Transcript Identification and characterization of genetic variants in

Identification and characterization of
genetic variants in the macrophage
expressed lysozyme gene and their
effect on lysozyme activity
• Contribution for the improvement of the disease
resistance in cattle
Historical back-ground of lysozyme.
Bovine Lysozyme Gene family (mLys and sLys genes).
Determination of serum lysozyme activity.
Pedigree chart of 2 heterozygote bulls for LZM+ activity
identified in Polish B&W dairy cattle population.
Lys-Mic genotyping in dairy cattle.
a) Electrophoresis pattern for Lys-Mic genotyping in various breeds
and species of dairy cattle.
b) Electrophoresis pattern for Lys-Mic genotyping in Polish Black
and White dairy cattle.
c) Effects of paternal allele (table-1).
Genomic organisation of mLys gene.
a) cDNA Structure of mLys gene (Steinhoff et al. 1994).
b) Gene structure of mLys gene (Hanke et al. 1996).
c) Location of microsatellite marker within mLys gene.
Concluding Remarks.
 Historical back-ground of lysozyme
 Sir Alexander Fleming discovered the
lysozyme (E.C.3.2.1.17) in 1922, a
remarkable bacteriolytic element found in
body tissues and secretions.
Definition
 Lysozymes are defined as 1,4--N-Acetylmuramidases (Lolles and Jolles, 1984).
The action of lysozyme
 In the bacterial peptido-glycan cell wall,
Lysozyme is responsible for the cleaving
the glycosidic bond between the c-1 of NAcetyl-muramic acid (MurNAc) and the c-4
of N-Acetyl-glucosamine (GlcNAc).
Figure 1: The action of lysozyme
Bovine lysozyme gene family (mLys and sLys)
 In dairy cattle, lysozyme is a multiple genes family.
 This gene family is present in the form of a gene cluster on chromosome 5(BTA5).
 This gene cluster is physically mapped to the chromosome at 5q23 (Gallagher
et al. 1993 and Brunner et al. 1994).
 As widely distributed in nature, lysozyme is expressed by various body tissues
and secretions. In dairy cattle, It is mainly expressed in stomach (sLys gene)
and macrophage (mLys gene)
 Importance of sLys Gene:
  sLys gene has much significance as a digestive enzyme.
  The recruitment of lysozyme as a digestive enzyme is a striking example of
adaptive evolution. (American group i.e., D.M. Irwin and co-workers: from
University of CA Davis, CA, USA).
 Importance of mLys gene: (Later in detail).
  (German group i.e., H.M. Seyfert and co-workers from FBN,
Dummerstorf, Germany).
Figure 2: Lysozyme cDNA
Coding region of lys protein
3’-untransalted region of mLys gene
5’-Terminal end
3’-Terminal end
TTTCTTA
11 aa Single peptide
Leader of mature lys
AAAGAAA
ACATTCAGTTCTT (h)
human retroposon element
ATCTTGAATCA(b)
Bovine retroposon ele.
130 aa
1
Glu-35 Asp-55
686 719
428
Pro-103
ATCTTGAATCA (b’)
bovine retroposon element
Bovine Retroposon(BDF)
Asp-102
36
AAAGAAC
699
991
conservity to human lys
1053
1242
Bov-sLys
1530
1335 1401
730
636 BP deletion in
bovine sLys gene
160 BP deletion in
human Lys gene
Figure 2: Important features of the cDNA structure of bovine mLys gene.
Genomic organisation of mLys gene
cDNA Structure of mLys gene (Steinhoff et al. 1994)
 cDNA of macrophaged expressed lysozyme gene revealed
following features:
 1) cDNA structure of 1530 bp.
 2) 5’-translated region: Covers a coding sequence region
for 130 aa’s including 11 aa single leader peptide.
 3) While, 3’-untranslated region includes: Human
retroposon element (13bp); Bovine reteroposon element
(11 bp); and also some conservity region for human
lysozyme gene and sLys.
Figure 3: mLys gene
Figure 3: Showing genomic organization of bovine mLys gene.
3’-untransalted region of mLys gene
Coding region of Lys gene
ATG
cDNA
**
TAA
*
5’-TTATTTGATATTAGGCCCACAGTG-3’
5’-utr
3’-GTTTTAGGGTGGGACACATCAT-5’
Leader Peptide
208 bp
[Exon 1]
K
E
164 bp
78 bp
[Exon 2]
1 B
E
1163 bp
[Exon 3]
2
[Exon 4]
K B
3
EV
E
4
Lys gene
1 Kb
2 Kb
3 Kb 4 Kb
5 Kb
6 Kb
7 Kb
8 Kb
9 Kb
10Kb
:Retroposon elements.
:Exon 1,2,3, and 4
* : Active area of AA.
K,E, B, EV: Sites for restriction enzyme.
ATG and TAA: Initiation and terminating codon
sequence for the transcription of mature lys gene.
