Biochem Genet (2008) 46:18–28 DOI 10.1007/s10528-007-9125-7
Sequence Characterization, Polymorphism, and Chromosomal Localizations of the Porcine Capz Genes E. Yang Æ H. Wang Æ X. X. Wu Æ Z. L. Tang Æ S. L. Yang Æ K. Li
Received: 4 February 2007 / Accepted: 23 July 2007 / Published online: 16 October 2007 Springer Science+Business Media, LLC 2007
Abstract CapZ is a widely distributed and highly conserved actin-binding protein that caps the barbed end of actin filaments and nucleates actin polymerization in a Ca2+-independent manner. In myofibrils, it is localized in the Z-lines. In this study, we cloned and characterized Capz subunit genes from the pig muscle. The nucleotide sequences and the predicted protein sequences share high sequence identity with other mammalian orthologs. The reverse transcriptase polymerase chain reaction (RT-PCR) revealed that porcine Capzb, Capza1, and Capza2 genes are expressed in all 11 tissues studied (liver, spleen, small intestine, large intestine, lymph node, kidney, heart, skeletal muscle, brain, fat, and lung) but in variable amounts. Radiation hybrid mapping data indicated that Capzb, Capza1, and Capza2 map to q2.1–2.6 of pig chromosome 6 (SSC6), q1.6–q2.2 of pig chromosome 4 (SSC4), and q1.3–2.3 of pig chromosome 18 (SSC18), respectively. An A/C single nucleotide polymorphism in Capzb intron 4 was identified with a HhaI PCR restriction fragment length polymorphism, which showed great allele frequency differences between Guizhou Xiang, Guangxi Bama, Wuzhishan, Tongcheng, Landrace, and Yorkshire pigs. The association analysis suggested that the Capzb genotype was associated with leaf fat (P \ 0.05) in our experimental population. Keywords Capz
Porcine Physical mapping Polymorphism Association analysis
E. Yang H. Wang Z. L. Tang S. L. Yang K. Li (&) Department of Gene and Cell Engineering, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, P.R. China e-mail:
[email protected] E. Yang H. Wang X. X. Wu Department of Animal Physiology and Biochemistry, School of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P.R. China
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Introduction Actin is a ubiquitous and abundant protein widely distributed in nearly all kinds of eukaryotic cells (Soeno et al. 1998). Actin capping proteins contribute to the organization and turnover of complex actin filament structure by regulating actin monomer dynamics at filament ends. In striated muscle cells, it is confirmed that the tight capping of the barbed ends by CapZ and of the pointed ends by tropomodulin (Tmod) contributes to the stabilization and the uniform length of actin (thin) filaments in myofibrils (Littlefield et al. 2001). CapZ plays a significant role in the structure of the Z-disk, where together with actinin it has been proposed to make up an anchoring complex for thin filaments in the Z-disk (Papa et al. 1999). CapZ is a heterodimer comprising an a-subunit and a b-subunit, and both subunits are required for effective barbed-end capping of filaments (Hart et al. 1997). There are at least two a-subunit isoforms in eukaryotic cells, each encoded by a separate gene, and three b-isoforms, produced by alternate splicing of mRNA from one gene (Pyle et al. 2002). CapZ, down-regulated during muscle aging in the rat, indicates a loss of actin and sarcomere function (Piec et al. 2005). As a structural protein, CapZ also has an influence on muscle biochemistry and meat quality, as its intensity changes a lot in the postmortem treatment in the pig, as shown by proteome analysis. Though the postmortem change of CapZ was not significantly related to pork tenderness (Lametsch et al. 2003), it is conceivable that CapZ is involved in muscle development, considering its critical role in these structures, which are also known to be important to meat quality. For comparative studies and as a step toward identifying mutations, we have isolated and sequenced cDNAs encoding the two a- and one b-subunit genes for porcine CapZ and mapped them to porcine chromosomes. Furthermore, we detected mRNA expression of Capzb, Capza1, and Capza2 in skeletal muscle during prenatal and postnatal development. We then identified several polymorphic sites and conducted preliminary association analysis in the experimental population in order to lay a basis for further study of porcine CapZ and to contribute to the understanding and improvement of pork quality.
