ANIMAL GENETICS • ORIGINAL PAPER

Buffalo alpha S1-casein gene 5′-flanking regionand its interspecies comparison

Amrutlal K. Patel & Mahavir Singh &

V. V. S. Suryanarayana

Received: 28 March 2013 /Revised: 4 September 2013 /Accepted: 19 September 2013 /Published online: 19 October 2013# Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2013

Abstract The expression of milk protein genes is tightly regu-lated in a spatio-temporal manner through the combinatorialinteraction of lactogenic hormones and a set of transcriptionfactors mediating developmental and tissue-specific gene expres-sion. The recruitment of a unique set of transcription factors isdetermined by the cis-regulatory motifs present in the genepromoter region. Here, we report the isolation, sequencing, struc-tural analysis and interspecies comparison of the 5′cis-regulatoryregion of the buffalo alpha S1 (αS1)-casein gene. The proximalpromoter region of the buffalo αS1-casein gene harbored theinsertion of a 72-bp fragment of long interspersed nuclear ele-ment of the L1_BT retrotransposon family. Among the core andvertebrate-specific promoter elements, the motifs for the bindingof Brn POU domain factors (BRNF), Lim homeodomain factors(LHXF), NK6 homeobox transcription factors (NKX6), nuclearfactor kappa B/c-rel (NFKB), AT-rich interactive domain factor(ARID), Brn POU domain factor 5 (BRN5), pancreatic and

intestinal homeodomain transcription factor (PDX1), Distal-less homeodomain transcription factors (DLXF), T-cell factor/lymphoid enhancer-binding factor-1 (LEFF) and GHF-1pituitary-specific POU domain transcription factor (PIT1) wereover-represented in the αS1-casein gene regulatory region(Z score >4.0). TheMultiple EM for Motif elicitation predictedthree motifs which consisted of the sequences known to bindmammary gland factor/signal transducer and activator of tran-scription 5 (MGF/STAT5), estrogen receptor-related alpha(ERα), steroidogenic factor 1 (SF1) and glucocorticoid recep-tor (GR), indicating their potential role in the mammary gland-specific gene expression. The interspecies comparison of theproximal promoter region revealed conserved sequences forTATA boxes and MGF/STAT5 in all species, whereas activatorprotein 1 (AP1), pregnancy-specific mammary nuclear factor(PMF), CCAAT/enhancer binding protein (C/EBP), double-stranded and single-stranded DNA-binding protein 1 (DS1and SS), ying and yang factor 1 (YY1), and GR half-sites wereamong ruminants. The functional significance of the L1_BTretrotransposon insertion on the buffalo αS1-casein gene ex-pression needs to be experimentally validated.

Keywords Milkproteins .αS1-caseingene .Murrahbuffalo .

cis-regulatory motifs . Repetitive elements

Introduction

Buffalo is one of themajor farm animal species contributing tothe dairy industry in India. The total protein contents in themilk of buffalo, cow, goat and sheep are 3.8, 3.4, 2.9 and5.5%, respectively, where casein constitutes 80–86% of thetotal proteins (Jenness 1982). The major milk-specific pro-teins in the milk of farm animals include the αS1-, αS2-,β- and κ- caseins, β-lactoglobulins and α-lactalbumin (Jenness1982). The relative proportions of αS1-, β-, κ- and αS2-caseins

Electronic supplementary material The online version of this article(doi:10.1007/s13353-013-0176-7) contains supplementary material,which is available to authorized users.

A. K. PatelDepartment of Animal Biotechnology, College of Veterinary Scienceand Animal Husbandry, AnandAgricultural University, Anand, India

M. Singh (*)Diabetes and Obesity Center, Institute of Molecular Cardiology(IMC), University of Louisville, School of Medicine, Louisville,KY 40202, USAe-mail: gene2genetics@yahoo.com

V. V. S. SuryanarayanaDepartment of Veterinary Biotechnology, Indian Veterinary ResearchInstitute, Izatnagar, UP, India

V. V. S. Suryanarayana (*)Molecular Virology Lab, Indian Veterinary Research Institute,Hebbal Campus, Bangaluru 560024, Indiae-mail: veluvarthy@yahoo.com

J Appl Genetics (2014) 55:75–87DOI 10.1007/s13353-013-0176-7

in the milk of Indian buffalo are approximately 40, 35, 12 and9%, respectively (Yamauchi et al. 1983). The caseins of cow(Holstein Friesian breed) milk are composed of 37.26 % αS1-,53.66% β- + κ-, and 9.07% αS2-caseins, whereas goat(Granadina breed) milk is composed of 22.87% αS1-, 66.81%β- + κ-, and 10.3%αS2-caseins (Ceballos et al. 2009). The levelof each protein in the milk is proportional to the degree oftranscription of a particular gene. The milk protein genes tran-scription is regulated by the complex interactions of transcriptionfactors and the composite response elements in the 5′-upstreamregion under the control of lactogenic hormones. The putativecis-regulatorymotifs in the promoter and/or enhancer region playimportant roles in recruiting the unique set of transcription factors(Rosen et al. 1999; Kabotyanski et al. 2006; Inman et al. 2005).

The analysis of promoter elements of the different milkprotein genes in various species showed significant differencesamong the species (Malewski 1998). The sequences of the 5′-upstream region of αS1-casein have been reported for cattle(Koczan et al. 1991; Kishore et al. 2013), sheep (Bhure andSharma 2008), goat (Ramunno et al. 2004) and rat (Yu-Lee et al.1986). The sequence analysis of αS1-casein gene promoters invarious species provided significant insights into the organisa-tion of the promoter structure and its conservation among spe-cies. The functional importance of the variouscis-regulatory elements of milk protein genes have beenestablished mainly through the study of the beta casein generegulatory region (Jiang et al. 2012; Lechner et al. 1997; Lee andOka 1992a, b; Buser et al. 2011; Chughtai et al. 2002; Doppleret al. 1989; Inman et al. 2005; Doppler et al. 1995; Meier andGroner 1994), demonstrating the regulation of casein geneexpression under the influence of lactogenic hormones such asprolactin, progesterone and glucocorticoid, as well as transcrip-tion factors such as MGF/STAT5, octamer-binding protein 1(Oct1), C/EBP and YY1 through combinatorial interaction withtheir cognate binding motifs in the proximal promoter region.

The characterisation of mammary tissue-specific promoterregion thus enables us to understand its developmental andtissue-specific regulation besides its application to drive the highlevel of expression of recombinant bioactive proteins in the milkof transgenic animals. Buffalo milk contains a relatively higherlevel ofαS1-casein compared to other ruminant species. In orderto understand the structure of the buffalo αS1-casein gene regu-latory region, we isolated the 2.2-kb 5′-regulatory region includ-ing exon I, sequenced and characterised by bioinformatics tools.

