determined at wavelengths 260
nm and 280 nm in Biophotometer (Eppendrof, Germany). Quantity of
DNA was calculated using the formula:
Quantity of DNA in g / ml =O.D.260
50
Dilution Factor
Samples showing an optical density ratio (260/280 nm)
between 1.7 and 1.9 considered as high purity were used for further
analysis.
3.3.2 Preparation of template DNA for PCR
The stock DNA was diluted with autoclaved distilled water to a
final concentration of 100ng/µl and used as the working solution.
33
Materials and Methods
3.4 Primer Designing
A total of six sets of primers table 3.1 were designed for Heat
Shock Protein 90 (HSP90AA1) gene using Primer3 software based on
the 5332 bp sequence for HSP90AA1 (NCBI GenBank NC-007319)
Oligonucleotide
content,
melting
Primer-dimer
temperature,
possibilities,
Oligonucleotide
PCR
product
size,
length
GC
Positional
constraints within the source sequence and miscellaneous constraints
were considered while designing the primers. The Wallace formula was
used to calculate working approximation of Tm value
Tm = 4(G + C) + 2(A + T) oC
The Designed primers were procured from Chromous Biotech
Bangalore. PCR primers, their product location in NC-007319 their
annealing temperature and the expected product sizes are summarized
in table 3.1.
3.5 Polymerase Chain Reaction- Single Strand Conformational
Polymorphism (PCR-SSCP)
PCR conditions were optimized for each Primer by testing a
number of variables such as master mix concentration, temperature
conditions etc. The combinations giving the best amplification were
used for further studies.
3.5.1 Composition of PCR mixture
The composition of PCR master mix used in study is given in
table 3.2 The contents were mixed thoroughly and spun for few
seconds in a spinner (Spinwin).
34
Materials and Methods
Table 3.1. Description of primers used for PCR amplifications of HSP90 AA1 gene
Fragments:
Fragment-1
(Exon 1 to 2)
Fragment-2
(Exon 3 to 4)
Amplicon
(bp)
689
839
Primer sequence 5`-3`
F-5’-GCT ACG CGT ACT CCC TCA GA-3’
R-5’-CTA CAG CAC CCC ACC CTG T-3’
F-5’-TGA TCA GGC CAT TGT GAT TG-3’
R-5’-CAT GTG CAG GGA TGG TAG TTT-3’
Location
541-1230
1441-2280
Annealing
temp.
60°C
56°C
35
Fragment-3
(Exon 5 to7)
Fragment-4
(Exon 8)
Materials and Methods
Fragment-5
(Exon 9)
Fragment-6
(Exon 10)
999
539
410
250
F-5’-CAC ATG TTT GAG GCA GCA TT-3’
R-5’-CAG AAG ACA CAC TCA ACT GTT CC-3’
F-5’-CCC ATG GGA ACA GTT GAG TG-3’
R-5’-GCT TTA AGC TCC TTT TAA GTT CG-3’
F-5’-GAC TAG AAC ATC TCT ATG CCC AGT T-3’
R-5’- CAC ATA GCA CTC GCG TAA GG-3’
F-5’- TAG TTC GCT CAG CCT TGA GA-3’
R-5’- AGA GCG CTG AAC ACA GCA G-3
2271-3270
3241-3780
3871-4281
4430-4680
56°C
55°C
56°C
57°C
Table 3.2. Composition of reaction mixture for PCR
S.No.
Components
Quantity
1
Sigma water
19 µl
2
10x Buffer (1X) with 1.5mM Mgcl2
2.5 µl
3
2.5 Mm dNTPs (100 µM each)
1.0 µl
4
Forward Primer (20 pmol/µl)
0.25 µl
5
Reverse Primer (20 pmol/µl)
0.25 µl
6
DNA Template (100 ng/ µl)
1.0 µl
7
Taq DNA Polymerase
1.0 µl
Total Volume
25.0 µl
3.5.2 Thermal cycling Conditions
The PCR amplifications were carried out in a 0.2 ml PCR tube in
a Master cycler (Eppendorf, Germany). The thermal cycling conditions
involved an initial denaturation at 94°C for 5 min, followed by 30
cycles with initial denaturation at 94°C for 1 min, primer specific
annealing temperatures of 60°C for 1 min (to specifically amplify
HSP90AA1 Fragment-1), 56°C for 1 min (to specifically amplify
HSP90AA1 Fragment-2, Fragment-3 and Fragment-5 regions), 55°C
for 1 min 30 sec (to specifically amplify HSP90AA1 Fragment-4 ), and
57°C for 1 min (to specifically amplify HSP90AA1 Fragment-6 ),
respectively, extension at 72°C for 1 min followed by a final extension
at 72°C for 5 min.
3.6 Agarose gel electrophoresis
The 1.5 gm of Agarose was transferred to a beaker and 100 ml
of 1X TBE buffer, was added to obtain 1.5% Agarose solution.
The
contents were placed in a microwave oven and heated to dissolve
Agarose into solution. The solution was cooled to 600C and 4µl of
36
Materials and Methods
Ethidium bromide (0.5
g/ml) was added and mixed thoroughly. The
gel was casted by positioning comb by 0.5 – 1 mm above the plate so
that a complete well was formed when the Agarose solution was
added. The gel was allowed to settle for 30-45 minutes at room
temperature, the comb and the tape was removed carefully and the
gel was mounted in the electrophoresis tank with 1X TBE buffer.
To confirm the specific PCR amplification, 8µl of PCR product
from each tube was mixed with 2µl of 6X gel loading dye and
electrophoresed in 1.5 per cent Agarose gel at a constant voltage of 80
Volts for 30 minutes in 1X TBE buffer. 100bp DNA ladder was used as
Molecular weight marker. The PCR products were visualized under the
UV trans-illuminator and documented using gel documentation system
(Bio-Rad, USA). The quality and quantity of the specific products were
assessed based on the presence of product of expected base pair in
the form of sharp DNA bands. The PCR amplified products were further
subjected to Polyacrylamide Gel Electrophoresis (PAGE) and visualized
after silver staining.
