„

2006 Zoological Society of JapanZOOLOGICAL SCIENCE

23

: 607–611 (2006)

Molecular Characterization of the Interferon-tau Gene of the Mithun (

Bos frontalis

)

Konadaka S. Rajaravindra

†

, Abhijit Mitra

†

*, Ajay K. Sharma,Sitangshu M. Deb and Arjava Sharma

Genome Analysis Laboratory, Animal Genetics Division, Indian VeterinaryResearch Institute, Izatnagar, Bareilly 243122 (U.P.), India

The mithun (

Bos frontalis

) not only remains one of the most neglected ungulate species due to itsremote range, but also has been identified as a vulnerable species due to its declining population.Augmenting its reproductive efficiency could be a strategy for reversing its population decline.Considering the importance of interferon-tau (IFNT) as a primary signal in establishing maternalrecognition of pregnancy (MRP), the present study was undertaken to characterize the IFNT geneof the mithun. A 588 bp mithun IFNT (mitIFNT) gene was PCR amplified using genomic DNA as thetemplate. Its nucleotide sequence comprised an entire open reading frame of 585 bp encoding a195 amino acid pre-protein. In nucleotide sequence, the mitIFNT gene was more than 85% similarto the homologous genes of domestic and wild ruminant species characterized to date. However,phylogenetic analysis placed mitIFNT into a clade containing IFNT of the red deer, but not IFNTsof cow, sheep, or goats, or other wild ruminant species. Our characterization of mitIFNT representsthe first complete sequence of any gene from the mithun.

Key words:

Mithun,

Bos frontalis

, Interferon-tau (IFNT), maternal recognition of pregnancy (MRP)

INTRODUCTION

The mithun (

Bos frontalis

), a semi-wild ruminant of thetropical evergreen rain forests of the hilly northeastern regionof India, is very important to local people for milk, meat, anddraft power, and is often referred to as “the cow of moun-tains”. Despite its usefulness, the mithun has remained oneof the most poorly known ungulate species because of itsremote range and other ecological and socio-political factors(Mondal

et al.,

2004). To date, a few studies documenting itshormonal profile (Mondal

et al.,

2004, 2005), growth perfor-mance (Mondal and Prakash 2004; Pal

et al.,

2004), bloodgroups and karyotype (Winter

et al.,

1984, 1986), carcasscharacteristics (Heli

et al.,

1994, 1996), and milk composition(Mondal

et al.,

2001) have been reported. However, very lim-ited information is available on its reproductive behavior(Gupta

et al.,

2004; Rajkhowa, 2005). The mithun is a poly-estrus seasonal breeder exhibiting an estrus cycle of 19–24days. The gestation period varies from 282 to 320 days, withan inter-calving period of 349–395 days. Although there is lit-tle information available on its fecundity, the mithun generallygives birth to a single calf.

Early embryonic mortality is the main source of repro-ductive wastage in domestic ruminants (Peters, 1996). Even

after optimum fertilization, the efficiency of the embryo-uterusinteraction during the pre-implantation period determines thesuccess of the establishment and maintenance of pregnancy.Impaired communication between the conceptus and thematernal uterine environment is one cause of early embryo-nic mortality (Thatcher

et al.,

1994). Consequently, maternalrecognition of pregnancy (MRP) is one of the most critical ofa cascade of events determining reproductive efficiency(Roberts and Schalue-Francis, 1990). In ruminant ungulates,MRP involves protection of the

corpora lutea

(CL) from luteol-ysis by inhibition of luteolytic pulses of PGF

2

a

(Roberts

et al.,

1996). Interferon-tau (IFNT), secreted constitutively by thetrophoectoderm for a limited period during pregnancy,especially during the stage of blastocyst elongation, acts asthe first signal in MRP (Knickerbocker

et al.,

1986; Stojkovic

et al.,

1999). IFNT exerts its antiluteolytic action by stabilizingor up-regulating progesterone receptors in the endometrium,inhibiting endometrial estrogen and oxytocin receptors, and/or inducing the synthesis of an enzyme inhibitor necessaryfor the synthesis of PGF

2

a

(Bazer

et al.,

1991).Advances in molecular biology, particularly functional

genomics, have led to several studies to unravel the intricatemechanisms involved in MRP. Considering its importance inMRP, the IFNT gene has been studied in many domesticruminant species including cattle (Meyer

et al.,

1995), sheep(Martal

et al.,

1979), and goats (Newton

et al.,

1996), andalso in wild ruminant ungulates such as the red deer (Dem-mers

et al.,

1999), American bison (Rasmussen

et al.,

2005), yak, musk ox (Leaman and Roberts, 1992), andgiraffe (Liu

et al.,

1996). Here, we report the characterizationof the IFNT gene from the mithun, providing the first com-plete sequence of any gene of this important species.

