Research Paper
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Acta Biochim Biophys Sin
2005,37: 843–850
doi:10.1111/j.1745-7270.2005.00113.x
cDNA Cloning, Sequence
Analysis of the Porcine LIM and Cysteine-rich Domain 1 Gene
Jun WANG, Chang-Yan DENG*,
Yuan-Zhu XIONG, Bo ZUO, Lei XING, Feng-E LI, Ming-Gang LEI, Rong ZHENG, and
Si-Wen JIANG
Key
Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of
Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China
Received: June 20,
2005
Accepted: October
10, 2005
This work was
supported by the grants from the Major State Basic Research Development Program
of China (G2000016105), the National High Technology Development Program of
China and the Natural Science Foundation of Hubei Province (2005ABA142)
*Corresponding author: Tel,
86-27-87287390; Fax, 86-27-87394184; E-mail, [email protected]
Abstract LIM domain proteins are important regulators in cell
growth, cell fate determination, cell differentiation and remodeling of the
cell cytoskeleton by their interaction with various structural proteins, kinases
and transcriptional regulators. Using molecular biology combined with in
silico cloning, we have cloned the complete coding sequence of pig LIM and
the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid
protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with
a pI of 8.39. It was found to be highly expressed in both skeletal muscle and
cardiac muscle. Alignment analysis revealed that the deduced protein sequence
shares 86%, 91% and 93% homology with that of its human, mouse and rat
counterparts, respectively. The LMCD1 protein was predicted by bioinformatics
software to contain a novel cysteine-rich domain in the N-terminal region, two
LIM domains in the C-terminal region, nine potential protein kinase C
phosphorylation sites, seven casein kinase II phosphorylation sites, a
tyrosine kinase phosphorylation site, seven N-glycosylation and
N-myristoylation sites and a single potential N-glycosylation site, which is
similar to the protein’s human counterpart. Phylogenetic tree was constructed
by aligning the amino acid sequences of the LIM domain from different species.
In addition, four base mutations were detected by comparing the sequences of
Large White pigs with those of Chinese Meishan pigs. The G294A mutation site
was confirmed by polymerase chain reaction-single-strand conformation
polymorphism analysis. Its allele frequencies were studied in five pig breeds.
Key words LIM and cysteine-rich domain 1 (LMCD1); SSCP; PCR; gene
expression; data analysis
LIM domain proteins are defined as proteins having a double zinc
fingers motif with a consensus amino acid sequence C-X2-C-X16–23-H-X2-C-X2-C-X16–23-C-X2-C, (where C represents
cysteine, and X represents other amino acids) [1]. They are important
regulators in cell growth, cell fate determination, cell differentiation and
remodeling of the cell cytoskeleton [2]. Bespalova and Burmeister [3] isolated
the human complete LMCD1 coding region and mapped this gene to 3p26-p24
by radiation hybrid mapping. Then the mouse LMCD1 gene was mapped to the
central region of chromosome 6 [3]. The predicted 365-amino acid LMCD1 protein
contains a cysteine-rich domain in the N-terminal region and two LIM domains in
the C-terminal region. It also has several potential phosphorylation and
N-myristoylation sites and a single potential N-glycosylation site. Northern
blot analysis detected a major 1.7 kb LMCD1 transcript in most of the
human adult and fetal tissues tested, with highest expression in skeletal
muscle. Little or no LMCD1 expression was found in fetal brain and
liver, or in adult brain, liver, thymus, small intestine, or peripheral blood
[3]. Recently, Ota et al. [4] and Strausberg et al. [5] have
isolated the human complete LMCD1 cDNA sequence, which contains six
exons and five introns. Furthermore, LIM protein was validated to have action
on the control of muscle genes [6]. The presence of LIM domains in the LMCD1
gene implies it may be involved in skeletal muscle protein-protein interactions
[1,7,8].
Therefore, the LMCD1 gene was selected as a candidate gene
for pig meat quality traits.
Materials and Methods
Tissue and blood samples
The tissue samples (heart, liver, lung, kidney, spleen, longissimus
dorsi muscle, fat, stomach and small intestine) were collected from
Large White, Landrace and Chinese local breed Meishan pigs at Jingping Pig
Station (Huazhong Agricultural University, Wuhan, China).
The blood samples of Yorkshire, Landrace and Chinese local breed
Meishan pigs were collected from Jingpin Pig Station. The Chinese local breeds
Exi Hei and Wannanhua pigs were from scattering farms in Enshi Municipality
(Enshi, China) and Anhui Province, respectively.
Total RNA extraction and
genomic DNA isolation
Total RNA was extracted from different tissues with a Trizol kit
(Gibco, Carlsbad, USA). In case where the samples were seriously contaminated
with genomic DNA, DNase I treatment on the total RNA was carried out before
first-strand cDNA synthesis.