3.6.1 PCR-SSCP analysis of HSP90AA1 gene fragments
Various factors such as amount of PCR product, denaturing
solution, voltage, running time, Acrylamide: bisacrylamide ratio,
Acrylamide concentration and temperature were optimized for SSCP
analysis. Two different Acrylamide : N,N,bisacrylamide composition
ratios of 29:1 and 19:1 were utilized for PCR-SSCP analysis depending
on the size of the HSP90AA1 gene amplicons and specific requirements
to achieve proper resolution of single stranded DNA fragments.
Acrylamide-bisacrylamide solution was dissolved completely using
magnetic stirrer and kept in room temperature till used. The specific
37
Materials and Methods
Table 3.3. Conditions of electrophoresis for SSCP analysis
Hsp90 Gene Acrylamide : bisacrylmide Acryl amide
DNA
Denaturing solution Duration
Ratio
38
Materials and Methods
Fragment-1
19:1
6%
8 µL
12 µL
12:00 h
Fragment-2
19:1
6%
8 µL
12 µL
15:00h
Fragment-3
19:1
5%
8 µL
12 µL
18:00h
Fragment-4
19:1
6%
10 µL
15 µL
10:00h
Fragment-5
29:1
8%
8 µL
12 µL
8:00h
Fragment-6
29:1
10%
15 µL
25 µL
6:00 h
gel conditions utilized for PCR-SSCP analysis of HSP90AA1 gene
fragments is shown in Table: 3.3.
The PCR-SSCP procedure involved PCR amplification of the gene
fragments, resolution in nondenaturing PAGE and visualization using
silver staining.
1.
The Single Strand Conformation Polymorphism (SSCP) analysis of
amplified gene fragments was carried out using vertical gel
electrophoresis unit (SCIE-PLAS, U.K). The two glass plates were
washed thoroughly using tap water with detergent and rinsed
thoroughly under running tap water. The plates were first wiped
two times with tissue paper soaked in distilled water, then wiped
70 percent alcohol and air-dried. The similar thorough cleaning
treatment was given to spacers and comb to ensure proper
alignment of 20 cm glass plates.
2.
The gel sandwich was assembled on a clean surface laying down
the long rectangular plate first, then two spacers of equal
thickness along the long edges of plate and the short plate was
placed on the rectangular plate. The two glass plates with spacers
between
them
were
fitted
well
with
proper
alignment
by
tightening the clamps. The sandwiched gel plates were fitted in
the stand with screw clamps. The cleaned comb (20 wells) was
inserted from the topside of the gel sandwich and immediately
clamps were applied over the plates containing comb to create
sharp wells.
3.
The native PAGE gel mix was prepared by adding ammonium
persulphate and N, N, N’, N’-tetramethylenediamine (TEMED) at
the same time and mixed well. This gel mix was filled smoothly
39
Materials and Methods
from upper side using syringe without any bubble. The gel was
kept undisturbed for at least 45 minutes for polymerization.
4.
After polymerization the comb was removed and wells were
flushed with 1X TBE buffer. The gel sandwich was placed in
electrophoresis tank with notched plate facing towards the buffer
reservoir. The reservoir of the electrophoresis tank was filled with
1X TBE and the gel was given pre-run at 200 volts at constant
temperature for a minimum of 30 minutes.
5.
Appropriate proportion of PCR product and formamide dye was
added in a 0.2ml PCR tube as indicated in table 3.3 were
denatured at 95°C for 10 minutes using the Eppendorf PCR
machine. After denaturation the samples were immediately kept in
ice-chilled box and the box was kept in -20°C freezer for 10
minutes.
6.
After completion of pre-run the wells were flushed again using
buffer. The samples were loaded on a nondenaturing acry1amide:
bisacrylamide
gel
using
gel
loading
tip
and
immediately
electrophoresis was performed in 1X Tris borate (pH 8.3)-EDTA
buffer at 10-12.5 volts/cm for 3-24 hr at room temperature
depending on the optimized conditions for each set of primer
(table 3.3).
7.
After completion of the electrophoresis the glass plates were
removed from the assembly and gels were subjected to silver
staining to visualize SSCP band patterns.
8.
In order to stain the gel it was immersed in a tray of appropriate
size filled with 10 per cent
acetic acid (500ml) for at least 10
40
Materials and Methods
minutes for fixing DNA bands so as to prevent diffusion of the DNA
bands (care was taken to see gel remains dipped well in solution).
The gel was agitated slowly for 10 minutes or until the tracking
dye is no longer visible.
9.
The acetic acid was decanted and 500 ml of distilled water was
poured to the tray and rinsed thoroughly by placing the tray on
oscillatory automatic shaker for 5 minutes.
10. Meanwhile 500 ml of 0.1 per cent silver nitrate solution was
prepared in amber color bottle and 500 µl of 37 per cent
formaldehyde was added and mixed. Distilled water was gently
decanted from tray. The gel was stained for 30 minutes in silver
nitrate solution containing formaldehyde solution with constant
shaking in a dark room or covering the tray with black cloth.
11. Then the gel was rinsed briefly for 30 seconds in distilled water.
12. Quickly distilled water was decanted from the tray. The freshly
prepared and chilled 3 per cent sodium carbonate solution (3 per
cent Na2Co3 and 750 µl of 37 per cent formaldehyde + 0.1 per
cent Sodium Thiosulfate) was added to the tray. The gel was kept
immersed until sharp bands were developed.
13. The gel was given 10 percent Acetic acid (stop solution) treatment
for 10 minutes. Then 500 ml distilled water was added to the tray.
14. The gel was finally transferred gently on blotting paper and
covered with the transparency sheet; excess water was soaked
with tissue paper, and dried in gel drier for 10 minutes.
41
Materials and Methods
Examination of Silver stained gels: The silver stained gels were
examined using transilluminator and photograph was taken using Sony
digital camera. The gels were also examined using Gel Doc system
(Bio Rad, USA) and SSCP variants were recorded. Then gels were
labeled and scanned for computer image analysis and documentation.
