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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 450-456 |
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doi:10.1111/j.1745-7270.2006.00187.x |
Identification of a
Differentially Expressed Gene PPP1CB between Porcine Longissimus�
dorsi of Meishan and Large White�Meishan Hybrids
Tao HUANG, Yuan-Zhu XIONG*,
Ming-Gang LEI, De-Quan XU, and Chang-Yan DENG
Key
Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, Huazhong Agriculture
University, Wuhan 430070, China
Received: March 8,
2006�������
Accepted: April 19,
2006
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30400313), the National High Technology Research and Development Program
of China and the Major State Basic Research Development Program of China
The nucleotide
sequence data reported in this paper have been submitted to GenBank under
accession numbers: DQ396471, DQ396472 and DQ398872
*Corresponding
author: Tel, 86-27-87282849; Fax, 86-27-87394184; E-mail,
[email protected]
Abstract������� To study the molecular basis of heterosis, suppression
subtractive hybridization was used to investigate the differences in gene
expression between porcine Longissimus dorsi of F1 hybrids Large White�Meishan and their female
parents Meishan. From two specific subtractive cDNA libraries, the clones selected
by reverse Northern high-density blot screening were chosen to clone
full-length cDNA by rapid amplification of cDNA ends. An expression-upregulated
gene for Meishan skeletal muscle, designated protein phosphatase 1, catalytic
subunit, beta isoform (PPP1CB), was identified. Porcine PPP1CB
contains an open reading frame encoding 327 amino acid residues with 13 and
1763 nucleotides in the 5' and 3' untranslated regions,
respectively. A DNA fragment of 721 nucleotides was amplified and a mutation
that creates/disrupts a restriction site for endonuclease RsaI was
found. The derived amino acid sequence of PPP1CB has high homology with
the PPP1CB of three species, Mus musculus (99%), human (99%) and
mouse (100%). The tissue expression analysis indicated that the swine PPP1CB
gene is generally expressed in most tissues. The possible role of PPP1CB
and its relation to porcine heterosis are discussed.
Key words������� differential gene expression; pig; heterosis; PPP1CB;
suppression subtractive hybridization
The use of heterosis has achieved great success in agriculture� and is considered essential to meet the world's food needs [1,2]. To reveal the mechanism of heterosis, many hypotheses have been advanced, such as the dominance, overdominance and epistasis hypotheses [3], intergenomic complementation [4], the nucleo-cytoplasmic� interaction and genetic equilibrium hypotheses [5], and the concerted effect of heterozymes [6]. But none of these can perfectly explain heterosis.
The phenomenon of heterosis is in fact the external exhibition� of gene expression and regulation in the heterozygote� [7]. It is necessary to go one step further to understand the molecular mechanisms of heterosis in terms of hybrids' differential gene expression relative to their parents. Recently, by differential display of mRNA and suppression subtractive hybridization (SSH), we have detected� significant differences in mRNA quantity and their expressed patterns between porcine F1 hybrids and their parents [8,9]. Thus, cloning and characterization of genes that are differentially expressed between hybrids and their parents should provide further insight into the molecular mechanisms responsible for heterosis. Here we report the identification of a differentially expressed gene PPP1CB in the Longissimus dorsi between F1 hybrids of Large White�Meishan and their female parents Meishan by SSH.
The reversible phosphorylation of proteins, catalysed by protein kinases and protein phosphatases, is a major mechanism for the regulation of almost all cellular functions, from metabolism to signal transduction, cell division and memory [10]. Phosphatases are classified into two major functional groups, protein tyrosine phosphatases and protein Ser/Thr phosphatases, although functional overlap of various extents is sometimes encountered [11,12].
The physiochemical properties of Ser/Thr phosphatases were used to classify these enzymes into four major classes: PPP1, PPP2A, the Ca2+-calmodulin regulated phosphatase� (PPP2B) and the Mg2+-dependent phosphatase (PPP2C). The catalytic subunits of PPP1 (PPP1C), PPP2A (PPP2AC) and PPP2B belong to the same gene family sharing approximately 40% sequence identity with no structural similarity to PPP2C or protein tyrosine phosphatases [13].