Genomic DNA was isolated from the blood samples of Yorkshire,
Landrace, Meishan, Exi Hei and Wannanhua pigs using phenol/chloroform
extraction and ethanol precipitation [9].
Primer design
A number of pig expressed sequence tags (ESTs) were initially
identified using the cDNA sequence of human LMCD1 (GenBank accession No.
NM_014583) by running a Blast search against the GenBank “EST-others”
databases. Two ESTs (GenBank accession No. BF198782 and BF442878) were
assembled into one contig with Sequencher 4.14 software. Primer L1 (forward, 5‘-AGCCTCTGTCTAAGAAGCAAA-3‘;
reverse, 5‘-CACGGGCTGCTTCTCCTT-3‘) was designed by the contig.
Primer L2 (forward, 5‘-CTCCAAGTATTCCACCCTCACA-3‘, locating the contig;
reverse, 5‘-CCTCAGGATGGCCTTAGCAC-3‘, locating the EST) was
designed by the contig and the EST (GenBank accession No. C94730). The structure
of the amplified sequence covers the coding region and all the exons. Primer L3
(forward, 5‘-CCTGGAAGATGATCGGAAAA-3‘; reverse, 5‘-TGATGGTGTCAAAGGTGGGA-3‘)
was designed by the porcine LMCD1 gene sequence (GenBank accession No.
AY821789) which we had submitted. L3 was used to validate the correctness of
mutation sites obtained by single-strand conformation polymorphism (SSCP) analysis
of polymerase chain reaction (PCR) products.
A number of pig expressed sequence tags (ESTs) were initially
identified using the cDNA sequence of human LMCD1 (GenBank accession No.
NM_014583) by running a Blast search against the GenBank “EST-others”
databases. Two ESTs (GenBank accession No. BF198782 and BF442878) were
assembled into one contig with Sequencher 4.14 software. Primer L1 (forward, 5‘-AGCCTCTGTCTAAGAAGCAAA-3‘;
reverse, 5‘-CACGGGCTGCTTCTCCTT-3‘) was designed by the contig.
Primer L2 (forward, 5‘-CTCCAAGTATTCCACCCTCACA-3‘, locating the contig;
reverse, 5‘-CCTCAGGATGGCCTTAGCAC-3‘, locating the EST) was
designed by the contig and the EST (GenBank accession No. C94730). The structure
of the amplified sequence covers the coding region and all the exons. Primer L3
(forward, 5‘-CCTGGAAGATGATCGGAAAA-3‘; reverse, 5‘-TGATGGTGTCAAAGGTGGGA-3‘)
was designed by the porcine LMCD1 gene sequence (GenBank accession No.
AY821789) which we had submitted. L3 was used to validate the correctness of
mutation sites obtained by single-strand conformation polymorphism (SSCP) analysis
of polymerase chain reaction (PCR) products.
Primers were designed with Primer 5.0 (http://www.premierbiosoft.com).
Reverse transcription (RT)-PCR
Primary cDNA synthesis was processed in a final volume of 25 ml containing 5?reaction buffer (5 ml), 1 mg of total RNA from certain tissue as the template, 0.5 mM of each
dNTP, 25 U of RNasin (40 U/ml), 2 ml of 10 mM oligo(dT15) and 200 U of M-MuLV reverse transcriptase
(200 U/ml; Promega, USA).
PCR amplification was carried out in a final volume of 25 ml containing
standard 1?PCR buffer with Mg2+ and 1 U Taq polymerase (Biostar International, Toronto,
Canada), 0.8 mM of each dNTP, 10 pmol of each primer and 1.0 ml of first-strand
cDNA product as the template. The PCR conditions were as follows: denaturation
at 94 ?C for 3 min; 94 ?C for 50 s, 57 ?C for 45 s for L1 (or 62 ?C for 50 s
for L2), 72 ?C for 50 s, 35 cycles; and an additional extension step at 72 ?C
for 10 min.
Cloning of PCR products and
sequencing
The PCR products were fractionated on 1.5% (w/v) agarose gel, and selected bands were purified using a
gel extraction kit (Sangon, Shanghai, China). The purified PCR products were
ligated into the pGEM-T vector (Promega) and transformed into DH5a competent
cells. Bacteria were grown in LB-ampicillin agar. Cloned PCR products were
sequenced by Sangon.
mRNA expression analysis
The tissue distribution of pig LMCD1 mRNA was determined by
semiquantitative RT-PCR [10,11]. The house-keeping gene
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an
internal control on the template level. Primers for GAPDH amplification
were: forward, 5‘-ACCACAGTCCATGCCATCAC-3‘; reverse, 5‘-TCCACCACCCTGTTGCTGTA-3‘.
The primer for LMCD1 was L1.