The different band patterns/variants were characterized based on the
number of bands and mobility shifts identified for the different
fragments of HSP90AA1 gene. Each pattern was represented by a
code.
3.7 Custom Sequencing
The unique SSCP patterns obtained for the representative PCR
products were segregated and further analyzed by direct sequencing
(Chromous Biotech Pvt. Ltd., Bangalore, India). The custom made
forward and reverse allele specific PCR primer sets used for PCR-SSCP
assay were utilized for direct sequencing. At least two individual
animal samples representative of each unique PCR-SSCP patterns were
given for direct sequencing to obtain representative sequences.
3.7.1 Sequence data analysis
The complementary sequences representative of unique PCRSSCP pattern were analyzed using Ridom trace edit sequence analysis
tool. The sequence chromatograms were first analyzed to resolve
ambiguous bases and to trim sequence ends of each chromatogram.
The finalized Chromatograms were carefully analyzed. The retrieved
sequences representing each of the unique PCR-SSCP patterns were
further analyzed using Clustal-W multiple sequence alignment tool for
detecting single nucleotide polymorphisms (SNP’s) and their respective
deduced amino acid variations.
42
Materials and Methods
Sequence data were analyzed using, Bioedit software (Hall,
1999) Clustal W multiple alignments for detecting single nucleotide
polymorphisms.
3.8 Reproductive performance data of Deoni Cows
The reproductive performance data viz. Age at First Calving
(AFC) and Calving Interval (CI) of Deoni breed of cattle was collected
from history-cum-pedigree sheets maintained at cattle yard, SRS of
NDRI, Bangalore.
3.9 Statistical Analysis
Statistical procedures were done as described by Snedecor and
Cochran (1994) and tests were performed using of SAS Version 9.2 to
find
out
any
significant
difference
(SAS
Inc.,
2003).
Test
of
significance chosen was at 1 per cent (P≤0.01). The Molecular data
were processed
using POPGENE v 1.3.2 (population genetic analysis
tool).
The Association between HSP90AA1 genetic variants/ patterns
and reproductive traits were analyzed with the General Linear model
(GLM) procedure of SAS Version 9.2, by using the model :
Yij = µ + Pi +eij
Where,
Yi = reproductive trait of jth animal belonging to ith pattern
µ
= Overall population mean
Pi
= Effect of ith pattern
eij= random error associated with
43
Yij observations
Materials and Methods
CHAPTER - 4
───────────────────────────
Results and Discussion
───────────────────────────
4.0 RESULTS AND DISCUSSION
The present investigation was carried out to characterize Heat
Shock Protein 90 (HSP90AA1) gene and to study the association of
HSP90 gene variants with reproductive performances in Deoni breed of
cattle.
4.1 Yield and purity of DNA
Blood samples collected from 72 Deoni cows were utilized for
genomic
DNA
isolation.
Miller’s
High
Salt
method
with
minor
modifications was used for the extraction of genomic DNA from WBC
cell pellet. The yield of DNA ranged from 375 to 1330 µg/ ml of blood
with a mean yield of 596.50 ± 36.50 µg / ml. The purity of DNA
(determined as O.D ratio at 260nm / 280nm) ranged between 1.7 and
1.9 in all the samples, with a mean of 1.80 ± 0.01 indicating high
purity of the extracted DNA. The DNA samples were diluted to get the
final concentration of 100 ng/ul in low Tris EDTA (TE) buffer for
utilizing them as DNA templates for further studies.
4.2 PCR amplification
PCR conditions were standardized for each Primer by testing a
number of factors such as primer concentration, number of cycles,
concentration of MgCl2, Taq DNA polymerase, template DNA and
annealing temperature to obtain optimum amplification. Six sets of
primers were designed in such a way that each of the 10 exons of
HSP90 gene was amplified and product size is below 1 kb (Table 4.1).
The PCR conditions were optimized for fragment specific amplification
of HSP90AA1 gene. The PCR products were electrophoresed in 1.5%
Agarose gel at 90V for 45 minutes along with 100 bp DNA ladder as
44
Results and Discussion
Molecular weight marker and visualized under Gel Doc System (BIO
RAD, USA)
The amplified HSP90AA1 PCR products (Figure 4.1-4.7), were
subjected to single-strand conformation polymorphism (SSCP) analysis
to determine the genetic variants.
Table 4.1. Annealing Temperature and product size of different
fragments
HSP 90 gene
fragments
Exons
Location
Amplicons
length (bp)
Annealing
temperature
Fragment-1
Exon 1 to 2
541-1230
689
60°C
Fragment -2
Exon 3 to 4
1441-2280
839
56°C
Fragment-3
Exon 5 to 7
2271-3270
999
56°C
Fragment-4
Exon 8
3241-3780
539
55°C
Fragment-5
Exon 9
3871-4281
410
56 °C
Fragment-6
Exon 10
4430-4680
250
57°C
4.3 Single Strand Conformation Polymorphism (SSCP) analysis
The
analyzed
amplified
by
Single
PCR
products
Strand
of
different
Conformation
Fragments
Polymorphism
were
(SSCP)
analysis. Various factors such as amount of PCR product, denaturing
solution, voltage, running time, Acrylamide: bisacrylamide ratio,
Acrylamide concentration and temperature were optimized for SSCP
analysis. Each PCR product was diluted in denaturing solution,
denatured at 95°C for 8 min, chilled on ice and resolved on optimized
concentration
of
non-denaturing
polyacrylamide
gels.
The
electrophoresis was carried out in a vertical electrophoresis chamber
(SCIE-PLAS, U.K) in 1× TBE buffer. The optimized Conditions for SSCP
analysis using vertical electrophoresis is summarized in Table4.2.