There are four PPP1 isoforms: PPP1a, PPP1b, and two forms of PPP1g, termed PPP1g1 and PPP1g2 [14]. PPP1 regulates many diverse cellular processes [15]. It is the major phosphatase that regulates glycogen meta�bolism in response to insulin and adrenalin [16]. PPP1 also controls� the activity of the sarcoplasmic reticulum Ca2+-ATPase [17], smooth muscle contraction and protein synthesis� [18]. However, relatively little is known about the porcine PPP1C.
In the present study, we describe the cloning and expression profile of porcine PPP1C. Additionally, part of the genomic sequence and polymorphisms of porcine PPP1C were cloned and characterized.
Materials and Methods
Animals
All animals used in the study were derived from the cross-experiments conducted in Huazhong Agriculture University Jingpin Pig Station (Wuhan, China).
SMART cDNA synthesis, SSH and
reverse Northern screening
Total RNAs were isolated with
Trizol Reagent (Gibco, Grand Island, USA) from the Longissimus dorsi of
six F1 hybrids of Large White�Meishan (three male and three female) of 4
months old, and six pigs (three male and three female) at the same age of
Meishan, the female parent of the cross-experiment, and were mixed to form RNA
pools. Poly(A)+ RNA was purified by biotinylated oligo(dT) probe and
streptavidin-bound magnetic particles (Promega, Madison, USA). SMART cDNA was
synthesized using the SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, USA)
for rapid amplification of cDNA end (RACE)-polymerase chain reaction (PCR). SSH
was carried out as described� in the PCR-Select cDNA Subtraction Kit
(Clontech). In the forward subtraction, the cDNA from Meishan was used as the
tester, and the cDNA from Large White�Meishan hybrid as the driver;
in the reverse subtraction, the cDNA from Large White�Meishan hybrid was used as the
tester, and the cDNA from Meishan as the driver. The differentially expressed
PCR products were inserted into pGEM-T vector (Promega) and transformed into Escherichia
coli DH5a, then cultured on LB plates
containing 50 mg/ml of ampicillin, 0.4 mM of
X-gal and 0.4 mM of isopropyl b-D-thiogalactopyranoside.
White colonies were picked up and 1 ml of
each bacterial LB culture was amplified in a 25 ml
PCR system using nested PCR primers 1 and 2R (Clontech). PCR products were then
denatured and alkaline blotted onto two Hybond-N+ positively charged
nylon membranes (Roche, Mannheim, Germany) in parallel [19]. The two nylon
membranes were hybridized with forward-subtracted and reverse-unsubtracted
probes. Reverse Northern high-density blot screening was carried out according
to the procedure of the DIG High Prime Labeling and Detection Starter Kit I
(Roche). The positive clones were sequenced using vector�-specific primers.
Reverse transcription (RT)-PCR
Total RNA derived from the Longissimus dorsi of additional three F1 hybrids of Large White�Meishan and their female parents Meishan was used as the starting material, and cDNA was synthesized as the PCR template using Moloney murine leukemia virus reverse transcriptase and the oligo(dT) primer (Promega). A specific primer pair G3PDH-F/G3PDH-R (Table 1) that amplified the 476 bp housekeeping gene G3PDH was used as the internal control. The differentially expressed clone, MS140, was detected by the specific primers 140F and 140R (Table 1).
For spatial expression analysis of MS140, total RNA was also isolated from various tissues (heart, liver, spleen, lung, kidney, adipose tissue, Longissimus dorsi, embryo, testis, uterus, ovary) and cDNAs were synthesized.