PCR was carried out in a final volume of 25 ml as above. The
conditions for PCR were in the exponential phase of amplification, as
judged by the use of different concentrations of cDNA template, which provided
a direct correlation between the amount of the amplification products
obtained and RNA template abundance in the samples: denaturing at 94 ?C for 4
min; 94 ?C for 50 s, 57 ?C for 45 s and 72 ?C for 50 s, 28 cycles; and an
additional extension step for 10 min at 72 ?C. To validate the results, we
repeated the RT-PCR three times.
Prediction and analysis of
putative LMCD1 domain
The cDNA sequence prediction was conducted with GenScan software (http://genes.mit.edu/GENSCAN.html).
Sequence similarity analysis in GenBank was performed using the Blast 2.1
search tool (http://www.ncbi.nlm.nih.gov/blast/).
ClustalW software (http://www.ebi.ac.uk/clustalw/)
was used for alignment of multiple sequences. Phylogenetic and molecular
evolutionary analyses were conducted using MEGA 3.1 software [12]. To predict
the biophysics characteristics of the putative protein of LMCD1,
software on the ExPASy Proteomics Server (http://au.expasy.org/)
was used. The prediction and analysis for the protein structural domain and
functional site were performed using Prosite software (http://www.expasy.org/prosite/).
The 3-D structure of the putative protein conserved domain was analyzed using
the 3-D Conserved Domain Architecture Retrieval Tool of Blast (http://www.ncbi.nlm.nih.gov/blast/).
SSCP analysis [13,14]
L3 amplified DNA product (amplification procedure was the same as L1
except for the anneling temperature was 60 ?C) was mixed with four volumes of
formamide loading dye [98% formamide, 0.3% bromophenol blue and xylene cyanol
(Sangon), 10 mM EDTA and 7% glycerin (Sunbiotech, Beijing, China)], then
denaturized at 98 ?C for 10 min, loaded onto a non-denaturing gel [12%
acrylamide/bisacrylamide (29/1, W/W), 1?TBE (25 mM Tris,
25 mM boric acid, 0.5 mM EDTA)] containing 5% glycerol (Sunbiotech), and
subject to electrophoresis at 4 ?C, 5 W for 10–25 h, depending on the size
of the PCR product analyzed and the gel composition. The gels were processed by
silver staining.
Results
RT-PCR, cloning of PCR
products and sequencing
Amplified cDNA products were fractionated on 1.5% (w/v) agarose gel, and clear amplified bands of primer L1
and L2 were obtained. RT-PCR products were cloned into vector pGEM-T and
sequenced. Sequencing results showed that the sizes of the L1 and L2 PCR
products were 479 bp and 945 bp, respectively (Fig. 1).
mRNA expression analysis
Fig. 2 shows that LMCD1 mRNA was
present at high levels in the Longissimus dorsi muscle and heart, and at medium
levels in the lung, kidney and spleen. The LMCD1 mRNA expression level
was low in fat and in the stomach, and lower in the liver and small intestine.
All expression patterns were, on the whole, in accordance with the results of
adult human LMCD1 mRNA expression [3].
Nucleotide sequence analysis
The sequences amplified by primers L1 and L2 were assembled into a
1216 bp sequence with Sequencher 4.14 software. This sequence was identified
and similarity aligned by running a BlastN search against the GenBank “nr”
databases. The analysis revealed that the sequence contains the complete coding
sequence of LMCD1. It was then submitted to the GenBank database with
the accession number AY821789. The porcine LMCD1 nucleotide sequence
shared high homology with those of four species by Blast analysis: mouse
(86%), pan troglodytes (91%), rat (82%) and human (89%). Comparative analysis revealed
that there was a six-base deletion in the coding sequence of porcine LMCD1
which results in a two-amino-acid deletion in the porcine protein, compared
with that of the human gene at position 825–830 (GenBank accession No.
14277673).
Four putative base substitutions (G294A as shown in Fig. 3,
C385T as shown in Fig. 4, A748G and A1099G) were detected in the exon
region by comparing sequences of Large White, Landrace and Meishan pig breeds.
A748G substitution changes a codon for Alanine into a codon for Threonine.
Prediction and analysis of
protein structural domain and functional site
Similarity comparison for the amino acid sequence of the LMCD1
gene in four species is shown in Fig. 5. The amino acid sequence of
porcine LMCD1 shares 86% identity with that of human, 91% with that of mouse
and 93% with that of rat. The amino acid sequence of LMCD1 shares significant
identity with those of mouse Tes1, mouse Tes2, human LMO6, and human triple-LIM
domain protein [3]. Based on the above results, Tes1, Tes2 and several kinds
of LIM domain proteins were collected to construct a combined phylogenetic tree
by Neighbor-Joining method and Unweighted Pair Group Method with Arithmetic
Mean (UPGMA) using MEGA 3.1 software (http://www.megasoftware.net/),
as shown in Fig. 6. Results revealed that porcine LMCD1 has a closer
genetic relationship with mouse and rat LMCD1 than with that of human. All of
the LIM domain proteins and the Tes1 and Tes2 genes have a similar location in
the phylogenetic tree, only their bootstrap values are different. These results
validate the correctness of the current classification of the LIM domain
protein.
