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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 1163 bp [Exon 3] E 2 3 Kb 4 Kb 5 Kb [Exon 4] K B 3 EV E 4 Lys gene 1 Kb 2 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 7 8 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.