Screening the partial coding region of metallothionein isoform-2 gene in Zebu cattle

Document Type : Full paper (Original article)


1 MVSc, Department of Animal Genetics and Breeding, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University (MAFSU), Seminary Hills, Nagpur-440006, Maharashtra State, India

2 Department of Animal Genetics and Breeding, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University (MAFSU), Seminary Hills, Nagpur-440006, Maharashtra State, India

3 3Environmental Health Division, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur-440020, Maharashtra State, India


Metallothionein (MT) is important because it binds tightly to heavy metals to decrease their toxicity. DNA was isolated from 30 toxic metal exposed and 30 toxic metal unexposed Zebu cows. The amplified metallothionein isoform-2 (MT-2) PCR product (489 bp) was further used for PCR-RFLP and DNA sequencing. MT-2 TaqI PCR-RFLP revealed homozygous genotype (AA) except for the E23 animal (AB). The genotype frequency of AA and AB (E23) genotypes in the exposed groups was 0.967 and 0.033 respectively. DNA sequencing was carried out for the toxic metal exposed sample (E23) and the control group sample (C13). Blast comparisons of the sequences were then aligned against a nucleotide database which revealed 150 nucleotide substitutions consisting of 70 transitions and around 80 transversions. DNA sequencing followed by PCR-RFLP for MT-2 revealed a higher number of nucleotide substitutions (150) for the AB genotype of E23 as compared to the AA genotype (38) of E21. The proportions of transversion mutations in the AB genotype were higher as compared to the MT-2 AA genotype. DNA sequencing was carried out based on random sampling for E21 and C13. Alignment analysis of the E21 and C13 sample revealed 38 nucleotide substitutions consisting of equal numbers of transition and transversion. BLAST analysis of the identified partial sequence revealed 89% identity with Bos taurus, 85% identity with sheep, 98% identity with buffalos and 100% identity with goat MT-2 sequences. Overall findings of the present study revealed DNA sequence variation in the coding region of the MT-2 gene of Zebu cattle which can be utilised to characterize and explore markers for heavy metal homeostasis in Zebu cattle.


Basu, N; Goodrich, JM and Head, J (2014). Ecogenetics of
mercury: from genetic polymorphisms and epigenetics to risk assessment and decision-making. Envir. Toxicol. Chem., 33: 1248-1258.
Clamp, PA; Feltes, R; Shalvevet, D; Beever, JE; Atac, E and Schook, LB (1993). Linkage relationship between ALPL, EN01, GPI, PGD TGFB1 on porcine chromosome 6. Genomics. 17: 324-329.
Doiphode, PU (2012). Metallothionein gene polymorphism study in cattle from Fly Ash zone. MVSc. Thesis submitted to Maharashtra Animal and FisherySciencesUniversity, Nagpur, India. PP: 1-47.
Egli, D; Domènech, J; Selvaraj, A; Balamurugan, K; Hua, H; Capdevila, M; Georgiev, O; Schaffner, W and Atrian, S (2006). The four members of the Drosophila Metallothionein family exhibit distinct yet overlapping roles in heavy metals homeostasis and detoxification. Genes Cells. 11: 647-658.
Jiang, ZH and Gibson, JP (1999). Genetic polymorphisms in the leptin gene and their association with fatness in four pig breeds. Mamm. Genome. 10: 191-193.
Klaassen, CD; Liu, J and Chaudhari, S (1999). Metallo-thionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol., 39: 267-294.
Leonard, JT; Grace, MB; Buzard, GS; Mullen, MJ and Barbagallo, CB (1998). Preparation of PCR products for DNA sequencing. Biotechniques. 24: 314-317.
Robbins, AH; McRee, DE; Williamson, M; Collett, SA; Xuong, NH; Furey, WF; Wang, BC and Stout, CD (1991). Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution. J. Mol. Biol., 221: 1269-1293.
Roggeman, S; de Boeck, G; De Cock, H; Blust, R and Bervoets, L (2014). Accumulation and detoxification of metals and arsenic in tissues of cattle (Bos taurus), and the risks for human consumption. Sci. Total Environ., 466-467: 175-184.
Sakulsak, N (2012). Metallothionein: an overview on its metal homeostatic regulation in mammals. Int. J. Morphol., 30: 1007-1012.
Untergrasser, A; Cutcutache, I; Koressaar, T; Ye, J; Faircloth, BC; Remm, M and Rozen, SG (2012). Primer3 - new capabilities and interfaces. Nucl. Aci. Res., 40: e115.
Wang, J; Zhang, LP; Yang, L; Fei, CH; Wang, L; Xie, C and Wu, JP (2008). Characteristics of MT-IV in sheep and goat. J. Genet. Genomics., 30: 1591-1596.
Wu, JP; Ma, BY; Ren, HW; Zhang, LP; Xiang, Y and Brown, MA (2007). Characterization of metallothioneins (MT-I and MT-II) in the yak. J. Animal Sci., 85: 1357-1362.
Yang, L; Zhang, LP; Wu, JP; Brown, MA; Liu, B; Ma, BY and Wang, L (2011). Metallothionein in yak characteri-zation of metallothionein-III in yak (Bos grunniens). J. Animal Vet. Adv., 10: 100-105.