Materials and methods

Isolation of genomic DNA and PCR amplificationof the αS1-casein gene 5′-regulatory region

Genomic DNA used in the study was isolated from the bloodof Murrah buffalo reared at Central Frozen Semen Production

and Training Institute, Hessarghatta, Bangaluru, India usingthe standard protocols (Sambrook et al. 1989). The amplifica-tion of the αS1 casein promoter region was performed bypolymerase chain reaction (PCR) using specific forward(CASI: 5′-GCGCGGTACCTTAAGAGGTGGCAAGAATAGTA-3′) and reverse (CASII: 5′-TTAGTGAAGCTTGAAAGATGAGACAGA-3′) primers designed based onthe published bovine sequence (Koczan et al. 1991). The50-μl reaction mixture consisted of 20 pmol of each primer,25 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2,200 μM each of four dNTPs, 100 ng tempate DNA and 1 unitof Taq DNA polymerase (AccuTaq, Sigma), and wassubjected to initial heating at 94°C for 5 min, followed by35 thermal cycles, each with denaturation at 94°C for 1 min,annealing at 60°C for 1 min and extension at 72°C for 3 minwith a final cycle having an extension at 72°C for 10 min in anEppendorf Mastercycler 5330, Germany. The amplified DNAfragments were purified from lowmelting point agarose as perstandard protocols (Sambrook et al. 1989).

Cloning and sequencing of the αS1-casein promoter region

The amplified DNA fragment was digested with KpnI andHindIII (sites incorporated in the primers) restriction enzymeand ligated to KpnI and HindIII digested pBlueScript KS+(Stratagene, USA). The recombinant DNAwas transformed inE. coli DH5α cells that were made competent for transforma-tion and the cells were plated on LB plates containing X-gal(20 μg/ml), IPTG (0.1 mM) and ampicillin (50 μg/ml). Thecolourless colonies thus obtained were subjected to colonyPCR using CASI and CASII primers. The clones showing thepresence of insert by PCR were further confirmed by releaseof the insert upon digestion with KpnI and HindIII. To gen-erate the overlapping clones, the restriction fragments of thefull-length clone were subcloned into plasmid pBluescriptKS+ vector. The DNA sequencing was performed by anABI PRISM 377 Perkin-Elmer automated DNA sequencerusing vector-specific T3 and T7 primers with labelled dNTPs.

Sequence analysis

The characterisation of repetitive elements was performedwith RepeatMasker version open-4.0.1 (RM database version20120418). The sequence was analysed by NCBI BLASTnusing discontinuous megablast to identify the best hits indifferent species. After removing the redundant sequences,the best hits for each species were selected and furthervisualised by a graphical option. The presence of knownregulatory motifs reported for milk protein gene expressionwas analysed by Regulatory Sequence Analysis Tools (RSAT)(van Helden 2003). The general core and vertebrate promoterelements were searched by MatInspector (Quandt et al. 1995)and the over-representation of transcription factor binding

76 J Appl Genetics (2014) 55:75–87

sites was analysed by the Genomatix RegionMiner tool(Genomatix Software GmbH, http://www.genomatix.de).The elicitation of novel motifs based on the conservationacross species was performed by Multiple EM for MotifElicitation (MEME) (Bailey et al. 2009) and its comparisonagainst the eukaryotic promoter databases was performed bythe Motif Alignment and Search Tool (MAST) (Bailey andGribskov 1997). The multiple sequence alignment of theproximal promoter region of buffalo (AF529305.2:1582-2187) with corresponding sequences of cow (Bos taurus :X59856.2:10019-10554 and Bos indicus : AY374986.1:133-652), yak (AF194983:421-953), sheep (AJ784891.1:1645-2185), goat (JN701804.1:600-1136), camel (AJ409277.1:73-638) and man (D85424.1:670-1229) was performed byClustalW integrated in BioEdit software using defaultparameters.

Results

Isolation and sequencing of the 5′-flanking regionof the αS1-casein gene

The 5′-region of the αS1-casein gene was amplified usingCASI and CASII primers designed from the 2.2 kb upstreamand intron I, respectively, of the bovine αS1-casein gene,which resulted in a single DNA band of approximately2.2 kb (data not shown). Since the primer sequences in theαS1-casein gene of bovine flank a region of 2.2 kb whichincludes promoter, exon I and part of intron I, the amplifiedproduct may correspond to the same region in case of buffalodue to the close relationship between these two species. Forsequencing of the full-length clone as well as nested clones byvector-specific primers, this resulted in a sequence of 2,246 bp(Fig. 1), about 72 bp larger than the expected product sizebased on the published bovine sequence. The 2,246-bp se-quence of the 5′-region of buffalo αS1-casein gene comprisedof a 2,138-bp 5′-flanking region, 53 bp of exon I and the first39 bp of intron I (GenBank accession number AF529305.2).

Repetitive elements in the αS1-casein 5′-flanking region

Interestingly, in the αS1-casein promoter region of buffalo, aninsertion of a 72-bp fragment between −308 and −379 com-pared to the bovine sequence, showing homology with 65 bp(from −315 to −379) with long interspersed nuclear element(LINE), corresponding to the L1_BT family, was observed(Figs. 1, 2 and 4; Table 1). In addition to the L-1, a 97-bp and347-bp LINE, corresponding to the BovB family and shortinterspersed nuclear element (SINE) of 228 and 177 bp and179 bp corresponding to the BovtA and ART2A/BovB familywas found at the −2042, −1879, −1459, −709 and −1699positions, respectively, in buffalo (Table 1).

Known regulatory motifs in the αS1-casein 5′-flanking region

Based on the reported motif sequences (Coll et al. 1995;Gerencsér et al. 2002), a number of consensus sequences arelocalised at 5′ to the putative transcription start site (Table 2).The buffalo sequence contained five AP1, seven C/EBP, oneCTF/NF1, five GR half, two MGF/STAT5, one DS1 and SS,two PMF, one PRL and eight YY1 binding motifs. Thebuffalo αS1-casein contains the same putative TATA box asthat described in the case of bovine (Koczan et al. 1991).Sequences for the TATA boxes with the consensus“TTTAAAT” were found at the −29 and −89 positions in thebuffalo. The DS1 and SS, which specifically bind to thesequences “AAATTAGCATNT” and “CCACAA”, respec-tively (Saito and Oka 1996), are present at −42 to −63 posi-tions in the buffalo.

General core and vertebrate-specific transcription factorbinding motifs in the αS1-casein 5′-flanking region

The analysis for the presence of core promoter and vertebrate-specific promoter elements by MatInspector in the buffaloαS1-casein gene revealed the presence of binding sites forseveral transcription factors, with 47 motifs for BRNF, 39 forLHXF, 37 for Paralog hox genes 1–8 from the four hoxclusters A, B, C, D (HOXF), 32 for SOX/SRY-sex/testisdetermining and related HMG box factors (SORY), 31 forhomeodomain transcription factors (HOMF), 30 for OCT1, 28for homeobox transcription factors (HBOX) and 27 for carti-lage homeoprotein 1 (CART), among others (SupplementaryTable 1). Among the motifs for the transcription factorsknown to be localised in breast tissue, the occurrence ofmotifsfor fork head domain factors (FKHD), human and murineETS1 factors (ETSF), STAT, glucocorticoid responsive andrelated elements (GREF), estrogen response elements (EREF)and Brachyury gene, mesoderm developmental factor(BRAC) was 24, 19, 12, 8, 3 and 1 in the buffalo αS1-casein promoter (Supplementary Table 2). We next lookedfor the over-representation of the motifs in the αS1-caseinpromoter over eukaryotic promoters. The motifs for the bind-ing of BRNF, LHXF, NKX6, ARID, BRN5, PDX1, DLXF,LEFF and PIT1 were over-represented in the αS1-caseinpromoter with Z score >4.0 over eukaryotic promoters(Table 3).

