Regulatory region of milk protein alpha S1-casein (αS1-CN) gene was sequenced, characterized, and analyzed to detect variations among 13 Indian cattle (Bos indicus) breeds. Comparative analysis of 1,587？bp region comprising promoter (1,418？bp), exon-I (53？bp), and partial intron-I (116？bp) revealed 35 nucleotide substitutions (32 within promoter region, 1 in exon-I, and 2 in partial intron-I region) and 4 Indels. Within promoter, 15 variations at positions ？1399 (A？>？G), ？1288 (G？>？A), ？1259 (T？>？C), ？1158 (T？>？C), ？1016 (A？>？T), ？941 (T？>？G), ？778 (C？>？T), ？610 (G？>？A), ？536 (A？>？G), ？521 (A？>？G), ？330 (A？>？C), ？214 (A？>？G), ？205 (A？>？T), ？206 (C？>？A), and ？175 (A？>？G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains. 1. Introduction Bovine caseins are distinguished into four protein fractions, namely, alpha S1-casein, alpha S2-casein, beta-casein, and kappa-casein encoded by genes: αS1-CN, αS2-CN, β-CN, and κ-CN, respectively [1]. Alpha S1-casein represents the major protein fraction (31%) among the bovine milk proteins (caseins and whey) and constitutes up to 40% of total casein [1]. It is a calcium sensitive and highly phosphorylated protein. It has an important role in the capacity of milk to transport calcium phosphate and is organized at 5′-terminus of casein cluster located on bovine chromosome 6 (BTA6). Till now, 9 variants (A-I) have been reported in the coding region of αS1-CN. Amongst these, B and C, differing in amino acid substitution (Glu/Gly) at position 192 of the mature protein, are the most common. The variant C has been reported to be common in zebu breeds, while other rare variants like A, D, and F have only been reported in European cattle [2]. These variants are well characterized and their associations with quantitative effects on milk performance/production parameters have been widely reported [3, 4]. The results on association studies involving only coding region variants are not always
References
[1]
H. M. Farrell Jr., R. Jimenez-Flores, G. T. Bleck et al., “Nomenclature of the proteins of cows' milk—sixth revision,” Journal of Dairy Science, vol. 87, no. 6, pp. 1641–1674, 2004.
[2]
A. M. Caroli, S. Chessa, and G. J. Erhardt, “Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition,” Journal of Dairy Science, vol. 92, no. 11, pp. 5335–5352, 2009.
[3]
E. M. Prinzenberg, C. Weimann, H. Brandt et al., “Polymorphism of the bovine CSN1S1 promoter: linkage mapping, intragenic haplotypes, and effects on milk production traits,” Journal of Dairy Science, vol. 86, no. 8, pp. 2696–2705, 2003.
[4]
M. Szymanowska, N. Strzalkowska, E. Siadkowska, J. Krzyzewski, Z. Ryniewicz, and L. Zwierzchowski, “Effects of polymorphism at -noncoding regions (promoters) of S1- and S2-casein genes on selected milk production traits in Polish Black-and White cows,” Animal Science Papers and Reports, vol. 21, pp. 97–108, 2003.
[5]
J. M. L. Heck, A. Schennink, H. J. F. van Valenberg et al., “Effects of milk protein variants on the protein composition of bovine milk,” Journal of Dairy Science, vol. 92, no. 3, pp. 1192–1202, 2009.
[6]
P. Martin, M. Szymanowska, L. Zwierzchowski, and C. Leroux, “The impact of genetic polymorphisms on the protein composition of ruminant milks,” Reproduction Nutrition Development, vol. 42, no. 5, pp. 433–459, 2002.
[7]
M. Szymanowska, T. Malewski, and L. Zwierzchowski, “Transcription factor binding to variable nucleotide sequences in -flanking regions of bovine casein genes,” International Dairy Journal, vol. 14, no. 2, pp. 103–115, 2004.
[8]
T. Malewski, “Computer analysis of distribution of putative cis- and trans- regulatory elements in milk protein gene promoters,” BioSystems, vol. 45, no. 1, pp. 29–44, 1998.
[9]
M. Szymanowska, E. Siadkowska, M. ？ukaszewicz, and L. Zwierzchowski, “Association of nucleotide-sequence polymorphism in the 5'-flanking regions of bovine casein genes with casein content in cow's milk,” Lait, vol. 84, no. 6, pp. 579–590, 2004.
[10]
E. M. Prinzenberg, H. Brandt, J. Bennewitz, E. Kalm, and G. Erhardt, “Allele frequencies for SNPs in the αs1-casein gene (CSN1S1) flanking region in European cattle and association with economic traits in German Holstein,” Livestock Production Science, vol. 98, no. 1-2, pp. 155–160, 2005.