45
Results and Discussion
Figure 4.1: Quality checking of DNA on 0.8% Agarose gel
Lane 1-8
: Working DNA samples
Figure 4.2: Resolution of PCR amplified product of fragment-1
on 1.5% Agarose gel
Lane 1-7: PCR product (689 bp), Lane M: 100bp ladder molecular marker
Figure 4.3: Resolution of PCR amplified product of fragment-2
on 1.5% Agarose gel
Lane 1-3: PCR product (839 bp), Lane M: 100bp ladder molecular marker
Figure 4.4: Resolution of PCR amplified product of fragment-3
on 1.5% Agarose gel
Lane 1-6 : PCR product (999 bp), Lane M: 100bp ladder molecular
marker
Figure 4.5: Resolution of PCR amplified product of fragment-4
on 1.5% Agarose gel
Lane 1-8: PCR Product (539 bp), Lane M: 100bp ladder molecular marker
Figure 4.6: Resolution of PCR amplified product of fragment-5
on 1.5% Agarose gel
Lane 1- 6: PCR product (410 bp), Lane M: 100bp ladder molecular marker
Figure 4.7: Resolution of PCR amplified product of fragment-6
on 1.5% Agarose gel
Lane 1-10: PCR product (250 bp) , Lane M: 100bp ladder molecular marker
Table 4.2. Optimized Conditions for SSCP analysis
Hsp90 gene
Fragments
Acrylamide :
bisacrylamide
Ratio
Acrylamide
(%)
Amplified
DNA
product
(µL)
Denaturing
solution
(µL)
Duration
(h)
Fragment -1
19:1
6
8
12
12:00
Fragment-2
19:1
6
8
12
15:00
Fragment-3
19:1
5
8
12
18:00
Fragment-4
19:1
6
10
15
10:00
Fragment-5
29:1
8
8
12
8:00
Fragment-6
29:1
10
15
25
6:00
The gels were silver stained. Silver stained SSCP gels were dried
and documented for detecting mobility shifts in different fragments of
HSP90AA1 gene in Deoni breed of cattle. The different band
patterns/variants were characterized by the number of bands and
mobility shifts identified for the different fragments of HSP90AA1 gene.
Each pattern was represented by a code.
4.3.1 Distribution of PCR-SSCP patterns
The PCR-SSCP analysis of HSP90AA1 gene amplicons revealed
varying degree of genetic polymorphisms with respect to each of the
HSP90AA1 gene fragments analyzed. PCR-SSCP analysis of amplicons
of the Fragment-1 comprising of Exons 1 to 2, Fragment-2 comprising
of Exons 3 to 4 and Fragment-3 comprising of Exons 5 to 7, showed
monomorphism in Deoni cattle. Thus the HSP90AA1 gene Fragment-1,
Fragment-2 and Fragment-3 showed absence of polymorphisms
indicating the probable absence/lack of mutation/s suggesting high
degree of conservation of HSP90 AA1 gene in Deoni breed of cattle.
The present findings are in agreement with the earlier reports of high
46
Results and Discussion
degree of conservation in HSP90 AA1 gene across breed and species
(Chen et al., 2006). PCR-SSCP analysis of Fragment-4 comprising
Exon 8 in HSP90AA1 gene revealed three unique SSCP patterns with
different mobility shifts (Figure 4.11), viz. pattern I, pattern II and
pattern III respectively. PCR-SSCP Pattern I showed one distinct band,
pattern II showed two distinct bands and pattern III showed six
distinct bands, respectively. Out of the total 72 Deoni animals
genotyped the genotypic frequency of pattern I, pattern II, and
pattern III were 0.2500, 0.639
and 0.111 respectively (Table 4.3).
The calculated Nei`s Gene diversity and Shannon’s information index
for the Fragment-4 were 0.5170 and 0.8769 respectively (Table 4.3) in
Deoni cattle. No earlier reports are available to compare or contrast
the present findings.
The fragment-5 comprising Exon 9 of HSP90AA1 gene in Deoni
breed of cattle revealed two PCR-SSCP patterns with different mobility
shifts (Fig.4.12) viz. namely pattern I and pattern II. PCR-SSCP
pattern I showed two distinct DNA band and pattern II showed three
distinct DNA bands. The genotypic frequency of pattern I and pattern
II among the 72 Deoni animals genotyped were 0.1530 and 0.847
respectively (Table 4.4). The calculated Nei`s Gene diversity and
Shannon’s information index for the fragment-5 were 0.2589 and
0.4275 respectively (Table 4.4) in Deoni cattle. No earlier reports are
available to compare or contrast the present findings.
The fragment-6 comprising Exon 10 of HSP90AA1 gene in Deoni
breed of cattle revealed two PCR-SSCP patterns with different mobility
shifts (Fig.4.13), viz. pattern I and pattern II. The HSP90AA1 gene
pattern I revealed two distinct bands while pattern II revealed three
distinct bands (Figure 4.13). Out of the total 72 Deoni animals
47
Results and Discussion
Table 4.3. Genotypic frequencies and genetic diversity in fragment-4 of HSP90 gene in Deoni cattle.
Pattern I
N
Pattern II
No of
Observations
Genotypic
frequency
No of
Observations
Genotypic
frequency
No of
observations
Genotypic
frequency
18
0.2500
46
0.639
8
0.111
72
h*
Pattern III
(N) Number of animals, (h*) Nei`s Gene diversity
I*
0.5170 0.8769
(I*) Shannon’s information index
48
Table 4.4. Genotypic frequencies and genetic diversity in fragment-5 of HSP90 gene in Deoni cattle.
N
Results and Discussion
72
Pattern I
Pattern II
No of
Observations
Genotypic
frequency
No of
Observations
11
0.153
61
h*
I*
Genotypic
frequency
0.847
0.2589
0.4275
(N) Number of animals, (h*) Nei`s Gene diversity & (I*) Shannon’s information index
Table 4.5. Genotypic frequencies and genetic diversity in fragment-6
of HSP90 gene in Deoni cattle.