Elicitation of novel motifs in the αS1-casein 5′-flankingregion

Further analysis of the proximal promoter region of the αS1-casein gene from eight species by MEME resulted in theidentification of three motifs, highly conserved in the αS1-casein gene promoter of all eight species (Fig. 3). We thenlooked for the presence of similar motifs in the eukaryotic

J Appl Genetics (2014) 55:75–87 77

-2138 ttaagaagaggtggcaagaatagtattacagaagaact -2101

gtatttaaaaggtcttaatgacccagatagccacagttgtgtagtctctcatctacagcttaaaactcaa -2031

tgttcaaaaaactaagttcatagcatactgccccatcacttcatggaaaactgtgggggagggggagaag -1961

gtggaagtagtgtcagattttattttcttggactcaaaatcactgcagacagtgattatagccatgaaat -1891

taaatgatgcttgctccttgaaaagaaagttctgaaaaacctagacagcatattaaaaagcagtgacatc -1821

actttactgataagtgtctttatagtcaaagctatggttcttccagtagccatgtacagatgtgagaatt -1751

ggactatgaagaaggatgaatgtcaaaggactgatgttttcaaattgtggtggatacactcctttgcgtg -1681

ccgtgctaagtcatttcaagtcatgtccaactctttgcgaacccagtggactgtggtctgccaggttccc -1611

tctgtccatgggattctccaggcaagaacaatggactgggttgccatttcctccaccaggggatcttccc -1541

aatccagatattgaacctgcatctctaatgtttcctgcattggcaggcaggttctttgccactagtgcca -1471

cctggaaagtccggattacactcctgggaaagacaaaagtagagtattacaatgcagcaaggatttttgt -1401

tctcagctccttgaataaattatagtgaatagaaaacactagtatcttgttgaaattgatgtgaaacaga -1331

cagtaaggaacataatatctaaagaaaacttcaatatgggaaattatagtcttttctatcttcaaagtgg -1261

agagcctgaacagttctgaaatttcttttaatacaaaataatggtcctgtcatacaactgtgaatacact -1191

gaaaatatcactatagattttttgaagtatataatatgattcctttcttataaacaatgagttgcaatca -1121

acaagtttttaaagccctcacttgtatagatatttttttagcacataatatttttctacaatgtacaatg -1051

ccagttaattctaggagtacaattaagaattggagagataagaatttttttcttttacttgtttacttta -981

aaagatggaaaatcagagttatggtttattttttgcaatatttaaaaattataattcttgaataactatt -911

aattttaattaaataatctgtaatgagaatcctcctaccaatgcaggagacatgagtttgatccctgggt -841

agggaagataccctgcagaaggaaatggcaacccactccaatattattacttgggaaatcccatggacag -771

aggagactggcaggctgcagtccatgggggtcacaaagaactggacatgacttagcaactaaacaacaac -701

aatttataccggaatgaatgaactagttaccacaactagtacacccaaaataaacaaagaatagcttggt -631

ggtataattaaaatgccaccaaagtttatacaataattatattttctttttgcaggaaaaagattagacc -561

acatataatgtaagttatttcaaaaggtaaataattataataaataatatggattaactgagttttaaga -491

ggtgaaataaataatgaattcttctaatggtcttgtatgttaataaaaattgaaaatttttgaagacccc -421

attttgtcccaggaatttcctttacaggtattgaatttttccggattcattttgatattcggcaaaacta -351

atacaattatgtaaaatttaaaaataaaataaaatttttaaaaaaagttacaaaggaaattttattgata -281

taataaatgcatgttctcataataaccataaatctagggttttgttggggggttttttgtatgttaattt -211

agaacaatgccattccatttcctgtataatgaatcacttctttgttgtaaactctccttagaatttcttg -141

ggagaggaactgaacagaacattgatttcctatgtgagagaattcttagaaTTTAAATAaacctattggt -071

taaactgaaaccacaaaattagcattttattaatcagtaggTTTAAATAgcttgaaagcaaaagtctgcc -1

ATCACCTTGATCATCAACCCAGCTTGCTGCTTCTTCCCAGTCTTGGGTTCAAG +53

gtattatgtatacgtataacaaaatttctatgattttcctctgtctcatctttca +108

Fig. 1 The nucleotide sequence of the 5′-flanking region, exon I and the first39 bp of intron I of the buffaloαS1-casein gene (EMBL/GenBank accessionnumber: AF529305.2). The numbering is relative to the transcription start

site (+1). Exon 1 is shown in capital letters. Sequences of the TATA boxesare shown in bold and capital letters . The repetitive elements areunderlined. The insertion element L1_BT in buffalo is bold and underlined

78 J Appl Genetics (2014) 55:75–87

promoter database, which resulted in significant hits to the twocasein gene promoter sequences (EP11090: Bos taurus αS1-casein; e-value: 1.1e−71, EP15030: Rattus norvegicus αS1-casein; e-value: 2.2e−10) available in the database, whereas weakhomology was observed to the four gene promoters ofDrosophila melanogaster (EP77099: LARK; e-value: 0.18,EP77868: CG2852; e-value: 0.31, EP77199: CG6512; e-value:0.32, EP78834: CG7217; e-value: 0.92) and adenovirus majorlate promoter (EP26036; e-value: 0.87). The region spanningthese motifs was searched for known mammary-specific reg-ulatory motifs, as well as those identified by the MatInspectoranalysis. These motifs consisted of the sequences known tobind MGF/STAT5, ERα, SF1 and GR (Table 4, Fig. 4).

Interspecies comparison of the αS1-casein 5′-flanking region

The proximal promoter region of buffalo from −557 to +49was compared with the corresponding sequences of cow,yak, sheep, goat, camel and man. The alignment (Fig. 4)indicates that the elements in the promoter region are highlyconserved. The TATA boxes and the sequences for the bind-ing of MGF were found to be conserved among all thespecies; however, variations were noticed in the AP1, PMF,C/EBP, DS1 and SS, YY1 and GR half sites. Besides knownregulatory motifs, the promoter region up to −180 upstreamand the region between −378 to −557 (positions correspond-ing to buffalo sequences) were found to be very much

conserved. The sequence of ruminant species showed morestructural identity as compared to other species.

