Acta agriculturae Slovenica, 86(december 2005)2, 99-102.
http://aas.bf.uni-lj.si
Agris category codes: L10                                                                            COBISS Code             1.03
GENETIC VARIATION OF THE MITOCHONDRIAL D-LOOP REGION
CONTAINING MITOCHONDRIAL TRANSCRIPTION FACTOR A (TFAM) BINDING
SITES IS NOT ASSOCIATED WITH MARBLING IN WAGYU X LIMOUSIN F2
CROSSES
Tanja KUNEJ a), Jennifer J. MICHAL b), Peter DOVČ c) and Zhihua JIANG d)
a) Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje 3, SI-1230 Domžale, Slovenia, Ph.D., e-mail: tanja.kunej@bfro.uni-lj.si
b) Washington    State    Univ.,    Dept.    of    Animal    Sciences,    Pullman,    WA    99164-6351,    M.Sc.,    e-mail: jennifer_michal@wsu.edu.
c) Same address as a), Prof., Ph.D., e-mail: peter.dovc@bfro.uni-lj.si.
d) Same address as b), Ph.D., e-mail: jiangz@wsu.edu.
Received November 03, 2005, accepted November 30, 2005. Delo je prispelo 03. novembra 2005, sprejeto 30. novembra 2005.
ABSTRACT
Mitochondrial transcription factor A (TFAM) is a nucleus-encoded protein that is essential for initiation of transcription and replication of mitochondrial DNA (mtDNA). It has been shown that TFAM binds the entire length of the mtDNA, but with higher affinity to sequences around both mitochondrial promoters located in 3’ domain of the D-loop region. The objectives of this study were to detect genetic polymorphisms in the sequence flanking TFAM binding sites of the mtDNA D-loop region and investigate their associations with marbling in Wagyu x Limousin F2 crosses. We identified six polymorphis sites in the TFAM binding sites of the D-loop mtDNA (G8A, T106C, A169G, A173G and C190T and one insertion/deletion at position 221 with one or two cytosines) with no significant differences in frequencies of polymorphic sites between extreme high/low marbling pools.
Key words:    cattle / breeds / Limousin / Wagyu / crosses / molecular genetics / mitochondrial transcription factor / mtDNA / muscles / marbling
GENETSKA RAZNOLIKOST MITOHONDRIJSKE D-LOOP REGIJE, KI VSEBUJE VEZAVNO MESTO ZA MITOHONDRIJSKI TRANSKRIPCIJSKI FAKTOR A (TFAM), NI POVEZANA Z MARMORIRANOSTJO PRI F2 WAGYU X LIMOUSIN KRIŽANCIH
IZVLEČEK
Mitohondrijski transkripcijski factor A (TFAM) je v jedru kodiran protein, udeležen v iniciaciji transkripcije in replikacije mitohondrijske DNA (mtDNA). Predhodne raziskave so pokazale, da se TFAM veže po vsej dolžini mtDNA, z večjo afiniteto pa na zaporedja v področju mitohondrijskih promotorjev, ki se nahajata na 3’ območju D-loop regije. Namen naše študije je bil identificirati genetske polimorfizme v območju vezave TFAM v D-loop regiji mtDNA in raziskati njihovo povezavo z marmoriranostjo mišic pri F2 Wagyu x Limousin križancih. V območju vezave TFAM smo v D-loop regiji mtDNA identificirali šest polimorfnih mest (G8A, T106C, A169G, A173G, C190T, insercija/delecija enega ali dveh C na poziciji 221), razporeditev alelov pa se ni statistično značilno razlikovala med skupinama živali z visokimi in nizkimi vrednostmi marmoriranosti.
Ključne besede:  govedo / pasme / limuzin / wagyu / križanci / molekularna genetika / mitohondrijski transkripcijski faktor / mtDNK / mišice / marmoriranost
100    Acta agriculturae Slovenica, 86(december 2005)2.
INTRODUCTION
Mitochondrial transcription factor A (TFAM) is a nucleus-encoded protein that is essential for initiation of transcription and replication of mitochondrial DNA (mtDNA). Decreased mitochondrial gene expression has been associated with onset of obesity in ob/ob mice (Wilson-Fritch et al., 2004). Therefore, genetic variants in the mtDNA flanking the TFAM binding sites might have influence on mitochondrial gene expression and biogenesis of mitochondria, and consequently affect energy metabolism and body fat deposition. Additionally, it has been shown that mtDNA substitution G2232A affect beef marbling score (Mannen et al., 2003). The complete bovine mitochondrial DNA contains 16338 bp (GenBank accession No. V00654) (Anderson at al., 1982). Evidence has shown that TFAM binds the entire length of the mtDNA, but with higher affinity to sequences around both mitochondrial promoters located in 3’ domain of the D-loop region (Kang et al., 2002; Garrido et al., 2003). The objectives of this study were to detect genetic polymorphisms in the sequence flanking TFAM binding sites of the mtDNA D-loop region and investigate their associations with marbling in Wagyu x Limousin F2 crosses.