N
72
Pattern I
Pattern II
No of
Observations
Genotypic
frequency
No of
Observations
Genotypic
frequency
17
0.236
55
0.764
h*
I*
0.3607
0.5466
49
(N) Number of animals, (h*) Nei`s Gene diversity & (I*) Shannon’s information index
Results and Discussion
Figure 4.8: PCR-SSCP patterns of HSP90 gene fragment-1 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-1
All monomorphic
Figure 4.9: PCR-SSCP patterns of HSP90 gene fragment-2 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-2
All monomorphic
Figure 4.10: PCR-SSCP patterns of HSP90 gene fragment-3 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-3
All monomorphic
Figure 4.11: PCR-SSCP patterns of HSP90 gene fragment-4 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-4.
Lane -3, 4, 6 and 7 shows rare allelic pattern P-1
Lanes 2, 5, 8, 9, 10, 12 &13 allelic pattern P-II
Lanes 1 and 11 rare allelic pattern P-III
Figure 4.12: PCR-SSCP patterns of HSP90 gene fragment-5 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-5
Lanes L6, L8 and L10 shows rare allelic pattern P-1
Lanes LI, 2 ,3, 4,5, 7,9,11,12,13,14 and 15 represent allelic pattern P-II
Figure 4.13: PCR-SSCP patterns of HSP90 gene fragment-6 in
Deoni Cattle
Silver stained PAGE representing PCR-SSCP band patterns for fragment-6
Lanes L1, L2 and L5 shows rare allelic pattern P-1
Lanes 3, 4, 6 7, 8,9,10 and 11, represent l allelic pattern P-II
genotyped the genotypic frequency of pattern I and pattern II
was 0.236 and 0.764 respectively (Table 4.5). The calculated Nei`s
Gene diversity and Shannon’s information index in Deoni cattle for the
fragment-6 were 0.3607 and 0.5466 (Table 4.5) respectively. No
earlier reports are available to compare or contrast the present
findings.
The genotype frequencies observed in the present investigation
suggest that the Deoni breed of cattle have a diverse type of SSCP
patterns for fragments 4, 5 and 6 of HSP90AA1 gene comprising of
Exons 8, 9 and 10 respectively in the sampled population indicating
the existence of variability.
4.4 Association
of
SSCP
patterns
with
reproductive
performance
Three SSCP patterns with different mobility shifts were observed
in fragment-4 namely pattern I, pattern II and pattern III respectively.
In case of fragment-5 and fragment-6 two SSCP patterns with
different mobility shifts were obtained, namely pattern I and pattern
II.
In order to unravel the effect of observed patterns in fragments
4, 5 and 6 of HSP90AA1 gene on reproductive traits viz. Age at First
Calving (AFC) and Calving Interval (CI) were analyzed for association
of different PCR-SSCP patterns of HSP90 gene using general linear
model (GLM) procedure of SAS System 9.2 VERSION (SAS Inc.,
2003).
The AFC in Deoni cattle ranged from 23 – 56 months with an
overall least square means of 40.72 ± 1.457 (table 4.6). The overall
50
Results and Discussion
LSM for AFC observed in the present study was higher than the earlier
reports of 38 months by Das et al. (2011), which was lower than
previous reports of 50 months by Deshpande and Singh (1977a) and
46 months by Singh et al. (2002).
The calving interval in Deoni cattle ranged from 320 – 616 Days
with an overall least squares mean of 432.875 ± 12.996 days, which
was lower than earlier reports of 447 days (Singh et al., 2002; Das et
al., 2011). Three distinct SSCP patterns were observed in fragment-4
of HSP90AA1 gene and their association with reproductive traits viz.
age at first calving and calving interval were analyzed and shown in
table 4.7 and 4.8 respectively. The animals with pattern III of
fragment-4 in HSP90AA1 gene had higher AFC (49.000±2.853
months) as compared to the animals with pattern I and pattern II
(p≤0.01). The Calving Interval was similar in cows with different
patterns (PI, PII, PIII) in fragment-4 of HSP90AA1 gene. No earlier
reports are available to compare or contrast the present findings.
Two
different
patterns
were
observed
in
fragment-5
of
HSP90AA1 gene. Least square means and ANOVA for age at first
calving and calving interval for two patterns in fragment-5 are shown
in table 4.9 and 4.10. The AFC and CI were similar in cows with
pattern I and pattern II. Fragment-6 of HSP90AA1 gene showed two
different patterns. Least square
means and ANOVA for age
at first
calving and calving interval of two patterns in fragment-6 HSP90AA1
gene are shown in table 4.11 and 4.12. There was no significant
difference among the least square means between different patterns in
age at first calving and calving interval. The association study of Hsp90
fragment-5 and 6 with reproduction parameters revealed no significant
51
Results and Discussion
difference in Deoni cows. There are no reports available to compare or
contrast the present findings.
Umaporn et al., 2006 reported association of HSP90 gene
polymorphism with heat tolerance traits in crossbred dairy cattle and
Thai native cattle. The polymorphism of HSP90 gene was reported by
them in Thai native and crossbred cattle. They used PCR-RFLP-SSCP
technique; PCR products were cut by restriction enzyme (Hpall) and
tested
for
polymorphism
using
Single-Strand
Conformation
Polymorphism (SSCP) technique. They obtained a PCR product of 600
bp in both native and cross bred Thai cattle. They reported 2 SSCP
patterns of HSP90 gene.
Recently the ovine HSP90AA1gene has been characterized by
Marcos-Carcavilla et al. (2008) and mapped to chromosome OAR18.
Further they reported several SNPs located in the 5′ flanking region
and intron 10, SNP found in this gene has been reported to be
associated with the response to scrapie in sheep (Marcos-Carcavilla et
al., 2008). SNP at position −660 in the HSP90AA1 gene 5′flanking
region is reported to be associated with the adaptation to differential
thermal
conditions
among
different
breeds
of
ovine’s
(Marcos-
Carcavilla et al., 2010) indicating the importance of HSP90AA1 gene in
heat stress tolerance/ susceptibility. No reports are available on the
influence
of
genetic
variants
in
HSP90
gene
on
reproductive
performance in cattle. As this gene variants influence tolerance/
susceptibility
to
thermal
stress,
they
are
likely
to
influence
reproductive performance.