Materials and Methods Sample Collection Eleven tissues, including lung, skeletal muscle, spleen, heart, large intestine, lymph node, small intestine, liver, brain, kidney, and fat, were obtained from four Wuzhishan mini pigs 18 to 24 months old, for spatial expression analysis. The genetic variability analysis within the porcine Capzb, Capza1, and Capza2 sequence employed genomic DNAs that represented three indigenous Chinese breeds (Wuzhishan, Laiwu, and Tongcheng pigs) and two introduced commercial breeds (Yorkshire and Landrace).
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cDNA Clone and Sequence Analysis Human mRNA sequences of Capzb, Capza1, and Capza2 (GenBank accession nos. NM004930, NM006135, and NM006136) were compared to all sequences available in the pig in the express tag (EST) databases using the Blast algorithm ( http://www.ncbi.nlm.nih.gov/BLAST). We selected the porcine ESTs that shared more than 80% sequence identity to the corresponding human cDNA in order to assemble the porcine gene using the DNAstar program (Madison, WI). To verify and clone the cDNA sequence of porcine Capzb, Capza1, and Capza2, RNA extraction, RT-PCR, and sequencing were performed as described previously (Wang et al. 2007).
Chromosome Mapping by IMpRH Radiation hybrid mapping was performed using the INRA-University of Minnesota 7,000 rads radiation hybrid panel (IMpRH), consisting of 118 hamster-porcine hybrid cell lines (Yerle et al. 1998). The PCR was performed as described previously (Wang et al. 2007).
Spatial Expression Analysis Gene expression patterns were determined by semiquantitative RT-PCR. Total RNA was extracted from 11 tissues, and reverse transcription was performed as described by Pan et al. (2003). PCR conditions were 5 min at 94C followed by 24 cycles of 30 s at 94C, 30 s at 62C, 30 s at 72C, and a final extension of 5 min at 72C. Amplification of b-actin cDNA was performed as a positive control. Electrophoresis was performed on 10 ll PCR products to determine the expression profile.
Experimental Populations and Association Analysis The animals used for association studies were 121 pigs from three pure-blood experimental populations, including Landrace (L, n = 25), Yorkshire (Y, n = 22), Tongcheng (T, n = 36), and two crossbred populations, including L (#) · YT ($) (LYT, n = 18) and Y(#) · LT ($) (YLT, n = 20). Tongcheng pigs were slaughtered at 75 kg body weight; the other pigs were slaughtered at 90 kg body weight. Phenotypic data were collected in a 1-year serial slaughter experiment at the Animal Husbandry Bureau of Tongcheng County (Hubei Province, China). Partial important production traits, including carcass traits (intramuscular fat, backfat, and eye area), percentage of leaf fat (leaf fat/carcass), and meat quality traits (meat color score, marbling score, tenderness, driploss, and fat) were measured according to the standard Chinese meat industry carcass measurements. To identify the single nucleotide polymorphism (SNP) sites in the three gene loci, PCRs were performed
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as previously described (Wang et al. 2007) to amplify the genomic DNA from different breeds. Then a preliminary association analysis was performed to determine whether the genotype of the polymorphism site was associated with certain phenotypes. The experimental population and statistical model were as reported previously (Wang et al. 2007).
Results Molecular Characterization of Porcine Capzb, Capza1, and Capza2 Genes Analysis of the cDNA sequences of the porcine Capz revealed the following: (1) The full-length cDNA of porcine Capzb consists of 1,677 bp that contains an ORF of 819 bp encoding a protein of 272 residues with a calculated molecular mass of 30.6 kDa and an isoelectric point (pI) of 5.76. It contains a 50 untranslated region of 104 bp (50 -UTR) and a 30 untranslated region of 754 bp (30 -UTR). (2) The fulllength cDNA of porcine Capza1 consists of 2,419 bp; computer analysis revealed an 861 bp ORF flanked by a 37 bp 50 -UTR and a 1,521 bp 30 -UTR, with a putative polyadenylation signal, AATAAA, located at 2,292–2,297 bp. The porcine Capza1 gene is predicted to encode a polypeptide of 286 amino acids with a molecular mass of 33.0 kDa and a pI of 5.63. (3) The deduced porcine Capza2 mRNA contained an 861 bp ORF flanked by a 100 bp 50 -UTR and a 1,282 bp 30 -UTR, with a consensus AATAAA polyadenylation signal 16 bp upstream of the poly (A) stretch. This ORF is predicted to encode a polypeptide of 286 amino acids, with an expected molecular mass of 33.0 kDa and pI of 5.67. The sequences of porcine Capzb, Capza1, and Capza2 were deposited in GenBank (accession nos. EF202986, EF202988, and EF202987).