E BE
B
EB
Lamda-17 (Clone)
Gene structure of mLys gene (Hanke et al. 1996)
 The important features of macrophaged expressed
lysozyme gene are as follows:
 1. The gene bank file number is: U25810; Hanke
et al. (1996).
 2. The complete mLys gene is of 10 kb in length.
 3. There are 4 exons of 208, 164, 78 and 1163 bp
in length.
 4. There are numerous restriction map sites and 4
different types of retroposon elements with in the
mLys gene.
 5. A highly polymorphic (TA)n type dinucleotide
repeat micro-satellite sequence is observed at
intron-2 of mLys gene.
Figure 8: Location of Lysmic marker in mLys gene
Figure 4: showing genomic organization of the microsatellite marker in bovine mLys gene.
Dairy cattle
Isolated DNA from blood or semen
1
2
3
Physically Mapped Lysozyme gene cluster
5q23
5’-terminal
3’- terminal
Isolated 1530 bp mLys cDNA
1 KB
5 KB
10 KB
About 10 Kb long mLys gene.
Exon 1
Exon 2
Exon 3
Exon 4
5 KB
1 KB
5’-terminal end
Exon 1
Forword Lys-mic Primer position at 5001-5024.
5’-ATAAGCATCAATATGCTCATCACT-3’
Exon 2
10 KB
Exon 3
Exon 4
Genomic DNA
3’- terminal end
Reverse Lys-mic Primer position at 5198-5175.
5’-GACAGTTTTTATATAGGAGCTGTG-3’
Determination of Serum Lysozyme Activity
(Phenotypic determination)
The blood serum lysozyme activity is measured as
% lytic activity in blood serum using Turbiditimetric method of Smolelis-Hartsell (Metzger
1970).
 On the basis of measured phenotypic values, one
can divide the animal group into two groups
i.e.,
Low lysozyme activity (LZM0) (% Lytic
values up to 20 units) and High lysozyme activity
(LZM+) ( Values above 20 units).
Distribution of lysozyme activity (Bi-model):
Lys-mic+/-
9
8
7
6
5
4
3
2
1
0
5
to
4
40
0
to
4
Percent lytic activity
35
5
to
3
30
0
to
3
25
5
to
2
20
0
to
2
15
5
to
1
10
0
Lys-mic-/-
5t
o1
0t
o5
No. of animals
Efeect of Lys-mic genotyping on fourth level of serum lysozyme activity.
Pedigree chart of 2 heterozygote bulls for LZM+
activity identified in Polish B&W dairy cattle population
 Two breeding bulls were identified as heterozygote for high LZM+ in
Polish black and white population,
Namely: CZORT 09312-4-5 and PARAN 51681-1-2.
 Bull Czort used in Polish B&W population as natural service, while
Paran used as AI bull.
 Inheritance of LZM+ gene in case of Czort came from maternal
side.
 Czort is basically utilised in active breeding population of Polish
B&W cattle for the mating with those cows which have infertility
problems.
 Second LZM+ bull Paran is actually eliminated from current breeding
program, but we are using this bull semen in experimentally designed
mating, particularly, to calve the homozygote animals for high LZM+.
 Previously identified LZM0 bull family.
 In Norwegian Red (NRF) dairy cattle (Olsaker et al., 1993)
Figure 5:
LYS-Mic genotyping in dairy cattle:
 Electrophoresis pattern for Lys-Mic genotyping
in various breeds and species of dairy cattle
(Weikard et al. 1996), explaining a distribution of
different polymorphic macro-satellite alleles in
different bovine species and breeds.
 A total of 12 polymorphic alleles were reported in
different bovine breeds and species.
 In Holstein breed, a total of 4 different alleles
were reported.
 We are following the same protocol for lys-mic
genotyping, as described by Weikard et al. 1996.
Location of microsatellite marker:
 A (TA)n dinucleotide-type microsatellite within the
intron 2 of the mLys gene.
Figure 6: Lysmic genotyping
1 2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 23
Genotyped animals
Lys-mic alleles
3
7
4 3 3 3 3 3 3 3 4 3 3
4 4 3 3 3 3 3 7 4 4 3
3 3 3 3 3 4 3 3 3 3
3 3 3 3 7 4 4 3 3 3
Lys-mic Genotypes
Figure 6: Showing the electrophoresis pattern of Lys-mic
genotyping in Polish Black-and-White dairy cattle.
Primer sequence & position of microsatellite
marker:
The primersequence:
5'-ATAAGCATCAATATGCTCATCACT-3‘
5'-GACAGTTTTTATATAGGAGCTGTG-3'
 positions 5001-5024 and 5198-5175 respectively,
from gene bank file: U25810
Electrophoresis pattern for Lys-Mic genotyping in
Polish Black and White dairy cattle (Pareek et al.
1998).