SNPs in the promoter region (base positions 895, 1125, and
1128) of the another gene of HSP family viz. Hsp70 have been
reported to be associated with calving percentages in bovines (Charles
52
Results and Discussion
et al., 2010). The deletion of cytosine at base position 895 had the
greatest effect on average calving date. Only 8% of cows homozygous
with the cytosine deletion calved, and those cows that calved had an
average calving date of 109 days which was approximately 35 days
longer
than
cows
without
the
deletion.
Single
nucleotide
polymorphisms (SNP) at 2,033 (G > C) of the HSP70 gene, results in
an amino acid change from glycine to alanine in the translated
products which has been
reported to have an effect on milk yield and
milk content (Lamb et al., 2007).
Table 4.6. Reproductive performances in Deoni breed of cattle.
TRAITS
Mean ± S.E
S.D
Age at First Calving
(Months)
40.722 ± 1.457
(23-56)
7.934
Calving Interval
(Days)
432.875 ± 12.996
64.884
(320-616)
Figures in parenthesis indicate range
Table 4.7. Effect of SSCP patterns of fragment-4 of HSP90
gene on reproductive performances in Deoni cattle.
Dependant
variables
Age at First Calving
(Months)
Calving Interval
(Days)
Patterns
No. of
observations (N)
I
18
40.500 ± 1.751
a
II
46
39.369 ± 1.690
a
III
8
49.000± 2.853
b
I
18
430.889 ± 15.615a
II
46
431.956 ± 15.072a
III
8
442.625 ± 25.446a
LSM ± S.E
Superscripts with different alphabets (a,b) differ significantly (P ≤ 0.01)
53
Results and Discussion
Table 4.8. ANOVA of SSCP patterns of fragment-4 of HSP90
gene on reproductive performances in Deoni cattle.
Source of variation
AFC
CI
d.f.
Mean Square Value F value
Between Groups
2
316.614
Within Groups
69
55.612
Total
71
Between Groups
2
435.155
Within Groups
69
4319.472
Total
71
5.693
0.101
Table 4.9. Effect of SSCP patterns of fragment-5 of HSP90
gene on reproductive performances in Deoni cattle.
Dependant
variables
Patterns
No. of
observations (N)
LSM ± S.E
Age at First Calving
(Months)
I
11
44.000 ± 2.390NS
II
61
40.131 ± 1.268NS
Calving Interval
I
11
447.617 ± 21.314NS
(Days)
II
61
422.191 ± 11.310NS
NS- non significant
Table 4.10. ANOVA of SSCP patterns of fragment-5 of HSP90
gene on reproductive performances in Deoni cattle.
Source of Variation
AFC
CI
d.f.
Mean Square Value F value
Between Groups
1
139.494
Within Groups
70
61.871
Total
71
Between Groups
1
5995.330
Within Groups
70
4184.551
Total
71
54
2.255
1.433
Results and Discussion
Table 4.11. Effect of SSCP patterns of fragment-6 of HSP90
gene on reproductive performances in Deoni cattle.
Dependant variables
Patterns
No. of
observations
(N)
Age at First calving
(Months)
I
17
37.529 ± 2.127NS
II
55
41.709 ± 1.428NS
I
17
407.882 ± 18.971NS
II
55
440.600 ± 12.734NS
Calving Interval
(Days)
LSM ± S.E
NS- non significant
Table 4.12. ANOVA of SSCP patterns of fragment-5 of HSP90
gene on reproductive performances in Deoni cattle
Source of Variation
AFC
CI
d.f.
Mean Square Value
F value
Between Groups
1
226.864
3.742
Within Groups
70
60.623
Total
71
Between Groups
1
13900.910
Within Groups
70
4071.614
Total
71
3.414
4.5 Sequencing and analysis
Representative samples in duplicate for each PCR-SSCP pattern
of fragment-4 comprising Exon 8, fragment 5 comprising Exon 9 and
fragment 6 comprising Exon 10 were carefully chosen and the PCR
products were custom sequenced (Chromous Biotech, Bangalore)
using the automated ABI DNA sequencer to confirm the mobility shift
in each pattern. Sequence data were analyzed using Bio edit software
55
Results and Discussion
Clustal
W
multiple
alignments
for
detecting
single
nucleotide
polymorphisms (SNPs).
In order to detect possible polymorphisms, we compared our
sequence to reference sequence of HSP90 AA1 in NCBI gene bank
accession
number NC-007319
for cattle. One single nucleotide
substitutions were detected in each of Exon 8, Exon 9 and Exon 10 of
HSP AA1 gene in Deoni
cattle (Tab.4.13). The analysis of fragment 4
comprising Exon 8 revealed T G transversion at position 3650 (Table
4.13) of HSPAA1 gene. The nucleotide sequences of pattern I and
pattern II in fragment 4 were found to be with two unique
homozygotic sequences, while the pattern III was observed to be
heterozyotic in nature.
The observed polymorphism at position 3650 (T
G) in exon 8
of HSP90 AA1 results in substitution of an amino acid from
Phenylalanine to Leucine which could potentially modify HSPAA1
expression. The observed polymorphisms at position 4111 (C
G) in
Exon 9 (NCBI GenBank accession number NC-007319) in fragment 5,
as well as at position 4578 (A
G) in Exon 10 (NCBI GenBank
accession number NC-007319) were silent mutations in the coding
region of the gene. The results indicated that the HSP90AA1 gene in
Bos indicus is highly conserved with high degree of homology with Bos
taurus cattle which is in agreement with the earlier report of Chen et
al. (2006).
The present findings confirm the suitability of HSP 90 gene as a
candidate gene for studying association between genetic variants with
reproductive performance in Deoni cattle.
56
Results and Discussion
The heterozygous (TG) cows (pattern III of fragment-4) had
higher
AFC
(49.000±2.853
months)
in
comparison
to
cows
homozygous for thymine (TT-pattern I) and homozygous cows for with
guanine (GG-pattern II); however, AFC was similar among both the
homozygous cows (Table 4.7). The Calving Interval was not different
among heterozygous (TG) cows and homozygous with thymine (TTpattern I) or homozygous with guanine (pattern II) cows (Table 4.7).