Spatial Expression Analysis of Porcine Capzb, Capza1, and Capza2 Genes RT-PCR was performed to determine the relative mRNA expression of Capzb, Capza1, and Capza2 in various pig tissues; the housekeeping gene b-actin was used as an endogenous control. The three subunit genes were expressed in all tissues examined, but in variable amounts (Fig. 1). Furthermore, the different subunit genes exhibit different expression patterns. Capzb is expressed strongly in lung, spleen, heart, lymph node, and brain, and the a-subunit genes were abundant in lung, spleen, and kidney.
Chromosomal Localization of the Porcine Capzb, Capza1, and Capza2 Genes Chromosomal locations of porcine Capzb, Capza1, and Capza2 were determined by PCR screening of the INRA–University of Minnesota 7,000 rads radiation hybrid panel (IMpRH) with gene-specific primers. The detailed mapping results are listed in Table 1.
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Fig. 1 RT-PCR tissue expression analysis of Capzb, Capza1, and Capza2 in lung, skeletal muscle, spleen, heart, large intestine, lymph node, small intestine, liver, brain, kidney, and fat
Polymorphism and Association Analysis After alignment of the pig mRNA sequence with the human DNA sequence (GenBank accession nos. NC_000001 and NC_000007), the exon/intron junction appeared. Then primer pairs were designed based on the mRNA sequence to amplify these gene fragments, employing the genomic DNA as templates, facilitating the location of valuable mutations. Comparative sequencing revealed four possible mutations in Capzb (GenBank accession no. EF202989), two in the fourth intron (C/T591 and A/C612), one in the sixth intron (G/C1501), and another mutation in the coding region (A/G921), but it is silent. For Capza2, two SNPs were found in the 30 -UTR (T/C/G1347, A/C1503) (Table 2). For further analyses, we genotyped the A/C612 polymorphism in the fourth intron of Capzb by RFLP in 121 unrelated pigs, representing five indigenous and introduced commercial breeds. The primer pairs were designed to obtain a 226 bp PCR product containing the A/C612 polymorphism recognized by the HhaI enzyme (Fig 2). Genotyping results showed great variation in the allele frequency between breeds (Table 3). Among our sample of 121 unrelated pigs, the Landrace, Yorkshire, and Chinese indigenous Bama pigs showed only the C allele, and the Chinese indigenous Xiang pig breed showed only the A allele. We performed a preliminary association study to determine whether this polymorphism has affected some muscle traits in the pig. The result was that the percent of leaf fat (leaf fat/ carcass) of pigs with the AA genotype differed significantly from that of pigs with the CC (P = 0.03) and the AC (P = 0.02) genotypes, but this polymorphism has no effects on other traits studied (Table 4).
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Capza-2
b
CTTACCAGTGAATCATACGC GAAAAGGATTCCACCAGAG