* Corresponding author. Phone: +91-581-2303384;Fax : +91-581-2303284;E-mail : drabhijitmitra@gmail.com

†

These two authors equally contributed to this work.Note: The nucleotide sequence of the interferon-tau gene isavailable in the DDBJ/EMBL/GenBank databases under accessionnumber AY665674doi:10.2108/zsj.23.607

K. S. Rajaravindra

et al

.608

MATERIALS AND METHODS

DNA isolation

Genomic DNA was isolated from venous blood using the stan-

dard phenol-chloroform extraction method (Sambrook

et al.,

1989).The integrity of the genomic DNA was checked by agarose gel(0.7%) electrophoresis and visualization of the gel under UV lightafter staining with ethidium bromide. The purity of the genomic DNA

Fig. 1.

Amino acid alignment of the mature IFNT protein (172 amino acids) of mithun with that of different domestic and wild ruminant ungu-lates. For domestic ruminants, the polymorphic variants of IFNT that exhibited highest and lowest similarity with mitIFNT were included in thealignment.

1

1

1

1

1

1

1

1

1

1

1

1

1

10 20 30 40

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ -

ＣＹＬＳＥＮＨＭＬＤＡＲＥＮＬＲＬＬＡＲＭＮＲＬＳＰＨＳＣＬＱＤＲＫＤＦＧＬＰＱＥＭＶＥ

Ｄ Ｄ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ -

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ -

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ -

Ｑ Ｑ ･ ･ Ｑ ･ Ｒ Ｒ ･ Ｋ ･ Ｒ ･

･ ･ ･ Ｈ Ｒ Ｒ Ｒ Ｒ Ｄ ･ ･ Ｒ Ｄ

･ ･ ･ ･ Ｌ Ｌ Ｌ Ｌ ･ ･ ･ Ｐ ･

･ Ｖ ･ ･ ･ ･ ･ ･ ･ Ｖ ･ Ｔ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｖ Ｌ Ｇ Ｇ ･ ･ ･ ･ Ｇ Ｖ Ｈ ･ Ｇ

Ｇ Ｇ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｖ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｑ ･ ･

Ｑ Ｑ ･ Ｑ ･ ･ ･ ･ ･ Ｑ Ｋ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｆ ･ ･ ･

･ Ｋ ･ ･ ･ Ｋ ･ ･ ･ ･ Ｋ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｇ Ｇ ･ ･ Ｄ Ｄ Ｄ Ｄ ･ Ｇ ･ Ｄ ･

Ｑ Ｑ Ｑ ･ ･ ･ ･ ･ Ｑ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｒ Ｔ ･ ･ ･ ･ ･ ･ ･ Ｓ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ Ｔ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｒ Ｒ ･ ･ ･ ･ ･ ･ Ｑ Ｑ ･ ･ ･

Ｆ Ｆ ･ Ｐ ･ ･ ･ ･ Ｐ Ｔ ･ ･ Ｐ

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｑ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ Ｇ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｋ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ａ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｆ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ Ｗ ･ ･ ･ ･ ･ ･ Ｒ Ｒ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｋ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｔ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

AY665674AJ000638AF238613M31558M73241AF158817AY357327AY357336AY643747DQ139308 U55050M73244 AY455289

Majority

Mit IFNT Reddeer IFNT Bov var c1 Bov var BOVTPH1C Ov var SHPOTP1B Ov var p8v1 Cap var 2a Cap var 6 Am. Bison IFNT Musk deer IFNT Giraffe IFNT Musk Ox IFNT Yak IFNT