Discussion

The expression of recombinant proteins in the milk of trans-genic animals offers potential benefits of high level of expres-sion, eukaryotic-specific post-translational modifications andease in protein purification over other available expressionsystems (Pittius et al. 1988; Bühler et al. 1990; Riego et al.1993; Persuy et al. 1995; Bagis et al. 2011). The characteri-sation of mammary gland-specific promoter elements hasgained interest due to its suitability for directing heterologousprotein expression besides adding to the understanding of theregulation of mammary tissue-specific gene expression. Theregulatory elements of milk protein genes from several specieshave been characterised and patented. Buffalo milk is rich inαS1-casein; hence, to test its suitability for transgene expres-sion, in this study, we isolated and characterised the buffaloαS1-casein gene 5′-regulatory region.

Interestingly, we observed an insertion of a 72-bp fragment(65-bp fragment of LINE/L1 element with 7-bp flankingrepeat) in the proximal promoter region at −308 bp of thebuffalo αS1 casein gene. This element showed 93.8% homol-ogy to the L1 (positions 8297–8361) and truncated at the 5′-ends, but the presence of the flanking 7-bp direct repeat

Fig. 2 BLASTn alignment of the αS1-casein gene promoter against theNCBI nr database using discontinuous megablast. The region spanningthe L1_BT retrotransposon insertion in buffaloαS1-casein gene proximalpromoter is shown. The accession number of the aligned sequences isindicated over the corresponding sequences. GU562878.1: Bosgrunniens , X59856.2: Bos taurus , AF529305.2: Bubalus bubalis ,

GU593719.1: Bubalus bubalis , AJ784891.1: Ovis aries , JN701804.1:Capra hircus , AY374986.1: Bos indicus , EU025875.1: Sus scrofa ,D85424.1: Homo sapiens , AJ409277.1: Camelus dromedarius ,FN386610.1: Equus asinus africanus. The red vertical lines indicatenucleotide substitution(s) compared to the buffalo αS1-casein gene pro-moter sequence (AF529305.2)

J Appl Genetics (2014) 55:75–87 79

sequence “AATTTTT” indicates that the truncation couldhave occurred before insertion. The lack of this element inother species indicates that the insertion of this element couldbe a recent evolutionary event. An insertion of such an ele-ment has been reported in 3′-UTR of goat kappa casein gene,which is associatedwith reduced protein synthesis (Pérez et al.1994). However, insertion of the L1 element in the promoterregion may not necessarily reduce transcriptional activity. The72-bp fragment inserted is comprised of AT-rich sequences(83%), which may have a positive effect on promoter unwind-ing during the initiation of transcription. However, such apossibility needs to be experimentally validated.

The role of putative sequence motifs in the mammarytissue-specific expression of casein genes has been experi-mentally demonstrated in several studies (Ball et al. 1988;Doppler et al. 1989; Schmitt-Ney et al. 1991; Groenen et al.

1992; Lee and Oka 1992a, b; Pierre et al. 1992; Altiok andGroner 1993; Happ and Groner 1993; Li and Rosen 1995;Raught et al. 1995; Saito and Oka 1996; Rosen et al. 1999;Kabotyanski et al. 2006; Buser et al. 2011). We observed theoccurrence of similar motifs in the buffalo αS1-casein pro-moter region (Table 2), which are highly conserved amongruminants but showed considerable variation with other spe-cies (Fig. 4). The high level of conservation in ruminantsexplains their close genetic relationships and shared regulato-ry mechanisms.

Besides tissue-specific regulatory motifs which restrictgene expression in specific tissue, the promoter region alsoconstitutes sequences for the binding of basal transcriptionalmachinery. The MatInspector analysis predicted several ofthese motifs (Supplementary Table 1), which constitute sev-eral overlapping sites and may not be occupied by all factors

Fig. 3 Prediction of motifs in the αS1-casein gene proximal promoterregion using Multiple EM for Motif Elicitation (MEME) and its compar-ison with the eukaryotic promoter databases. a Motifs predicted byMEME based on the sequences of the proximal promoter region of eightspecies. Position relative to the transcription start site is indicated over thegraph. b Positional location of motifs on the αS1-casein promoter invarious species. c The presence of predictedmotifs in the promoter region

of the sequences available in the eukaryotic promoter database using theMotif Analysis and Search tool. EP11090: Bos taurus αS1-casein,EP15030: Rattus norvegicus αS1-casein, EP77099: Drosophilamelanogaster LARK, EP77868: Drosophila melanogaster CG2852,EP77199: CG6512, EP26036: Ad12 major late, EP78834: Drosophilamelanogaster CG7217. Position relative to the transcription start site isindicated over the graph

80 J Appl Genetics (2014) 55:75–87

as predicted by the computational methods. Hence, we lookedfor the over-represented transcription factor binding sites com-pared to other eukaryotic promoters, which revealed that thesites for the binding of BRNF, LHXF, NKX6, ARID, BRN5,PDX1, DLXF, LEFF and PIT1 were significantly over-represented in the αS1-casein promoter (Table 3). The soft-ware available so far have been designed to identify the over-representation of motifs in a set of co-regulated genes.

However, based on the functional association of these tran-scription factors in the milk protein gene expression, theanalysis of the promoter sequence of a single gene by suchan approach may also prove to be as useful as the over-represented motifs for transcription factor PIT1, which hasbeen observed to be associated with milk protein gene expres-sion (Heidari et al. 2012; Renaville et al. 1997). The BRNF,which includes BRN2, 3, 4 and Oct 6, are brain-specific POU

Fig. 4 Multiple sequence alignment of the promoter region of the αS1-casein gene among eight species. Positions are relative to the point oftranscription initiation. Putative transcription factor binding sites and

TATA boxes are boxed . The asterisks indicate positions where thehomology is 100% among the eight species. Motifs predicted by MEMEare underlined

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Table 2 Summary of regulatory motifs known to regulate milk protein gene expression identified in the buffalo αS1-casein gene promoter region

Factora Strand Patternb Startc Endc Matching sequence References

AP1 + TGA[AGCT]T[AC]A −1389 −1383 tcctTGAATAAatta Lee et al. (1987)+ TGA[AGCT]T[AC]A −922 −916 ttctTGAATAActat

+ TGA[AGCT]T[AC]A −181 −175 ataaTGAATCActtc

− TGA[AGCT]T[AC]A −181 −175 gaagTGATTCAttat

− TGA[AGCT]T[AC]A −41 −35 ctacTGATTAAtaaa

C/EBP + [AC]TT[AGCT]C[AGCT][AGCT][AC]A −2114 −2106 tagtATTACAGAAgaac Raught et al. (1995)+ [AC]TT[AGCT]C[AGCT][AGCT][AC]A −544 −536 agttATTTCAAAAggta