Polymorphisms identified in the coding region of the HSP90AA1 gene
could be a step towards identification of genetic markers for selecting
Bos indicus cattle with a propensity for lower AFC. However, further
studies using large number of cows from different breeds of Bos
indicus cattle are warranted before using them in the selection
programmes.
57
Results and Discussion
Table 4.13. Single Nucleotide Polymorphisms observed in HSP90 Gene in Deoni cattle
Gene
Segment
Positiona
Variationb
Polymorphic
Amino acid
allelic
changec
frequency
HSP90AA1
gene
Exon 8
3650
Phen
Leu
TTTTTT/GTCTTT
0.431
AAAGGC/G AGGAG
0.139
Gly (no change)
GAGGAA/GTCCAC
0.236
Lys (no change)
Fragment - 4
Exon 9
Fragment- 5
4111
58
Exon 10
Fragment- 6
4578
a based on the sequences from the NCBI GenBank accession number NC-007319 for cattle
b polymorphic residues underlined ( the common nucleotide followed by the variant)
c Phen-Phenylalanine; Leu-Leucine; Gly-Glycine; Lys-Lysine
Results and Discussion
CHAPTER - 5
───────────────────────────────
Summary and Conclusions
───────────────────────────────
5. SUMMARY AND CONCLUSIONS
The investigations were undertaken in Deoni breed of cattle to
characterize HSP90AA1 gene and to evaluate association between
genetic
variants
of
the
HSP90AA1
gene
with
reproductive
performances. The present investigation is the first study aimed at
molecular characterisation of HSP90AA1 gene in Bos indicus cattle. In
the present study, blood samples from seventy-two Deoni cows from
Southern Campus of NDRI herd were collected and DNA was extracted
using Miller’s High Salt method with minor modifications.
A total of six sets of overlapping primers were designed to cover
the entire coding sequences of HSP90AA1 gene using Primer 3
software. The PCR amplification of six fragments covering all the 10
exons were carried out after optimizing PCR conditions for each
fragment. The amplified PCR products were analyzed by Single-Strand
Conformation Polymorphism (SSCP) analysis and the genetic variants
in Deoni breed of cattle were determined after silver staining.
The
different band patterns in PCR fragments of HSP90AA1 gene were
characterized by the number of bands and mobility shifts.
The results of the present study indicated that the fragment 1
comprising Exons 1 to 2, fragment 2 comprising Exons 3 to 4 and
fragment 3 comprising Exons 5 to 7 showed
monomorphic patterns
with no mobility shifts indicating absence of any single nucleotide
polymorphism. The exons 1 to 7 showed monomorphism with similar
pattern in all the 72 animals studied.
PCR-SSCP analysis of fragment 4 comprising Exon 8 of
HSP90AA1 gene revealed three unique SSCP patterns. The pattern I,
pattern II and pattern III were observed with a genotypic frequency of
59
Summary and Conclusions
0.250, 0.639 and 0.111, respectively. The calculated Nei`s Gene
diversity and Shannon’s information index for fragment 4 was 0.5170
and 0.8769, respectively. The fragment 5 comprising of Exon 9 of
HSP90AA1 gene revealed two unique SSCP patterns (pattern I, pattern
II) with a genotypic frequency of 0.153 and 0.847, respectively. The
Nei`s Gene diversity and Shannon`s information index was 0.2589
and 0.4275, respectively. The PCR-SSCP analysis of fragment 6 of
HSP90AA1 gene comprising of Exon 10 revealed two unique SSCP
patterns (pattern I, pattern II) with a genotypic frequency of 0.236
and 0.764, respectively. The Nei`s Gene diversity and Shannon's
information index for fragment 6 was 0.3607 and 0.5466, respectively.
The heterozygous (TG) cows (pattern III of fragment-4) had
higher age at first calving in comparison to cows homozygous for
thymine (TT-pattern I) and homozygous cows for with guanine (GGpattern II). The cows with pattern I and pattern II had similar age at
first calving. There was no difference in calving interval in cows with
different SSCP patterns in Exon 8.
The two patterns observed in fragment 5 had no effect on AFC
and CI in Deoni cows. The age at first calving and calving interval were
similar among the cows with pattern I and pattern II for fragment 6
which comprises exon 10.
On the basis of differences in the SSCP patterns, three allelic
patterns of fragment 4 comprising Exon 8, two allelic patterns each
from fragment 5 and 6 comprising Exon 9 and 10, respectively of
HSP90AA1 gene were selected and corresponding PCR products in
duplicate were processed subsequently for custom sequencing.
60
Summary and Conclusions
CLUSTAL-W multiple sequence analysis was carried out to find
out polymorphisms. Our sequences were compared to sequence of
HSP90AA1 in GenBank accession number NC-007319 for cattle. The
analysis of fragment 4 comprising Exon 8 revealed T G transversion
at position 3650 of HSPAA1 gene. The nucleotide sequences of pattern
I and pattern II in fragment 4 were found to be two unique
homozygotic sequences, while the pattern III was observed to be
heterozyotic in nature. The observed polymorphism (T
G) at position
3650 in Exon 8 of HSP90 AA1 results in substitution of an amino acid
from Phenylalanine to Leucine. Polymorphisms at
G) in Exon 9 and at position 4578 (A
position 4111 (C
G) in Exon 10 were also
detected which were found to be silent mutations in the coding region
of the gene. The heterozygous TG cows (pattern III of fragment-4)
had
higher
age
at
first
calving
(49.000±2.853
months)
when
compared with homozygous thymine (TT) cows (pattern I) and
homozygous (GG) cows (pattern II) while calving interval was similar
among cows with different SSCP patterns. Preliminary studies have
shown no effect of SNPs in Exon 9 and 10 on reproductive
performance in Deoni cows.
The results indicated that the HSP90AA1 gene in Deoni breed of
cattle is highly conserved. Further, studies on SNPs in exon 8 of
HSP90AA1 using large number of animals from different breeds and
2D and 3D protein structure predictions and analysis may reveal some
of the important characteristics of the observed single nucleotide
polymorphism at structure and function level.