45

45

45

45

45

45

45

45

45

45

45

45

41

50 60 70 80

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

ＧＤＱＬＱＫＤＱＡＩＳＶＬＨＥＭＬＱＱＣＦＮＬＦＨＴＥＨＳＳＡＡＷＮＴＴＬＬＥＱＬＣＴＧ

Ｓ Ｇ ･ Ｎ ･ ･ ･ ･ Ｎ Ｎ Ｓ ･ Ｎ

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｅ ･ ･ ･ Ｅ ･ ･ ･ ･ ･ ･ ･ ･

Ａ ･ ･ ･ Ａ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ Ｆ Ｆ Ｆ Ｆ ･ ･ ･ Ｌ ･

･ ･ ･ ･ Ｃ Ｐ ･ Ｙ ･ ･ ･ ･ ･

･ ･ ･ Ｌ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ Ｙ Ｙ Ｙ Ｙ ･ ･ ･ Ｙ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｓ ･ ･ ･ Ｓ Ｓ Ｔ Ｉ ･ ･ ･ Ｒ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ Ｙ ･ Ｙ Ｙ Ｒ ･ Ｎ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ Ｉ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｒ Ｒ ･ ･ Ｒ ･ ･ Ｒ ･ Ｒ Ｒ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｌ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ａ ･ Ｃ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｄ Ｄ ･ ･ ･ Ｄ Ｄ ･ ･ Ｄ Ｄ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ａ Ｎ ･ ･

･ ･ ･ ･ ･ ･ ･ Ｉ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｅ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｒ Ｒ ･ ･ ･ ･ Ｒ Ｈ ･ Ｒ ･ Ｒ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

AY665674AJ000638AF238613M31558M73241AF158817AY357327AY357336AY643747DQ139308 U55050M73244 AY455289

Majority

Mit IFNT Reddeer IFNT Bov var c1 Bov var BOVTPH1C Ov var SHPOTP1B Ov var p8v1 Cap var 2a Cap var 6 Am. Bison IFNT Musk deer IFNT Giraffe IFNT Musk Ox IFNT Yak IFNT

89

89

89

89

89

89

89

89

89

89

89

89

85

100 110 120 130

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｈ Ｈ Ｈ ･ ･ ･ ･ ･ ･ ･ Ｈ Ｈ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｄ Ｄ Ｄ ･ ･ Ｄ ･ ･ ･ ･ Ｄ ･ ･

･ ･ ･ ･ ･ Ｈ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｅ ･ ･ ･

･ ･ ･ ･ Ｔ Ｔ Ｔ Ｔ ･ ･ ･ Ｔ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｒ ･ ･

･ ･ ･ ･ Ｒ Ｒ Ｒ Ｒ ･ ･ ･ Ｒ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ａ ･ ･ ･

Ｑ Ｑ Ｑ ･ ･ Ｑ ･ Ｌ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｌ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｔ ･ ･ ･

･ ･ Ｅ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ Ｋ ･ ･ ･ ･ ･ ･ ･ ･ Ｑ ･ ･

Ｅ Ｅ ･ ･ ･ Ｅ ･ ･ ･ Ｅ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ａ Ｄ Ａ Ｄ ･ ･ ･ ･ Ｄ Ａ Ｇ ･ Ｄ

･ ･ ･ Ｍ ･ ･ ･ ･ Ｍ ･ Ｍ ･ Ｍ

･ Ｒ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ Ｋ Ｎ Ｋ Ｋ ･ ･ ･ Ｋ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ Ｄ Ｄ Ｄ Ｄ ･ ･ ･ Ｄ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｔ Ｔ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ Ｖ Ｖ Ｖ Ｍ ･ ･ ･ Ｖ ･

Ａ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｍ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｒ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ Ｅ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ Ｄ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ Ｙ ･ ･ ･ Ｋ ･ Ｙ ･

･ ･ ･ ･ Ｆ Ｄ Ｄ Ｄ ･ Ｉ ･ Ｄ ･

･ ･ ･ ･ ･ Ｌ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ Ｑ ･ ･ ･ Ｑ Ｑ Ｑ ･ Ｑ ･ Ｑ ･