RACE-PCR
To obtain the 5' full-length
cDNA, an oligonucleotide primer 140-1R (Table 1) complementary to the 3'
end of the cDNA was used in combination with the SMART 5' primer (Table
1), designed against SMART II oligo�nucleotide from the SMART PCR cDNA
Synthesis Kit to amplify the 5' end of the gene, using SMART amplified
cDNA from pigs' Longissimus dorsi as the template. PCR was carried out
in a GeneAmp PCR System 9600 (Perkin Elmer, Ramsey, USA) with the following
cycling parameters: 95 �C initial denaturation for 4 min; 35 cycles of 95 �C
denaturation� for 40 s, 56 �C annealing for 40 s, and 72 �C extension for 1.5
min; followed by a 10 min extension at 72 �C.
To obtain the 3' full-length cDNA, primer 140-1F (Table 1) was used in combination with the SMART 3' primer (Table 1), designed against CDS III 3' PCR Primer from the SMART PCR cDNA Synthesis Kit to amplify the 3' end of the cDNA. PCR was carried out with the cycling parameters: 95 �C initial denaturation for 4 min; 35 cycles of 95 �C denaturation for 50 s, 54 �C annealing for 50 s, and 72 �C extension for 2 min; followed by a 10 min extension� at 72 �C.
Database and sequence analysis
The full-length nucleotide sequence of the porcine PPP1CB was compared with GenBank at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/Genbank/index.html) using BLASTN and BLASTX searches of the "nr" database. The deduced amino acid sequence was analyzed using the ExPASy Molecular Biology Server (http://cn.expasy.org/). Multiple sequence alignments and construction of the unrooted phylogenetic tree were carried out using the CLUSTALW 1.83 program (http://www.ebi.ac.uk/clustalw/index.html).
Genomic PCR and identification
of gene polymorphisms
One primer pair (140-2F and 140-2R) was designed (Table 1) to amplify a fragment of the PPP1CB gene from the porcine genome. The optimal cycling para�meters were 95 �C initial denaturation for 4 min; 35 cycles of 95 �C denaturation for 40 s, 55 �C annealing for 40 s, and 72 �C extension for 45 s; followed by a 10 min extension at 72 �C. The products were cloned into the pGEM-T cloning� vector and sequenced using vector-specific primers. Polymorphisms� were detected based on sequence comparison.
Results
Identification of MS140
as an upregulated gene in Longissimus dorsi from Meishan
In order to isolate differential genes between F1 hybrids� of Large White�Meishan and their female parents Meishan, forward and reverse subtractive cDNA libraries were constructed. By reverse Northern high-density blot screening� of more than 600 clones randomly picked up from subtractive libraries, one clone, designated MS140, demonstrated high-level expression in Longissimus dorsi for Meishan [Fig. 1(A)]. RT-PCR analysis was used to confirm differential expression of MS140 [Fig. 1(B)].
Cloning of the full-length
cDNA of porcine skeletal muscle
The differentially expressed clone (MS140) was found to have 97% homology to the mRNA sequence of Bos taurus gene for Ser/Thr protein phosphatase PPP1b catalytic subunit. Further sequence analysis indicated that the cDNA fragment of MS140 (GenBank accession No. DQ396472) contained part of the CDS and part of the 3' untranslated region (UTR) of the cDNA (72 bp of predicted translated sequence+525 bp UTR=597 bp in total). One PCR fragment of approximately 1.5 kb was amplified by 5'-RACE [Fig. 2(A)]. There were several fragments amplified by 3'-RACE and the brightest one of approximately 1.7 kb was reamplified [Fig. 2(B)]. These products� were then cloned to T vectors and sequenced. A 2759 bp complete cDNA sequence of PPP1CB was identified (GenBank accession No. DQ396471) and shared a 100% similarity with the CDS of swine PPP1CB (NM214184). Porcine PPP1CB cDNA contains an open reading frame of 984 nucleotides, encoding a protein of 327 amino acids (Fig. 3). We inferred the ATG codon at nucleotide 14-16 to be the true start site of translation, because it begins the longest reading frame, which is homological to that of other species. In addition, the putative methionine initiation� codon occurs in a favorable sequence context for the initiation� of translation with a purine in the -3 position and a G in the +4 position [20]. A polyadenylation signal, AATAA, was found at nucleotide 2272-2276 located upstream of the poly(A) tract. Another polyadenylation signal, AGTAAA, was found just before the poly(A) tract. Five AU motifs (ATTTA) associated with the instability of mRNA were found in the 3' UTR.