Primary structure analysis revealed that the molecular weight of the
putative LMCD1 protein is 40.788 kDa and its theoretical pI is 8.39. Topology
prediction showed there was a novel cysteine-rich domain in the N-terminal
region and two idiocratic LIM domain structural sequences in the C-terminal
region, but no transmembrane helices [15,16]. Nine potential protein kinase C
phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine
kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites
and a single potential N-glycosylation site were also found by prediction (Fig.
6), similar to the structure of human LMCD1 [3]. Based on the single
potential N-glycosylation site in the protein, it can be inferred that
the LMCD1 protein may be a glycoprotein.
The putative domain of the protein encoded by porcine LMCD1 and the
3-D structure of the conserved domain of this putative protein are shown in Figs.
7 and 8, respectively. From these two figures, we can find two LIM
zinc-binding domains and a PET domain that is suggested to be involved in
protein-protein interactions, which further validates the correctness of the
current classification of the putative LMCD1 protein.
SSCP analysis
The correctness of the G294A substitute was confirmed by PCR-SSCP.
The size of the primer L3 PCR product was 164 bp. The PCR-SSCP results are
shown in Fig. 9. The distribution of the polymorphism in five different
pig breeds is given in Table 1. We can conclude that only allele G was
found in Large White and Landrace pigs. c2 analysis of three
genotypes in different pig populations showed that the frequency of genotype
was significantly different (c2=128.1200>c20.01(8), P<0.01) in Large White, Landrace, Meishan, Exi Hei and Wannanhua pigs.
Discussion
Muscle LIM protein is composed of two neighboring LIM domains and a
glycine-rich domain [6,17]. It is an important regulator in the development of
skeletal muscle and cardiac muscle. According to the current classification of
LIM domain proteins, LMCD1 belongs to the group 3 proteins, which
contain one or more LIM domains at the C-terminal region [3]. This gene family
is characterized by idiocratic LIM domains and a conserved cysteine-rich motif
mostly expressed in musculature [3,8].
When a correct gene has been identified, there may be more than one
polymorphism within that gene. Polymorphism in the coding sequence that does
not change the amino acid (synonymous mutation) is unlikely to have an effect
on phenotype [18,19]. Two clues are used to predict whether or not a
non-synonymous polymorphism will affect phenotype. First, a mutation that
causes a radical change in the amino acid is more likely to affect the properties
of the protein than a conservative amino acid substitution. Second, amino acids
that are conserved across species are likely to be needed for protein function,
so mutations that change them are likely to affect phenotype [19,20]. Because of
SNPs (single nucleotide polymorphisms) have high density and stability in
genomes. They were widely used in the identification of a functional gene and
location of quantitative trait loci (QTL) SNP occurring in the coding region
of a gene (cSNP) maybe belong to QTL to affect the level of gene expression and
the protein structure, so taking cSNP as a marker would be more available for
maker-assisted selection [14,21]. In our study, three cSNPs were detected by
aligning the cDNA sequences of different porcine breeds, and the correctness
of the G294A substitute was validated by PCR-SSCP. In addition, the
distribution of three genotypes, based on the G294A substitute, was studied in
five pig breeds. The results showed that allele A did not exist in Large White
and Landrace pigs, which may have been affected by long-term breeding and
selection or the limited number of animals in this study. It may also be that
allele A has a special function that affects porcine meat quality. Further
studies must be conducted to confirm whether this site can be regarded as a molecular
marker or not.
Bioinformatics analysis showed the LMCD1 protein was highly
conserved among the different species in this study. Some potential functional
sites predicted in pig LMCD1 were also found in human LMCD1. These results
offer some evidence to further understand the function of porcine LMCD1. Two
means that constructing phylogenetic tree have different substitution models
resulted in dissimilar bootstrap values, all LIM domain protein genes have a similar
location in the two trees, which further validates the correctness of the
classification of the LMCD1 gene.
This is the first report on the LMCD1 gene in pig. We have
obtained its complete coding sequence; we will continue our research to obtain
its full-length sequence, then carry out a functional analysis. Like other LIM domain
proteins with predominant expression in skeletal muscle, the LMCD1 protein
might be involved in protein-protein interactions during muscle development and
remodeling [1,3,8], so further research based on these primary results is
needed.
Acknowledgements
We would like to thank the staff at Jingping Pig Station
(Huazhong Agricultural University) and
teachers and graduate students at the Key Laboratory of Swine Genetics and
Breeding, Ministry of Agriculture for managing and slaughtering experimental
animals.
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