ＬＱＱＱＬＥＤＬＤＡＣＬＧＰＶＭＧＥＫＤＳＥＬＧＲＭＧＰＩＬＴＶＫＫＹＦＱＧＩＨＶＹＬＫ

90

AY665674AJ000638AF238613M31558M73241AF158817AY357327AY357336AY643747DQ139308 U55050M73244 AY455289

Majority

Mit IFNT Reddeer IFNT Bov var c1 Bov var BOVTPH1C Ov var SHPOTP1B Ov var p8v1 Cap var 2a Cap var 6 Am. Bison IFNT Musk deer IFNT Giraffe IFNT Musk Ox IFNT Yak IFNT

133

133

133

133

133

133

133

133

133

133

133

133

129

140 150 160 170

･ ･ Ｋ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ Ｇ ･ ･ ･ ･ Ｋ Ｇ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｖ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｖ ･ ･

･ ･ Ｉ Ｉ ･ ･ ･ ･ Ｉ Ｉ Ｉ ･ ･

･ Ｑ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ Ｍ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

Ｆ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ Ｔ Ｔ Ｔ ･ Ｔ ･ Ｔ ･

･ ･ ･ ･ ･ Ｖ ･ ･ ･ ･ ･ ･ ･

･ Ｉ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ Ｓ ･ ･ ･ ･ ･ ･ ･ Ｉ ･ ･ ･

Ｓ Ｒ Ｓ ･ Ｓ ･ ･ ･ ･ Ｓ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｌ ･ ･ ･

Ｅ ･ Ｅ ･ Ｅ ･ ･ ･ ･ Ｅ Ｅ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ Ｔ Ｔ Ｔ ･ ･ Ｋ Ｋ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ Ｉ ･ ･ ･ ･ ･ ･ ･ ･ Ｔ ･

Ｄ ･ ･ ･ ･ ･ ･ ･ ･ Ｄ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ Ｎ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ Ｔ ･ ･ ･

･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･ ･

･ ･ Ｓ Ｌ ･ ･ ･ ･ Ｌ ･ ･ ･ ･

ＥＫＥＹＳＤＣＡＷＥＩＶＲＶＥＭＭＲＡＬＳＳＳＴＴＬＱＫＲＬＲＫＭＧＧＤＬＮＳＰ

AY665674AJ000638AF238613M31558M73241AF158817AY357327AY357336AY643747DQ139308 U55050M73244 AY455289

Majority

Mit IFNT Reddeer IFNT Bov var c1 Bov var BOVTPH1C Ov var SHPOTP1B Ov var p8v1 Cap var 2a Cap var 6 Am. Bison IFNT Musk deer IFNT Giraffe IFNT Musk Ox IFNT Yak IFNT

Interferon-tau Gene of the Mithun 609

was checked by means of spectrophotometric readings at OD

260

and OD

280.

PCR amplification of the IFNT gene

Degenerate primers (IFNT/startF, 5’ATGGCCTTCGTGCTCTC-TCT3’ and IFNT/stopR, 5’TCAARGTGAGTTCAGATCT3’) designedon the basis of available IFNT sequences from cattle (Acc. No.AF238611-13), sheep (Acc. No. M88773), and goat (Acc. No.M73243) were used. PCR amplification was carried out in a totalvolume of 25

m

l of reaction mixture containing approximately 100 ngof genomic DNA, 1X PCR buffer (100 mM Tris-HCl pH 8.8 at 25

∞

C,500 mM KCl, and 0.8% Nonidet P40), 1.5 mM MgCl

2

, 200

m

M eachdNTP, 2

m

M each primer, and 1.0 unit of

Taq

DNA polymerase. ThePCR protocol involved an initial denaturation at 95

∞

C for 3 min; 30cycles of denaturation (95

∞

C for 30 sec), annealing (56

∞

C for 30sec), and extension (72

∞

C for 45 sec); and one cycle of final exten-sion (72

∞

C for 10 min). The PCR product was checked by 1% aga-rose gel electrophoresis in 1X TAE buffer after staining with ethid-ium bromide.

Sequencing and sequence analysis of IFNT gene

Both strands of the amplicon were directly sequenced on anautomated sequencer (Sequence Analyzer Version 2.0, ABI Prism)by Sanger’s dideoxy chain termination method. The sequenceobtained was subjected to BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) to verify that it was IFNT. The nucleotide and deducedamino acid sequences of the mithun IFNT gene were comparedwith those of other species available in GenBank using the Clustaloption in MegAlign (Lasergene Software, DNASTAR). A phylogramwas also constructed.