MATERIAL AND METHODS
Primer sequences and PCR amplification
Primers were designed to amplify a fragment of 459 bp from positions 16 292 to 412 in the bovine mitochondrial DNA (Anderson et al., 1982). The primer sequences were: forward, 5'-CATCTAAAACGGTCCATTCTTTCCTC-3’ and reverse, 5’-ACTCATCTAGGCATTTTCA-GT-3’. Genomic DNA (50 ng) was amplified in a final volume of 10 µl that contained 1 x PCR buffer, 1 mM MgCl2, 200 µM dNTPs, 0.5 U Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA) and 5 pmol of each primer. PCR was performed in an Applied Biosystems Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) under the conditions: initial denaturation at 94 °C for 2 min, followed by touchdown amplification (1 °C temperature decrease per cycle for eight cycles from 66 °C to 59 °C) and followed by 28 cycles of denaturation at 94°C (30 sec), annealing at 59 °C (30 sec) and extension at 72 °C (30 sec) with a final extension step at 72 °C for 7 min. The PCR products were examined by electrophoresis on a 1.5% agarose gel with 1 X TBE buffer. The gels were stained with ethidium bromide and photographed.
PCR-RFLP analysis
The 459 bp PCR products were digested with 2U of NlaIII at 37 °C overnight and analysed on 3% agarose gels. The PCR product included two NlaIII sites (recognition motif CATGˇ) on positions 22 and A169G yielding the products of 69 bp and 390 bp in case of no NlaIII recognition on 169 site, or products of 69 bp, 149 bp and 241 bp in case of NlaIII recognition. In case of heteroplasmy at 227 site, we expected the appearance of additional two NlaIII-digestion products of 205 bp and 185 bp in case of no recognition on 169 site or additional NlaIII-digestion products of 56 bp and 185 bp in case of NlaIII recognition at 169 site.
RESULTS
We used 31 animals at the two tails of marbling score distribution for mutation detection and initial association screening, including 15 samples with low marbling scores (ranging from 4 to 4.5) and 16 samples with high marbling scores (ranging from 7.5 to 9.5). Sequencing was done
Kunej T. et al. Genetic variation of the mitochondrial D-loop region … in Wagyu × Limousin F2 crosses.           101
directly and individually on PCR products using the BigDye Terminator Cycle Sequencing method (Applied Biosystems, Foster City, CA, USA). A total of six polymorphic sits were identified, including five substitutions at positions G8A, T106C, A169G, A173G and C190T and one insertion/deletion at position 221 with one or two cytosines (Table 1). Fisher’s exact test revealed no significant differences in frequencies of all polymorphic sites between extreme high/low marbling pools (P > 0.05) (Table 1).
Table 1.            Variants identified in the 3’ mtDNA D-loop region that contains two TFAM
binding sites Preglednica 1.   Polimorfizmi, identificirani v 3’ D-loop področju mtDNA, ki vsebuje dve
vezavni mesti za TFAM
Position*
Mutation
Low marbling        High marbling         Significance
106
169
173
190
221**
G	15	15	P = 0.516129
A	0	1	
T	13	15	P = 0.373748
C	2	1	
G	13	11	P = 0.174416
A	2	5	
A	14	12	P = 0.160672
G	1	4	
C	13	16	P = 0.225806
T	2	0	
CC	0	1	P = 0.205381
C -	9	11	
- -	6	4	
* Nucleotide positions were based on GenBank accession number V00654.
**No insertion of cytosine (- -), insertion of one cytosine (C -) or two cytosines (CC) on the nucleotide position 221.
2       3
8       9       10     11      12
:¦::: -:?;1;.':;-;.
High marbling
Low marbling
Figure 1. Restriction patterns of samples representing high (1–6) and low (7–12) marbling score.
On the right hand side of each block is molecular marker. Slika 1.    Restrikcijska analiza vzorcev živali  z  visoko  (1–6)  in nizko  (7–12)  oceno  za
marmoriranost. Na desni strani vsake skupine vzorcev je molekularni marker.
8
102    Acta agriculturae Slovenica, 86(december 2005)2.
DISSCUSION
Previous studies showed the relationships between mtDNA sequence variations and economic traits as milk yield, milk fat yield and milk fat percentage (Schutz et al, 1994; Boettcher et al., 1996) and beef marbling score (Mannen et al., 2003). Identified six polymorphic sites in the bovine mtDNA were also reported previously by different groups (Steinborn et al., 1998; Mannen et al., 1998; Mannen et al., 2003). In the present study, none of these polymorphic sites showed any significant association with marbling in the Wagyu x Limousin cross population, because there were no significant genetic differences observed between high and low marbling animals (Table 1). In dairy cattle, it was also found that there were no significant relationships established between D-loop polymorphism and any traits, such as mature equivalent yield of milk, fat, SNF, and milk energy as well as concentrations of fat, SNF, and milk energy (Boettcher et al., 1996). The major reason might be that none of these polymorphisms occurs within the TFAM binding sites.
ACKNOWLEDGEMENTS
This work was supported by the bilateral scientific exchange program between USA and Slovenia (project number: BI-US/03-04/17). We thank Dr. Michael D. MacNeil, USDA Agricultural Research Service, Fort Keogh Livestock and Range Research Laboratory for providing us materials and performance data for the project.
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