AGTCCTTGCATATGTACGTG TCCACCATTACCAGTTGTC
GCAGCAGTGTTAAGCCTCT TGCGGAGAGTTCTGATAGC
Primer squence (50 -30 )
Position deduced from the closest marker available on the cytogenetic map
From the Web site http://www.ncbi.nlm.nih.gov/Locuslink/
EF202987
Capza-1
a
EF202986
EF202988
Capzb
GenBank Acc. No. (pig)
Gene
Table 1 Chromosomal assignment of three porcine Capz genes
289
541
241
Size (bp)
7q31.2–q31.3
1p13.2
1p36.1
Human cytogenetic positiona
27.1
30.5
19.5
Retention (%)
SW787
SW2435
SW446
Closest mark
73
56
35
Dist (cR)
5.39
7.63
10.06
LOD score
Porcine IMpRH mapping results
18q1.3–18q2.3
4q1.6–4q2.2
6q2.3–6q2.6
Porcine cytogenetic positionb
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Table 2 Primers employed in these experiments Gene
Primer
Primer sequence (50 -30 )
Binding region
Capzb
SNP-1 PLa
TGCGGTGTCTCGTCTGTCTAC
Exon 4
SNP-1 PR
GTGGAGGTCGGCTTGTAATG
Exon 5
SNP-2 PL
ACGAGACTGTGAGCGACTGC
Exon 6
SNP-2 PR
CTCAGCCCGTTGATGATGTC
Exon 7
SNP-3 PL
TAGAAGGCTGGAGACGGATC
Exon 4
SNP-3 PR
TCGGTGACTACTGTGTTCCTG
Intron 4
CDS PLb
TGGATGTTCACTCCTGGGC
50 UTR
CDS PR
GACAAGACGGATTCACTGG
Exon 8
Mapping PLc
GCAGCAGTGTTAAGCCTCT
Exon 8
Mapping PR
TGCGGAGAGTTCTGATAGC
30 UTR
Express PLd
AGCCTCTGCTTCCCACTAAC
30 UTR
Express PR
GACAAGACGGGATTCACTGG
30 UTR
Genomic PLe
CTCTAAGGCCTATGTGA
Exon 5
Capza-1
Capza-2
a
Size (bp)
PCR (TM)
514
60
662
60
225
60
1102
60
241
60
180
59
932
58
541
60
2054
59
310
60
1270
61
289
61
1022
60
289
60
0
Genomic PR
TTCGCAGCATAGCCTCTT
3 UTR
Mapping PL
AGTCCTTGCATATGTACGTG
30 UTR
Mapping PR
TCCACCATTACCAGTTGTC
30 UTR
CDS PL
ATGGCCGACTTCGAGGATC
Exon 1
CDS PL
CGCCACCATTACCAGTTGTC
30 UTR
Express PL
AGTCCTTGCATATGTACGTG
30 UTR
Express PR
TCCACCATTACCAGTTGTC
30 UTR
Genomic PL
CGACAGTTGCCAGTTACAC
Exon 10
Genomic PR
GAAAAGGATTCCACCAGAG
30 UTR
Mapping PL
CTTACCAGTGAATCATACGC
30 UTR
Mapping PR
GAAAAGGATTCCACCAGAG
30 UTR
CDS PL
ATGGCGGATTTGGAAGAGC
Exon 2
CDS PR
GCTAAGCTGTGTCCTCTAACC
30 UTR
Express PL
CTTACCAGTGAATCATACGC
30 UTR
Express PR
GAAAAGGATTCCACCAGAG
30 UTR
SNP primer for polymorphism genotyping
b
CDS primer for amplifying coding region of the CapZ
c
Mapping primer for radiation hybrid mapping
d
Express primer for various expression
e
Genomic primer for amplifying genomic cDNA
Discussion In our present study, we cloned and characterized porcine Capzb, Capza1, and Capza2 genes. Both the nucleotide sequences and the predicted protein sequences share high sequence identity with other mammalian orthologs. The porcine Capza1 and Capza2 genes share the same structure and are similar in size, with 10 exons of approximately the same length and 9 introns of different size, but it was reported
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Fig. 2 RFLP analysis of porcine Capzb polymorphism, an RFLP of the 226 bp Capzb fragment containing the A612C polymorphism recognized by the HhaI enzyme (AA 226 bp, CC 124/102 bp, and AC 226/124/ 102 bp)
Table 3 Allele frequency of Capzb SNP (A/C) in different pig breeds Breed
No.