RESULTS

Using the degenerate primer pair, a fragment of theexpected 588 bp length was amplified. BLAST analysis ofthe amplicon sequence retrieved IFNT and IFN-likesequences of different ungulate species (33, 24, 11, and 3sequences from cattle, sheep, goat, and buffalo, respec-tively, and one sequence each from musk ox, bison, reddeer, yak, and giraffe) showing more than 85% similarity.Accordingly, the sequence was submitted to GenBank asmithun IFNT (mitIFNT) under accession number AY665674.The deduced amino acid sequence indicated that the ampl-icon contained the entire 585-bp open reading frame encod-ing 195 amino acids of pre-IFNT. After translation, the first23 residues constituting the signal peptide are cleaved toyield a mature IFNT of 172 amino acids.

The nucleotide sequence of mature IFNT of the mithun

was compared with homologous sequences from other spe-cies and the degree of similarity was established. ThemitIFNT gene showed the highest similarity (91.7%) to thatof Red deer (GenBank Acc. No. AJ000638). In a compari-son with nucleotide sequences of 17 polymorphic variantsof bovine IFNT, mitIFNT exhibited the highest similarity(89.8%) to variant c1 (GenBank Acc. No. AF238613) andthe lowest (88.4%) to variant BOVTPH1C (GenBank Acc.No. M31558). Among 21 polymorphic variants of ovineIFNT, variant SHPOTP1B (GenBank Acc. No. M73241) wasmost similar (88.6%) and variant p8v1 (GenBank Acc. No.AF158817) was least (85.9%). When mitIFNT was com-pared with the 10 variants of caprine IFNT, it exhibited thehighest similarity (87.5%) to variant cifnt2a (GenBank Acc.No. AY357327) and the lowest (86.3%) to variant cifnt6(GenBank Acc. No. AY357336). Compared to wild ruminantspecies, mitIFNT demonstrated 88.8, 88.4, 86.3, 85.9, and87.4% similarity to sequences from American Bison (Gen-Bank Acc. No. AY643747), musk deer (GenBank Acc. No.DQ139308), giraffe (GenBank Acc. No. U55050), musk ox(GenBank Acc. No. M73244), and yak (GenBank Acc. No.AY455289), respectively.

Comparison of the deduced amino acid sequence ofmature IFNT from the mithun with that of other species (Fig.1) demonstrated a similar trend as shown by the nucleotidesequences. Phylograms constructed on the basis of IFNTnucleotide sequences (Fig. 2), as well as deduced aminoacid sequences (not shown in figure), from various speciesshowed that sheep, goat, and musk ox comprise one clade,and cow, bison, yak, and giraffe comprise another. IFNTfrom the mithun and the red deer comprise a third clade,whereas IFNT from the musk deer stands alone and repre-sents an entirely different clade.

DISCUSSION

The sequence of mitIFNT is the first and the only com-plete coding sequence of a gene reported from the mithun.Like other type I IFN genes, the IFNT genes of all ruminantungulates, including mitIFNT, are intronless. IFNT genesevolved from a common ancestral IFN-

w

gene by a duplica-tion event that occurred 36 million years ago, and have beenretained in ungulate ruminants without any change in genelength (Roberts

et al.,

1997). The coding sequence ofmitIFNT comprises of a 585 base pair ORF that encodes a

Fig. 2.

Phylogram depicting the evolutionary relationships among IFNT genes, based on the nucleotide sequence of the mature-peptideregion.

AY357327AY357336M73244AF158817M73241AY643747AY455289M31558AF238613U55050AY665674AJ000638DQ139308

Cap var 2a Cap var 6 Musk Ox IFNT Ov var p8v1 Ov var SHPOTP1B Am. Bison IFNT Yak IFNT Bov var BOVTPH1C Bov var c1 Giraffe IFNT Mit IFNT Reddeer IFNT Musk Deer IFNT

12.5

12 10 8 6 4 2 0

K. S. Rajaravindra

et al

.610

pre-IFNT of 195 amino acids. The first 23-amino-acid signalpeptide is cleaved off, yielding a mature polypeptide of 172amino acids. Unlike most IFN-

a

s, which have a standardlength of 166 amino acids (Capon

et al.,

1985), all reportedIFNTs, including mitIFNT and the ancestral progenitor IFN-

w

, are 172 amino acids long and possess an additional six-amino-acid extension at the carboxyl terminus. Roberts

etal.