− [AC]TT[AGCT]C[AGCT][AGCT][AC]A −1418 −1410 aatcCTTGCTGCAttgt

− [AC]TT[AGCT]C[AGCT][AGCT][AC]A −1353 −1345 atcaATTTCAACAagat

− [AC]TT[AGCT]C[AGCT][AGCT][AC]A −1247 −1239 agaaATTTCAGAActgt

− [AC]TT[AGCT]C[AGCT][AGCT][AC]A −950 −942 aaatATTGCAAAAaata

− [AC]TT[AGCT]C[AGCT][AGCT][AC]A −790 −782 tgggATTTCCCAAgtaa

CTF/NF1 − GCCAAT −1502 −1497 gcctGCCAATgcag Li and Rosen (1995)

GR-half + TGTTCT −1403 −1398 ttttTGTTCTcagc Welte et al. (1993)+ TGTTCT −269 −264 tgcaTGTTCTcata

− TGTTCT −1586 −1581 ccatTGTTCTtgcc

− TGTTCT −210 −205 gcatTGTTCTaaat

− TGTTCT −125 −120 tcaaTGTTCTgttc

MGF/STAT5 +/− TTC[AGCT][AGCT][AGCT]GAA −1991 −1983 tcacTTCATGGAAaact Wakao et al. (1994)+/− TTC[AGCT][AGCT][AGCT]GAA −98 −90 agaaTTCTTAGAAttta

DS1&SS + AAACCACAAAATTAGCATTTTA −63 −42 actgAAACCACAAAATTAGCATTTTAttaa

Saito and Oka (1996)

PMF +/− ATCAN(0,8)TGAT −1823 −1810 tgacATCACTTTACTGATaagt Lee and Oka (1992a)+/− TGATN(0,8)ATCA +1

PRL - CTGATTA −40 −34 cctaCTGATTAataa Schuster et al. (1988)

YY1 + CCAT[AGCT]T −1771 −1766 gtagCCATGTacag Meier and Groner (1994)+ CCAT[AGCT]T −1567 −1562 gttgCCATTTcctc

+ CCAT[AGCT]T −422 −417 gaccCCATTTtgtc

+ CCAT[AGCT]T −196 −191 cattCCATTTcctg

− CCAT[AGCT]T −1297 −1292 tttcCCATATtgaa

− CCAT[AGCT]T −978 −973 ttttCCATCTttta

− CCAT[AGCT]T −818 −813 gttgCCATTTcctt

− CCAT[AGCT]T −514 −509 taatCCATATtatt

a Sequence-specific transcription factors. Abbreviations: AP1 , activator protein 1; C/EBP, CCAAT/enhancer binding protein; CTF/NF1, nuclear factor 1;GR , glucocorticoid receptor;MGF, mammary gland factor;PMF, pregnancy-specific mammary nuclear factor;PRL , prolactin; YY1 , ying and yang factor 1b Published consensus sequence for each element in milk protein gene. N , any nucleotide. N(0,8) indicates that up to eight nucleotides are allowedc Positions of the motifs as numbered in Fig. 1

Table 1 List of repetitive elements in the buffalo αS1-casein promoter region

Query Position in query Repeat Repeat class/family Position in repeat % Diversity % Deletion % Insertion

Beginning End Beginning End

Buffalo αS1-casein promoter −2138 −2042 BovB LINE/RTE-BovB 2234 2326 20.5 0 4.3

−2043 −1879 BovB LINE/RTE-BovB 3155 3314 18.9 0.6 3.8

−1877 −1699 ART2A SINE/RTE-BovB 1 178 23.0 0 0.6

−1686 −1459 Bov-tA1 SINE/BovA 224 1 15.2 0 1.8

−885 −709 Bov-tA2 SINE/BovA 32 206 14.9 0 1.1

−379 −315 L1_BT LINE/L1 8297 8361 6.2 0 0

82 J Appl Genetics (2014) 55:75–87

domain transcription factors known to be involved in devel-opment and differentiation (Ryan and Rosenfeld 1997). BRN5is widely expressed in the developing brain but was also foundto be expressed in the kidney, lung, heart, adrenal, skin, testisand anterior pituitary (Andersen et al. 1993). Although not yetdemonstrated, these transcription factors may positively ornegatively regulate the casein gene expression in order toorchestrate their expression only during lactation. The syner-gistic effects of NKX6 and PDX1 on the insulin gene expres-sion in pancreatic beta cells have been reported (Gefen-Haleviet al. 2010) and represent the possible candidates to study theireffect on milk protein gene expression. Although studiesdemonstrating the effect of these enrichedmotifs on the caseingene expression are lacking, they represent an importantsource of information for further evaluation of their possiblerole in the milk protein gene expression.

The computational search for transcription factor bindingmotifs more often results in the identification of motifsrecognised by the basal transcription machinery common toeukaryotic promoters and less often to the tissue-specificgene expression. The transcription factor binding motifsobserved in the αS1-casein gene promoter are also presentin the genes expressed in other tissue. The search for novelmotifs in αS1-casein by MEME resulted in the identification

of three motifs (Table 4), which are highly conserved acrossthe species. Interestingly, all three motifs consisted of thesequences known to bind MGF/STAT5, ERα, SF1, PR andGR (Table 4), indicating their potential role in the mammarygland-specific gene expression. The significant homology tothese motifs were only identified in the casein genes pro-moter sequence available in the eukaryotic promoter data-base (e-value < 0.1), whereas the weak homology was ob-served with Drosophila gene promoters and adenovirus ma-jor late promoter (e-value > 0.1), which suggests that thesemotifs may confer mammary gland-specific expression ofcasein genes. However, the role of these motifs in conferringmammary gland-specific expression needs to be experimen-tally validated.

The comparative analysis of proximal promoter amongvarious species revealed a high level of conservationamong ruminant species compared to non-ruminants. A15-bp insertion at −182 bp upstream was observed innon-ruminant species, which indicates that the divergence ofruminants may have occurred before the insertion. The se-quence of man and camel also shows deletion of sequencemotifs at various places, which is conserved in other species,indicating that the specific modifications may have occurredafter speciation.

Table 3 List of over-represented transcription factor binding sites in the buffalo αS1-casein gene promoter region

TF families Detailed family information Number of matchesin input

Expected inpromoters

Standarddeviation

Over-representationin promoters

Z-score(promoters)