Genotype AA
Allele frequency CC
AC
A
C 0.49
Tongcheng pig
36
7
6
23
0.51
Xiang pig
44
44
0
0
1.00
0.00
Bama mini pig
34
0
34
0
0.00
1.00
Wuzhishan mini pig
30
20
0
10
0.83
0.17
Landrace
25
0
25
0
0.00
1.00
Yorkshire
22
0
22
0
0.00
1.00
Total
191
that the combination of a 1/b heterodimer exhibited a higher affinity for actin (about four-fold higher) than a 2/b heterodimer (Hug et al. 1992) when examined with in vitro translation products of RNA transcripts derived from the cDNAs. We also aligned the predicted protein sequences encoded by Capzb, Capza1, and Capza2 genes. The deduced amino acid sequence of porcine Capzb shows 100% identity with the corresponding human proteins, including the actin-binding site that affects the ability to bind the barbed ends of actin filaments and nucleate actin polymerization (Barron-Casella et al. 1995). The protein sequences of pig Capza1 and Capza2 shared 97% and 99% homology with the human and 96% and 98% homology with the mouse. Like the orthologous genes in the human and the mouse, porcine Capz displays homologies in its coding regions. CapZ is a heterodimer composed of an a-subunit and a b-subunit; we found different mRNA expression patterns for the Capz subunit genes among the porcine tissues investigated. This is in accordance with the observations in other species (Hart et al. 1997). In addition, the two isoforms of the a-subunit displayed different mRNA expression patterns, and the CapZ protein containing each isoform showed different binding affinities to actin, indicating their different roles in regulation of
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50
9
AC
AA
Intramuscular fat (%)
5.646 ± 0.141
5.565 ± 0.203
6.424 ± 0.413
Leaf fat (%)a
3.116A ± 0.094
3.071A ± 0.135
3.751B ± 0.276
75.298 ± 0.699
76.069 ± 0.343
75.812 ± 0.239
Carcass (%)
3.023 ± 0.192
3.246 ± 0.094
3.144 ± 0.065
Backfat (cm)
32.288 ± 1.538
32.351 ± 0.755
32.340 ± 0.525
Eye area (cm2)
Values with different superscript letters show statistically significant differences (P \ 0.05)
67
CC
a
No.
Genotype
Table 4 Association analysis of the Capzb A/C polymorphism
3.387 ± 0.135
3.119 ± 0.067
3.080 ± 0.046
Meat color
2.308 ± 0.187
2.413 ± 0.092
2.359 ± 0.064
Marble
44.963 ± 4.187
41.136 ± 2.061
44.879 ± 1.428
Tenderess
2.067 ± 0.364
1.968 ± 0.180
1.993 ± 0.125
Driploss (%)
2.346 ± 0.317
2.207 ± 0.158
2.542 ± 0.109
Fat (%)
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actin assembly. All these suggest that the biological function of CapZ protein could be regulated through mechanisms that control the transcription of individual CapZ subunit genes. We mapped the porcine Capzb, Capza1, and Capza2 genes to SSC6, SSC4, and SSC18, respectively. We determined that the porcine Capza2 gene was closely linked with the marker SW787, and this result is consistent with the location obtained previously by linkage mapping and FISH, though a rather small location discrepancy exists between the two methods (Campbell et al. 2001). Porcine Capzb maps to q2.3–q2.6 of SSC6 within the confidence intervals of several putative quantitative trait loci for carcass traits and meat quality traits (Mohrmann et al. 2006), thus facilitating the identification of positional candidate genes. In addition, the human Capza1 gene has been mapped to chromosome 1 region p13.2, the Capza2 gene has been mapped to chromosome 7 position q31.2–q31.3, and the Capzb gene to chromosome 1 region p36.1 (Barron-Casella et al. 1995), and our assignments of the three porcine genes are in agreement with the comparative map between human and pig chromosomes (Yue et al. 2003). A polymorphism site located in the fourth intron of Capzb was used to assess the allele variation and its relationships with the economic traits in the experimental population. The allele frequency was obviously different among breeds. A previous study found the porcine Capz protein increased during postmortem storage, indicating that it may have an influence on pork quality (Lametsch et al. 2003). We have not found, however, any association of the polymorphism site with the carcass and meat traits studied. Instead, an association of the Capz polymorphism with the percent of leaf fat was evident in this study. Further association analyses should be performed in other populations to determine whether this gene is involved in the fat deposition process. Though the SNP does not induce any amino acid alteration, it may be linked to other loci controlling the traits of interest. In conclusion, we cloned the porcine Capzb, Capza1, and Capza2 genes, assigned them to porcine chromosomes, and analyzed the distinct spatial expression patterns of the three subunit genes. Several SNPs were identified in these gene loci, and an SNP located in the fourth intron of Capzb gene was associated with the percent of leaf fat in our experimental populations. All these provide a foundation for further research on Capz. Acknowledgments The authors wish to express their thanks to Dr Martine Yerle of INRA for providing the RH panel. This research was supported by the National High Science and Technology Foundation of China (20060110Z1039), National Natural Science Foundation of China (30500359), Key Project of National Basic Research and Developmental Plan of China (G2006CB102105), Key Project of National Natural Science of China (30330440), State Platform of Technology Infrastructure (2005DKA21101), and Key Project of Scientific Research Foundation of Ministry of Human Resources of China for Returned Chinese Scholars.
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