(1997) speculated that the signature tail might haveevolved from a frame shift mutation or a mutation in the stopcodon after divergence of the IFNT/IFN-

w

progenitor genefrom IFN-

a

genes about 130 million years ago. However, itis interesting that this signature carboxyl-terminal tail inIFNT presumably does not serve any biological role, andparticularly is not involved in the antiluteolytic function (Ealy

et al.,

1998a).Bovine IFNT is glycosylated with N-linked oligosaccha-

rides and has molecular masses of 22 and 24 kDa (Bazer

et al.,

1994). Ovine IFNT is not glycosylated, but caprineIFNT exists in both glycosylated and non-glycosylated form(Baumbach

et al.,

1990). Like bovine IFNT, mitIFNT alsocontains a potential site for n-glycosylation at Asn78, andthe molecular mass of the deduced mature protein is esti-mated to be 20 kDa. The deduced mature mitIFNT pos-sesses four conserved cysteine residues (Jarpe

et al.,

1994)at positions 1, 29, 99, and 139 (Fig. 1). In addition, mitIFNTalso has a conserved Ser-Leu-Gly residue (Imakawa

et al.,

1987) preceding the first-position cysteine.IFNT is encoded by a cluster of genes. A total of 12, 18,

and 10 polymorphic variants have been identified in cattle(Ealy

et al.,

2001), sheep (Nephew

et al.,

1993; Winkelman

et al.,

1999; Alexenko

et al.,

2000), and goat (Ealy

et al.,

2004), respectively. These variants differ significantly in anti-luteolytic activity as well as in antiviral (Winkelman

et al.,

1999) and antiproliferative activities (Ealy

et al.,

1998b).While characterizing the IFNT gene in buffalo (

Bubalusbubalis

), we also cloned more than four variants (unpub-lished data). Recently, two new isoforms of the IFNT genehave been reported in bison (Rasmussen

et al.,

2005).Therefore, it is likely that the IFNT molecule in the mithun isalso encoded by multiple genes, and that the sequenceidentified in our study is just one among them.

Simoons (1984) has suggested that the mithun des-cended 8,000 years ago from one race of gaur,

Bos gaurusgaurus

Smith, a close relative of cattle. Nevertheless, thenucleotide and protein sequences of mitIFNT were found tobe more similar to those of the buffalo (

Bubalus bubalis

)characterized in this laboratory (GenBank Acc. No.AY535404, AY665673; unpublished data) than those of cat-tle. Interestingly, a DNA fingerprinting study using randomamplified polymorphic DNA (RAPD) markers also indicatedmore similarity of mithun with buffalo than with cattle(NRCM, 2002).

IFNT also possesses antiviral, antiproliferative andimmunomodulatory activities. However, the semi-wild natureof the mithun has restricted our ability to characterize andevaluate the biological potency of other possible allelic/poly-morphic variants of mitIFNT using RNA isolated from theconceptus. The use of recombinant IFNT synthesized

invitro

has produced promising results in augmenting repro-ductive performance in domesticated ruminant ungulatespecies (Homeida and al-Afaleq, 1994; Asselin

et al.,

1997;

Ott

et al.,

1997; Peiris

et al.,

1998; Nagaya

et al.,

2004).Similarly, further research can be carried out to producerecombinant mitIFNT for therapeutic purposes. In conclu-sion, we have characterized the IFNT gene, which is the firstand most important signal in the early embryo-maternalcommunication, in the mithun, a semi-wild ruminant species.

ACKNOWLEDGMENTS

Financial assistance provided to KSR in the form of a JuniorResearch Fellowship (ICAR) is duly acknowledged. The authors aregrateful to the Director and Dr Gyanendra Singh (Scientist),National Research Centre on Mithun, Jharnapani, Nagaland, Indiafor providing blood samples, and to Dr. S. K. Singh (Scientist, Ani-mal Reproduction, IVRI, Izatnagar) and Dr. Sukanta Mondal (Scien-tist, Animal Physiology, NIANP, Bangalore) for critical reading ofthis manuscript.

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(Received September 23, 2005 / Accepted January 7, 2006)