BRNF Brn POU domain factors 47 11.89 3.44 3.95 10.07

LHXF Lim homeodomain factors 39 10.97 3.3 3.55 8.33

NKX6 NK6 homeobox transcription factors 21 4.7 2.17 4.47 7.29

ARID AT-rich interactive domain factor 22 5.32 2.3 4.13 7.02

BRN5 Brn POU domain factor 5 24 6.97 2.64 3.44 6.27

PDX1 Pancreatic and intestinal homeodomain transcriptionfactor

14 3.3 1.82 4.24 5.62

DLXF Distal-less homeodomain transcription factors 15 3.9 1.97 3.84 5.37

LEFF T-cell factor/lymphoid enhancer-binding factor-1 17 4.92 2.22 3.45 5.22

PIT1 GHF-1 pituitary-specific POU domain transcriptionfactor

11 2.9 1.7 3.79 4.46

ATBF AT-binding transcription factor 8 1.95 1.4 4.11 3.98

GFI1 Growth factor independence transcriptional repressor 7 1.78 1.33 3.94 3.54

PAXH Paired box homeodomain binding sites 8 2.22 1.49 3.6 3.54

SATB Special AT-rich sequence binding protein 6 1.61 1.27 3.72 3.06

NFKB Nuclear factor kappa B/c-rel 9 3.3 1.82 2.72 2.86

TEAF TEA/ATTS DNA binding domain factors 5 1.57 1.25 3.19 2.35

CIZF CAS-interacting zinc finger protein 4 1.31 1.14 3.05 1.91

HAML Human acute myelogenous leukemia factors 5 2.01 1.42 2.48 1.75

ZF05 C2H2 zinc finger transcription factors 5 3 1.02 1.01 2.93 1.46

PPAR Peroxisome proliferator activated receptorhomodimers

2 0.68 0.82 2.94 0.99

GTBX GT box 1 0.37 0.61 2.68 0.21

HNFP Histone nuclear factor P 1 0.45 0.67 2.22 0.08

J Appl Genetics (2014) 55:75–87 83

Tab

le4

Novelmotifspredictedby

Multip

leEM

forMotifElicitatio

n(M

EME)inthebuffaloαS1

-caseingene

prom

oterregion

andthetranscriptionfactor

bindingsiteswith

inthesemotifsidentifiedby

MatInspectoranalysis

Motif

Pattern

Matrix

family

Detailedfamily

inform

ation

Anchor

positio

nStrand

Core

similarity

Matrix

similarity

Matchingsequence

Motif1

−15:[G

A][GA]A

A[G

A][CA]A

[AG]A

[AG][G

A]TCTGCCATCACCTTGATCATCAACC

CAGCTTGCTGCTTC:+

33

V$E

REF

Estrogenresponse

elem

ents

1−

10.88

gatcAAGGtgatggcagactttt

V$B

RAC

Brachyury

gene,m

esoderm

developm

entalfactor

5−

10.88

tgatgatcaaGGTGatggcagac

V$S

F1F

Vertebratesteroidogenicfactor

6−

10.95

tgatCAAGgtgatgg

V$L

EFF

LEF1/TCF

8−

10.82

tgatgaTCAAggtgatg

V$S

NAP

snRNA-activatingproteincomplex

10+

0.96

0.76

aTCACcttgatcatcaacc

V$H

NF1

Hepaticnuclearfactor

111

−0.76

0.77

ggtTGATgatcaaggtg

V$A

BDB

Abdom

inal-B

type

homeodomaintranscriptionfactors

14+

0.80

0.83

cttgatcaTCAAcccag

V$G

TBX

GTbox

21−

10.71

gcagcaagctgG

GTTgatg

V$Z

ICF

Mem

bersof

theZIC

family,zincfinger

proteinof

the

cerebellu

m22

+1

0.88

caaccC

AGCttg

ctg

V$Z

F01

C2H

2zinc

finger

transcriptionfactors1

29−

0.87

0.82

actGGGAagaagcagcaagctgggt

Motif2

−141:G

GGAGAGGAA[CT]TG[A

G]A

CAG

AA[CA]ATT[G

A]ATTTCCTA

T[G

T]TGA

GAGAATTCTTA

G:-92

V$E

TSF

Hum

anandmurineETS1

factors

−132

+1

0.97

ttgggagaG

GAActgaacaga

V$M

OKF

Mouse

Krüppel-likefactor

−130

−0.75

0.74

gttctgttcagttC

CTCtccc

O$INRE

Coreprom

oter

initiator

elem

ents

−130

−1

0.95

gtTCAGttcct

V$C

LOX

CLOXandCLOXhomology(CDP)

factors

−119

−0.87

0.84

ggaaATCAatgttctgttc

V$G

REF

Glucocorticoidresponsive

andrelatedelem

ents

−117

+1

0.82

acaG

AACattgatttccta

V$G

REF

Glucocorticoidresponsive

andrelatedelem

ents

−117

−1

0.91

taggaaatcaaT

GTTctgt

V$L

EFF

LEF1/TCF

−117

−1

0.84

aggaaaTCAAtgttctg

V$N

FAT

Nuclear

factor

ofactiv

ated

T-cells

−116

−1

0.83

ataG

GAAatcaatgttctg

V$G

FI1

Growth

factor

independence

transcriptionalrepressor

−117

−1

0.94

ggaA

ATCaatgttct

V$C

LOX

CLOXandCLOXhomology(CDP)

factors

− 114

+0.88

0.95

gaacattGATTtcctatgt

V$H

NF6

Onecuth

omeodomainfactor

HNF6

−115

−1

0.95

ataggaaaTCAAtgttc

V$E

TSF

Hum

anandmurineETS1

factors

−112

−1

0.95

ctcacataGGAAatcaatgtt

V$H

EAT

Heatshock

factors

−94

−1

0.96

atttaaattctaAGAAttctctcac

V$S

TAT

Signaltransducer

andactiv

ator

oftranscription

−94

−1

0.97

taaaTTCTaagaattctct

V$S

TAT

Signaltransducer

andactiv

ator

oftranscription

−92

+1

0.97

agaaTTCTtagaatttaaa

V$B

CL6

POZdomainzinc

finger

expressedin

B-cells

−92

+0.79

0.89

gaaT

TCTtagaatttaa

Motif3

−429:G

AAGACCC[CT][AG]TTTTGTCC

CAAG[A

G]ATTTCCTTTA

[CT]A

GGTA

TTGAATTTTTC:-380

V$A

IRE

Autoimmuneregulatory

elem

entb

inding

factor

−415

−0.91

0.86

aaattcctgggacaaaaT

GGGgtcttc

V$H

EAT

Heatshock

factors

−409

+0.97

0.79

ccattttgtcccaggaatTTCCttt

V$S

ORY

SOX/SRY-sex/testisdeterm

iningandrelated

HMGboxfactors

−409

−1

0.92

aaaggaAATTcctgggacaaaatgg

V$S

TAT

Signaltransducer

andactiv

ator

oftranscription

−410

−1

0.95

gaaaTTCCtgggacaaaat

V$S

TAT

Signaltransducer

andactiv

ator

oftranscription

−408

+1

0.88

tttgtcccaGGAAtttcct

V$E

TSF

Hum

anandmurineETS1

factors

−406

+1

0.86

ttgtcccAGGAatttccttta

V$IKRS

Ikaros

zinc

finger

family

−407

+1

0.84

tcccaG

GAAtttc

84 J Appl Genetics (2014) 55:75–87

Conclusions

The structural characterisation of the αS1 casein gene pro-moter of buffalo resulted in the identification of an insertionelement in the proximal promoter region. The bioinformaticsanalysis suggested the presence of known mammary tissue-specific and core regulatory motifs in the promoter region,which are highly conserved among ruminants and predictedthree motifs which appear to be unique in the αS1 casein genepromoter region. However, further study is required in order toanalyse the functional importance of these conserved motifs inthe regulation of the αS1-casein gene. The insertion of the L-1fragment in the proximal promoter may alter the degree oftranscription initiation of the αS1-casein gene in buffalo. Thestructural characterisation of the αS1-casein gene regulatoryregion may contribute to a better understanding of the devel-opmental and tissue-specific regulation of milk protein geneexpression.