61
Summary and Conclusions
CONCLUSIONS
1. PCR-SSCP
analysis
is
a
powerful
technique
to
detect
polymorphisms in HSP90AA1 gene in Deoni cattle.
2. The amplified fragments of exons 1 to 7 of HSP90AA1 gene was
found to be highly conserved as revealed by PCR-SSCP analysis.
3. The SSCP variants were observed in fragments comprising Exons
8, 9 and 10 of HSP90AA1 gene in Deoni breed of cattle.
4. The presences of SNPs were confirmed by direct sequencing and
the possible amino acid change due to the observed SNPs were
predicted.
5. Associations
between
HSP90AA1
gene
polymorphism
with
reproductive performance (age at first calving and calving
interval) were analysed. The T G transversion at position 3650
of HSPAA1 gene was found to influence age at first calving. The
heterozygous TG cows had higher age at first calving as
compared to both the homozygous cows.
62
Summary and Conclusions
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Appendix
APPENDICES
Composition of reagents and buffers used in the study.
I. EDTA (10 per cent) solution
a.
Ethylene Diamine Tetra Acetic Acid (EDTA)
-
1g
b.
Triple glass distilled water to make up to
-
10 ml
II. RBC Lysis Buffer
a.
Ammonium Chloride
-
8.0235 g
b.
Potassium chloride (10 mM)
-
0.7455 g
c.
EDTA
-
0.0372 g
d.
Triple glass distilled water to make up to
-
1000 ml
e.
Autoclaved and stored at 40C
III. Tris Buffer Saline
a.
Potassium chloride
-
0.0373 g
b.
Sodium chloride (0.4 M)
-
8.1800 g
c.
Trishydro xymethyl aminomethane HCl
-
0.0303 g
d.
Magnesium chloride (10 mM)
-
1 ml
e.
Triple glass distilled water to make up to
-
1000 ml
f.
pH adjusted to
-
7.4
g.
Autoclaved and stored at 40C
-
1.2114 g
IV. Tris-EDTA buffer (TE buffer)
a.
Tris hydroxymethyl aminomethane HCl
b.
(10 mM) pH 7.6
i
c.
EDTA (0.1 mM)
-
0.3722 g
d.
Triple glass distilled water to make up to
-
1000 ml
e.
pH adjusted to
-
8.0
f.
Autoclaved and stored at 40C
g.
Sodium dodecyl sulphate (10 per cent SDS)
h.
Sodium dodecyl sulphate
-
10 g
i.
Triple glass distilled water to make up to
-
100 ml
V. Saturated sodium chloride
a.
Sodium chloride
-
29.22 g
b.
Triple glass distilled water to make up to
-
100 ml
a.
EDTA
-
18.612g
b.
Triple glass distilled water to make up to
-
100 ml
a.
Proteinase K
-
20 mg
b.
Triple glass distilled water to make up to
-
1 ml
c.
Stored at -20 C
VI. 0.5M EDTA
VII. Proteinase K
VIII. Agarose (0.8 per cent)
a.
Agarose
-
0.64 g
b.
TAE (50X) buffer
-
1.6 ml
c.
Triple glass distilled water to make up to
-
80 ml
ii
VIX. Ethidium bromide staining solution (10 per cent)
d.
Ethidium bromide
-
100 mg
e.
Triple glass distilled water to make up to
-
1 ml
X. Gel loading buffer (6X)
f.
Glycerol
-
5 ml
g.
Bromophenol Blue
-
125 mg
h.
Xylene cyanol
-
125 mg
i.
Triple glass distilled water to make up to
-
500 ml
XI. TBE 5X buffer
IX. ix
j.
Tris 445 mM
-
54 g
k.
Boric acid 445 mM
-
27.5 g
l.
EDTA 10mM
-
3.7224 g
m.
Dissolve in 1000ml Double distilled water
Ethidium bromide staining solution )
a.
Ethidium bromide
-
10 mg
b.
Triple glass distilled water to make up to
-
1 ml
-
4g
-
0.005 g
X. Gel loading buffer (6X)
for 10 ml solution
a.
Sucrose 40% W/V
b.
Bromophenol Blue
c.
EDTA 0.1 M
-
2 ml of 0.5M EDTA
d.
SDS
-
0.05 g
e.
Triple glass distilled water to make up to
-
make up to 10 ml
0.05% W/V
0.5% W/V
iii
XI.
30% Acrylamide/Bis‐Acrylamide (37.5:1)
a.
Acrylamide
‐
150 g
b.
Bis‐acrylamide
‐
4g
-
0.1 g
c.Ammonium Persulfate
XII.
XIII.
XIV.
500 mL
10% (w/v) Ammonium Persulfate (APS)
a.
Ammonium persulfate
b.
Make 50 mL aliquots. Store at ‐20°C.
500 mL
‐
50 g
Denaturing Solution
‐
1L
a.
NaCl
‐
87.66 g
b.
NaOH
‐
20.0 g
SSCP electrophoresis gel
a.
30 % Poly acrylamide gel solution
‐
7.5 ml
b.
Glycerol
‐
l 3 ml
c.
TBE buffer
‐
3.6 ml
d.
Autoclaved distilled water
‐
15.9 ml
e.
TEMED
‐
15 μl
f.
Ammonium per sulphate (10%)
‐
150 μl
g.
(Prepared fresh)
‐
50 ml
XV.
Silver staining
a) Acetic acid solution( Fixative)
Glacial acetic acid
iv
Distilled water
‐
450 ml
Silver nitrate
‐
0.5 gram
Formaldehyde
‐
750 μl
Distilled water
‐
500 ml
Sodium carbonate
‐
15 gram
Formaldehyde
‐
750 μl
Sodium thiosulphate (10%)
‐
10 μl
‐
500 ml
Glacial acetic acid 10 per cent
‐
10ml
Distilled water
‐
100ml
b) Silver nitrate solution
c) Sodium carbonate solution (Developer solution)
(freshly prepared
Distilled water
d) Stop Solution
v