This eletrophoresis pattern is explaining
about the co_segregation of the paternal
alleles 3&7 in the half-sib family group of
Czort.
Two new gene variants i.e., nr7 & nr 10
were added to the previous publication of
Weikard et al.1996.
Figure 7: Lysmic genotyping in Czort’s halfsib progenies

1
2
3
4
5
6
8
7
9
10
11
12
13
14
15

P 
P 
P 
P 
P 
P 
P 
P 
P 
P 
P 
P 
P 
P
P

16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
P 
P 
P 
P 
P 
P 

P 
P 
P 
P 
P 
P 
P 
P 
P
P

32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
 P P P 
P
P P P
P 
P 
P 
P 
P 
P 

P

P 
P 
P 
P
P
Figure 33: Lys-mic genotyping in the analyzed half-sib family of Czort-09312-4-5.
Symbols: Czort-09312-4-5 ( ), 50 Dams () and 50 progenies (P).
[PS: Genotype records from 4 different gels].
Effects of paternal allele (table result)
Highly significant of paternal allele 7 was
observed on lysozyme activity at different
levels and, it was concluded that paternal
allele 7 is completely associated with high
serum lysozyme activity thus it can be
considered as a marker allele for the LZM+.
Concluding Remarks





Identification of two rarely existed LZM+ family in Polish B&W cattle
is the second incidence after Norwegian (NRF) cattle in the worldwide dairy cattle population.
Co-segregation of lys-mic allele 7 at a microsatelllite locus has been
positively associated with the LZM+.
A notorious very low conc. of lysozyme revealed that further
investigations and analysis of such rarely existed bovine families are
the essential pre-requisite:
To identify more LZM+ animals in the general dairy cattle population
through lys-mic genotyping.
To search out more new genetic variants for LZM+ within mLys gene.
A trait associated (viz., SCC, Leukocyte number etc.) study together
with Lys-Mic genotyping is also required for better understanding of
mLys gene and to further prove that lysozyme could be as a candidate
gene for the mastitis resistance.
So far, the reason for low expression of mLys gene in mammary gland
tissue is unknown, but the identification of casual mutation with in
the promoter region of mLys gene may help in solving out this
problem.