Acknowledgements The authors wish to thank the Director, IVRI,Izatnagar and Joint Director, IVRI, Bangalore for providing facilitiesand IVRI, Izatnagar for providing fellowship to Patel AK to carry outthis work. The authors wish to thank Dr. C.G. Joshi, Professor, AnimalBiotechnology, Anand Agricultural University, Anand for the criticalreading of the manuscript.

References

Altiok S, Groner B (1993) Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones. Mol CellBiol 13(12):7303–7310

Andersen B, Schonemann MD, Pearse RV 2nd, Jenne K, Sugarman J,Rosenfeld MG (1993) Brn-5 is a divergent POU domain factorhighly expressed in layer IV of the neocortex. J Biol Chem268(31):23390–23398

Bagis H, Aktoprakligil D, Gunes C, Arat S, Akkoc T, Cetinkaya G,Kankavi O, Taskin AC, Arslan K, Dundar M, Tsoncheva VL,Ivanov IG (2011) Expression of biologically active human interfer-on gamma in the milk of transgenic mice under the control of themurine whey acidic protein gene promoter. BiochemGenet 49(3–4):251–257. doi:10.1007/s10528-010-9403-7

Bailey TL, Gribskov M (1997) Score distributions for simultaneousmatching to multiple motifs. J Comput Biol 4(1):45–59

Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J,Li WW, NobleWS (2009) MEME SUITE: tools for motif discoveryand searching. Nucleic Acids Res 37(Web Server issue):W202–W208. doi:10.1093/nar/gkp335

Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B (1988)Prolactin regulation of beta-casein gene expression and of a cyto-solic 120-kd protein in a cloned mouse mammary epithelial cell line.EMBO J 7(7):2089–2095

Bhure SK, Sharma B (2008) The PCR amplification, sequencing andcomputer-aided analysis of ovine αS1-casein gene promoter. IndianJ Biotechnol 7:478–481

Bühler TA, Bruyère T, Went DF, Stranzinger G, Bürki K (1990) Rabbitbeta-casein promoter directs secretion of human interleukin-2 intothe milk of transgenic rabbits. Biotechnology (N Y) 8(2):140–143T

able4

(contin

ued)

Motif

Pattern

Matrix

family

Detailedfamily

inform

ation

Anchor

positio

nStrand

Core

similarity

Matrix

similarity

Matchingsequence

V$E

TSF

Hum

anandmurineETS1

factors

−402

−1

0.94

cctgtaaaGGAAattcctggg

V$N

FKB

Nuclear

factor

kappaB/c-rel

−404

+1

0.98

ccaggaatTTCCttt

V$N

FKB

Nuclear

factor

kappaB/c-rel

−404

−1

0.93

aaaggaaaTTCCtgg

V$IRXF

Iroquoishomeoboxtranscriptionfactors

−394

−0.757

0.86

aataCCTGtaaag

V$IRXF

Iroquoishomeoboxtranscriptionfactors

−393

+0.757

0.86

tttaC

AGGtattg

V$H

NF6

Onecuth

omeodomainfactor

HNF6

−386

−1

0.90

ggaaaaatTCAAtacct

J Appl Genetics (2014) 55:75–87 85

Buser AC, Obr AE,Kabotyanski EB,GrimmSL, Rosen JM, Edwards DP(2011) Progesterone receptor directly inhibits beta-casein gene tran-scription in mammary epithelial cells through promoting promoterand enhancer repressive chromatin modifications. Mol Endocrinol25(6):955–968. doi:10.1210/me.2011-0064

Ceballos LS, Morales ER, de la Torre Adarve G, Castro JD, Martínez LP,Sampelayo MRS (2009) Composition of goat and cow milk pro-duced under similar conditions and analyzed by identical method-ology. J Food Compos Anal 22(4):322–329

Chughtai N, Schimchowitsch S, Lebrun JJ, Ali S (2002) Prolactin inducesSHP-2 association with Stat5, nuclear translocation, and binding tothe beta-casein gene promoter in mammary cells. J Biol Chem277(34):31107–31114. doi:10.1074/jbc.M200156200

Coll A, Folch JM, Sanchez A (1995) Structural features of the 5′ flankingregion of the caprine kappa-casein gene. J Dairy Sci 78(5):973–977.doi:10.3168/jds.S0022-0302(95)76712-1

Doppler W, Groner B, Ball RK (1989) Prolactin and glucocorticoidhormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line.Proc Natl Acad Sci U S A 86(1):104–108

Doppler W, Welte T, Philipp S (1995) CCAAT/enhancer-binding proteinisoforms beta and delta are expressed in mammary epithelial cellsand bind to multiple sites in the beta-casein gene promoter. J BiolChem 270(30):17962–17969

Gefen-Halevi S, Rachmut IH, Molakandov K, Berneman D, Mor E,Meivar-Levy I, Ferber S (2010) NKX6.1 promotes PDX-1-induced liver to pancreatic beta-cells reprogramming. Cell Repro-gram 12(6):655–664. doi:10.1089/cell.2010.0030

Gerencsér A, Barta E, Boa S, Kastanis P, Bösze Z,Whitelaw CBA (2002)Comparative analysis on the structural features of the 5′ flankingregion of kappa-casein genes from six different species. Genet SelEvol 34(1):117–128. doi:10.1051/gse:2001007

Groenen MA, Dijkhof RJ, van der Poel JJ, van Diggelen R, Verstege E(1992) Multiple octamer binding sites in the promoter region of thebovine alpha s2-casein gene. Nucleic Acids Res 20(16):4311–4318

Happ B, Groner B (1993) The activated mammary gland specific nuclearfactor (MGF) enhances in vitro transcription of the beta-casein genepromoter. J Steroid Biochem Mol Biol 47(1–6):21–30

Heidari M, Azari MA, Hasani S, Khanahmadi A, Zerehdaran S (2012)Effect of polymorphic variants of GH, Pit-1, and beta-LG genes onmilk production of Holstein cows. Genetika 48(4):503–507

Inman CK, Li N, Shore P (2005) Oct-1 counteracts autoinhibition ofRunx2 DNA binding to form a novel Runx2/Oct-1 complex on thepromoter of the mammary gland-specific gene beta-casein. Mol CellBiol 25(8):3182–3193. doi:10.1128/MCB.25.8.3182-3193.2005

Jenness R (1982) Inter-species comparison of milk proteins. In: Fox PF(ed) Developments in dairy chemistry, volume I. ACS, New York,pp 87–114

Jiang S, Ren Z, Xie F, Yan J, Huang S, Zeng Y (2012) Bovine prolactinelevates hTF expression directed by a tissue-specific goat beta-casein promoter through prolactin receptor-mediated STAT5a acti-vation. Biotechnol Lett 34(11):1991–1999. doi:10.1007/s10529-012-1009-1