[11]
K. Sanders, J. Bennewitz, N. Reinsch et al., “Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population,” Journal of Dairy Science, vol. 89, no. 8, pp. 3164–3174, 2006.
[12]
E. M. Ibeagha-Awemu, E. M. Prinzenberg, and G. Erhardt, “High variability of milk protein genes in Bos indicus cattle breeds of Cameroon and Nigeria and characterization of a new αs1-casein promoter allele,” Journal of Dairy Research, vol. 72, no. 1, pp. 1–9, 2005.
[13]
A. W. Kuss, J. Gogol, H. Bartenschlager, and H. Geldermann, “Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression,” Journal of Dairy Science, vol. 88, no. 6, pp. 2246–2252, 2005.
[14]
A. W. Kuss, T. Peischl, J. Gogol, H. Bartenschlager, and H. Geldermann, “ S1-casein yield and milk composition are associated with a polymorphic regulatory element in the bovine S1-casein gene,” in Indicators of Milk and Beef Quality, J. F. Hocquette and S. Gigli, Eds., pp. 301–305, EAAP Publication, 2005.
[15]
W. L. Bai, R. H. Yin, Q. L. Dou et al., “A single nucleotide polymorphism and sequence analysis of CSN1S1 gene promoter region in Chinese BOS Grunniens (YAK),” Animal Biotechnology, vol. 21, no. 1, pp. 36–41, 2010.
[16]
L. Chianese, M. Quarto, F. Pizzolongo et al., “Occurrence of genetic polymorphism at the s1-casein locus in Mediterranean water buffalo milk,” International Dairy Journal, vol. 19, no. 4, pp. 181–189, 2009.
[17]
L. Ramunno, G. Cosenza, A. Rando et al., “The goat S1-casein gene: gene structure and promoter analysis,” Gene, vol. 334, no. 1-2, pp. 105–111, 2004.
[18]
S. K. Bhure and B. Sharma, “The PCR amplification, sequencing and computer-aided analysis of ovine S1-casein gene promoter,” Indian Journal of Biotechnology, vol. 7, no. 4, pp. 478–481, 2008.
[19]
T. A. Schild and H. Geldermann, “Variants within the -flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins s1, s2 and ,” Theoretical and Applied Genetics, vol. 93, no. 5-6, pp. 887–893, 1996.
[20]
J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, NY, USA, 1989.
[21]
A. E. Kel, E. G？？ling, I. Reuter, E. Cheremushkin, O. V. Kel-Margoulis, and E. Wingender, “MATCH: a tool for searching transcription factor binding sites in DNA sequences,” Nucleic Acids Research, vol. 31, no. 13, pp. 3576–3579, 2003.
[22]
V. Matys, E. Fricke, R. Geffers et al., “TRANSFAC: transcriptional regulation, from patterns to profiles,” Nucleic Acids Research, vol. 31, no. 1, pp. 374–378, 2003.
[23]
N. Grabe, “AliBaba2: context specific identification of transcription factor binding sites,” In Silico Biology, vol. 2, no. 1, pp. S1–S15, 2002.
[24]
M. Stephens and P. Scheet, “Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation,” American Journal of Human Genetics, vol. 76, no. 3, pp. 449–462, 2005.
[25]
M. Stephens, N. J. Smith, and P. Donnelly, “A new statistical method for haplotype reconstruction from population data,” American Journal of Human Genetics, vol. 68, no. 4, pp. 978–989, 2001.
[26]
P. Librado and J. Rozas, “DnaSP v5: a software for comprehensive analysis of DNA polymorphism data,” Bioinformatics, vol. 25, no. 11, pp. 1451–1452, 2009.
[27]
L. Excoffier and H. E. L. Lischer, “Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows,” Molecular Ecology Resources, vol. 10, no. 3, pp. 564–567, 2010.
[28]
K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar, “MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods,” Molecular Biology and Evolution, vol. 28, pp. 2731–2739, 2011.
[29]
M. Rijnkels, P. M. Kooiman, P. J. A. Krimpenfort, H. A. De Boer, and F. R. Pieper, “Expression analysis of the individual bovine -, (s2)- and -casein genes in transgenic mice,” Biochemical Journal, vol. 311, no. 3, pp. 929–937, 1995.
[30]
D. Koczan, G. Hobom, and H. M. Seyfert, “Characterization of the bovine S1-casein gene C-allele, based on a MaeIII polymorphism,” Animal Genetics, vol. 24, no. 1, p. 74, 1993.