Analysis of the PPP1CB
protein sequence
The conceptual translation product of the porcine PPP1CB transcript is a peptide of 327 amino acids. Comparison with three reported molecules of other species shows an extraordinary high level of similarity (99%-100% identity overall). The amino acid sequence of PPP1CB is highly conserved among species. The differences between the protein encoded by this gene in pig and three other spieces are shown in Table 2.
Analysis of phylogenetic tree
A phylogenetic tree was constructed using the porcine PPP1CB sequence from our study and other recently available� sequences in the database, using DNASTAR software, as shown in Fig. 4. The phylogenetic tree analysis revealed that the swine PPP1CB has a closer genetic relationship� with the PPP1CB of gallus, rabbit, rat, chick, than with those of human, mouse, dog, frog and danio.
Tissue expression profile
The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of pigs as the templates, and the result revealed that the swine PPP1CB gene was not only expressed in Longissimus dorsi muscle, but also expressed in the heart, liver, spleen, lung, kidney, adipose tissue, testis, uterus and embryo (Fig. 5).
Polymorphisms of the porcine PPP1CB
To search for different PPP1CB alleles within the pig populations, part of the 3' UTR of PPP1CB was investigated in three different individuals representing three pig breeds (Large White, Landrace and Chinese Meishan). A DNA fragment of 721 nucleotides was amplified and sequenced� (GenBank accession No. DQ398872). Two potential� polymorphic sites were identified and a mutation created/disrupted a restriction site for endonuclease RsaI (Fig. 6). We analysed this polymorphism in purebreds and crossbreds (Large White, Meishan, Landrace and Large White�Meishan) by means of the PCR-restriction fragment� length polymorphism technique (Fig. 7) using primer pairs 140-WF and 140-WR (Table 1). Allele AA was not found. Allele BB was highly frequent in Meishan and Landrace. Allele AB was highly frequent in F1 hybrids Large White�Meishan (Table 3).
Discussion
SSH is a recently developed method for identifying differentially� expressed genes between two different mRNA populations. The efficiency and reproducibility of SSH has been proven in different studies for differentially expressed genes. SSH combined with the high throughput reverse Northern screening method permits the efficient and rapid cloning of dozens to hundreds of differentially expressed genes in one experiment [21]. It was reported that the rate range of the positive cDNA clones obtained by SSH was from 10% to 90% [22], which is higher than differential display PCR [23] and representational difference analysis [24] methods.
The level of protein phosphorylation is controlled by the opposing and coordinated activities of protein kinases and phosphatases that catalyse protein phosphorylation and dephosphorylation, respectively. PPP1 is a major eukaryotic protein Ser/Thr phosphatase that regulates diverse cellular processes such as cell-cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling [25]. Therefore the activity of PPP1C including PPP1CB is very important for protein phosphorylation and the regulation of many physiological processes. Thus, the downregulated expression� observed in the F1 hybrids suggests that the phosphorylation process, mediated through PPP1CB, might be a candidate molecular pathway for influencing porcine heterosis. In addition, in Meishan pigs, there is a high frequency of the B allele, and 22 of 24 animals are BB genotypes (Table 3). Meishan is also the breed showing the higher level of expression of PPP1CB, so it is also possible that there is a link between the presence of the B allele and the upregulation of gene expression in Meishan pigs. Of course, it is also possible that PPP1CB down�regulated expression is linked to a quantitative trait locus and that the amount of PPP1CB mRNA has no direct effect on porcine heterosis. All of these points need further verification.
It would be premature to speculate how changes in PPP1CB in hybrids might affect heterosis, but alterations in the reversible phosphorylation system might be important for other modifications in patterns of gene expression in hybrids that affect heterosis in their turn. To further understand the function of the gene, more research based on these primary results is needed.
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