Kabotyanski EB, Huetter M, Xian W, Rijnkels M, Rosen JM (2006)Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specifictranscription factors and chromatin modifiers. Mol Endocrinol20(10):2355–2368. doi:10.1210/me.2006-0160

Kishore A, Mukesh M, Sobti RC, Mishra BP, Sodhi M (2013) Variationsin the regulatory region of alpha S1-casein milk protein gene amongtropically adapted Indian native (Bos Indicus) cattle. ISRNBiotechnol. doi:10.5402/2013/926025

Koczan D, Hobom G, Seyfert HM (1991) Genomic organization of thebovine alpha-S1 casein gene. Nucleic Acids Res 19(20):5591–5596

Lechner J, Welte T, Tomasi JK, Bruno P, Cairns C, Gustafsson J, DopplerW (1997) Promoter-dependent synergy between glucocorticoid

receptor and Stat5 in the activation of beta-casein gene transcription.J Biol Chem 272(33):20954–20960

Lee CS, Oka T (1992a) A pregnancy-specific mammary nuclear factorinvolved in the repression of the mouse beta-casein gene transcrip-tion by progesterone. J Biol Chem 267(9):5797–5801

Lee CS, Oka T (1992b) Progesterone regulation of a pregnancy-specifictranscription repressor to beta-casein gene promoter in mouse mam-mary gland. Endocrinology 131(5):2257–2262

Lee W, Mitchell P, Tjian R (1987) Purified transcription factor AP-1interacts with TPA-inducible enhancer elements. Cell 49(6):741–752

Li S, Rosen JM (1995) Nuclear factor I and mammary gland factor(STAT5) play a critical role in regulating rat whey acidic proteingene expression in transgenic mice. Mol Cell Biol 15(4):2063–2070

Malewski T (1998) Computer analysis of distribution of putative cis- andtrans-regulatory elements in milk protein gene promoters.Biosystems 45(1):29–44

Meier VS, Groner B (1994) The nuclear factor YY1 participates inrepression of the beta-casein gene promoter in mammary epithelialcells and is counteracted by mammary gland factor during lactogen-ic hormone induction. Mol Cell Biol 14(1):128–137

Pérez MJ, Leroux C, Bonastre AS, Martin P (1994) Occurrence of aLINE sequence in the 3′ UTR of the goat alpha s1-casein E-encoding allele associated with reduced protein synthesis level.Gene 147(2):179–187

Persuy MA, Legrain S, Printz C, Stinnakre MG, Lepourry L, Brignon G,Mercier JC (1995) High-level, stage- and mammary-tissue-specificexpression of a caprine kappa-casein-encodingminigene driven by abeta-casein promoter in transgenic mice. Gene 165(2):291–296

Pierre S, Jolivet G, Devinoy E, Théron MC, Maliénou-N’Gassa R,Puissant C, Houdebine LM (1992) A distal region enhances theprolactin induced promoter activity of the rabbit alpha s1-caseingene. Mol Cell Endocrinol 87(1–3):147–156

Pittius CW, Hennighausen L, Lee E, Westphal H, Nicols E, Vitale J,Gordon K (1988) A milk protein gene promoter directs the expres-sion of human tissue plasminogen activator cDNA to the mamma-ry gland in transgenic mice. Proc Natl Acad Sci U S A 85(16):5874–5878

Quandt K, Frech K, Karas H,Wingender E, Werner T (1995) MatInd andMatInspector: new fast and versatile tools for detection of consensusmatches in nucleotide sequence data. Nucleic Acids Res 23(23):4878–4884

Ramunno L, Cosenza G, Rando A, Illario R, Gallo D, Di Berardino D,Masina P (2004) The goat alphas1-casein gene: gene structure andpromoter analysis. Gene 334:105–111. doi:10.1016/j.gene.2004.03.006

Raught B, Liao WS, Rosen JM (1995) Developmentally and hormonallyregulated CCAAT/enhancer-binding protein isoforms influencebeta-casein gene expression. Mol Endocrinol 9(9):1223–1232

Renaville R, Gengler N, Vrech E, Prandi A, Massart S, Corradini C,Bertozzi C, Mortiaux F, Burny A, Portetelle D (1997) Pit-1 genepolymorphism, milk yield, and conformation traits for ItalianHolstein-Friesian bulls. J Dairy Sci 80(12):3431–3438. doi:10.3168/jds.S0022-0302(97)76319-7

Riego E, Limonta J, Aguilar A, Pérez A, de Armas R, Solano R, RamosB, Castro FO, de la Fuente J (1993) Production of transgenic miceand rabbits that carry and express the human tissue plasminogenactivator cDNA under the control of a bovine alpha S1 caseinpromoter. Theriogenology 39(5):1173–1185

Rosen JM, Wyszomierski SL, Hadsell D (1999) Regulation of milkprotein gene expression. Annu Rev Nutr 19:407–436. doi:10.1146/annurev.nutr.19.1.407

Ryan AK, Rosenfeld MG (1997) POU domain family values: flexibility,partnerships, and developmental codes. GenesDev 11(10):1207–1225

Saito H, Oka T (1996) Hormonally regulated double- and single-strandedDNA-binding complexes involved in mouse beta-casein gene tran-scription. J Biol Chem 271(15):8911–8918

86 J Appl Genetics (2014) 55:75–87

Sambrook J, Fritsch EF,Maniatis T (1989)Molecular cloning: a laboratorymanual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

Schmitt-Ney M, Doppler W, Ball RK, Groner B (1991) Beta-casein genepromoter activity is regulated by the hormone-mediated relief oftranscriptional repression and a mammary-gland-specific nuclearfactor. Mol Cell Biol 11(7):3745–3755

Schuster WA, Treacy MN, Martin F (1988) Tissue specific trans-actingfactor interaction with proximal rat prolactin gene promoter se-quences. EMBO J 7(6):1721–1733

van Helden J (2003) Regulatory sequence analysis tools. Nucleic AcidsRes 31(13):3593–3596

Wakao H, Gouilleux F, Groner B (1994) Mammary gland factor (MGF)is a novel member of the cytokine regulated transcription factor

gene family and confers the prolactin response. EMBO J 13(9):2182–2191

Welte T, Philipp S, Cairns C, Gustafsson JA, Doppler W (1993)Glucocorticoid receptor binding sites in the promoter regionof milk protein genes. J Steroid Biochem Mol Biol 47(1–6):75–81

Yamauchi K, Shimizu M, Takamiya Y, Kawakami H, Ganguli NC (1983)Studies on milk proteins from two breeds of Indian Buffalo. Jpn JZootech Sci 54:329–335

Yu-Lee LY, Richter-Mann L, Couch CH, Stewart AF, Mackinlay AG,Rosen JM (1986) Evolution of the casein multigene family: con-served sequences in the 5′ flanking and exon regions. Nucleic AcidsRes 14(4):1883–1902

J Appl Genetics (2014) 55:75–87 87