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Expression of TPM1κ, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle.

Dube S, Panebianco L, Matoq AA, Chionuma HN, Denz CR, Poiesz BJ, Dube DK - Mol Biol Int (2014)

Bottom Line: We have cloned the PCR amplified DNA and determined the nucleotide sequences.Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1 κ is significantly lower compared to TPM1 α in both mouse heart and skeletal muscle.To the best of our knowledge, this is the first report of the expression of TPM1 κ in mammalian skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

ABSTRACT
We have investigated the expression of TPM1 α and TPM1 κ in mouse striated muscles. TPM1 α and TMP1 κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1 κ . However, the deduced amino acid sequences of human TPM1 α and mouse TPM1 α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1 κ is significantly lower compared to TPM1 α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1 κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1 κ in mammalian skeletal muscle.

No MeSH data available.


Alternate splicing of the TPM1 gene and exon composition of various TM isoforms. (a) Splicing pattern of the TPM1 gene and the strategy for amplification of TPM1α and TPM1κ. Arrows indicate the primer pairs used for amplification of TPM1α and TPM1κ by RT-PCR and subsequent southern hybridization. The nucleotide sequences of various primer pairs and probes are listed in Table 1. (b) Exon composition of various TPM isoforms.
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fig1: Alternate splicing of the TPM1 gene and exon composition of various TM isoforms. (a) Splicing pattern of the TPM1 gene and the strategy for amplification of TPM1α and TPM1κ. Arrows indicate the primer pairs used for amplification of TPM1α and TPM1κ by RT-PCR and subsequent southern hybridization. The nucleotide sequences of various primer pairs and probes are listed in Table 1. (b) Exon composition of various TPM isoforms.

Mentions: Figure 1 illustrates the pattern of alternative splicing of the two sarcomeric TPM1 isoforms. TPM1α contains exons 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, whereas TPM1κ has all the same exons except it contains exon 2a instead of exon 2b. The figure also depicts the strategy for amplification of the two TPM1 isoforms. Primer pair P1(+)/P2(−) was used to amplify both TPM1α and TPM1κ. Amplification by the primer pairs P3(+)/P2(−) and P4(+)/P2(−) will yield TPM1α and TPM1κ, respectively. Subsequently, an isoform-specific [32P]-labeled probe was used for southern hybridization of TPM1α or TPM1κ. The sequences of various primer pairs as well as probes are shown in Table 1. We amplified TPM1α and TPM1κ by RT-PCR with cDNA from mouse heart muscle using the mus TPM1α/κ primer pair. The amplified DNA was run on a 1.5% agarose gel and the DNA band of ~850 bp was extracted using MinElute Gel Extraction kit of QIAGEN. The eluted DNA was ligated to T/A cloning vector (Invitrogen) following the manufacturer's specification as described earlier [10]. The ligation mix was used for transformation of competent one shot E. coli cells (Invitrogen) using the protocol supplied by Invitrogen. Appropriate hybridization positive colonies were picked up from the plates after filter hybridization with [32P]-labeled probe. Exon 2a specific probe was used for TPM1κ and exon 2b specific probe was used for TPM1α. Colonies were grown overnight with LB medium containing appropriate concentration of antibiotic and plasmid DNA was isolated using QIAprep Spin Miniprep kit (QIAGEN). Isolated plasmid DNA was sequenced at the Cornell University DNA sequencing facility (Figure 5).


Expression of TPM1κ, a Novel Sarcomeric Isoform of the TPM1 Gene, in Mouse Heart and Skeletal Muscle.

Dube S, Panebianco L, Matoq AA, Chionuma HN, Denz CR, Poiesz BJ, Dube DK - Mol Biol Int (2014)

Alternate splicing of the TPM1 gene and exon composition of various TM isoforms. (a) Splicing pattern of the TPM1 gene and the strategy for amplification of TPM1α and TPM1κ. Arrows indicate the primer pairs used for amplification of TPM1α and TPM1κ by RT-PCR and subsequent southern hybridization. The nucleotide sequences of various primer pairs and probes are listed in Table 1. (b) Exon composition of various TPM isoforms.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Alternate splicing of the TPM1 gene and exon composition of various TM isoforms. (a) Splicing pattern of the TPM1 gene and the strategy for amplification of TPM1α and TPM1κ. Arrows indicate the primer pairs used for amplification of TPM1α and TPM1κ by RT-PCR and subsequent southern hybridization. The nucleotide sequences of various primer pairs and probes are listed in Table 1. (b) Exon composition of various TPM isoforms.
Mentions: Figure 1 illustrates the pattern of alternative splicing of the two sarcomeric TPM1 isoforms. TPM1α contains exons 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, whereas TPM1κ has all the same exons except it contains exon 2a instead of exon 2b. The figure also depicts the strategy for amplification of the two TPM1 isoforms. Primer pair P1(+)/P2(−) was used to amplify both TPM1α and TPM1κ. Amplification by the primer pairs P3(+)/P2(−) and P4(+)/P2(−) will yield TPM1α and TPM1κ, respectively. Subsequently, an isoform-specific [32P]-labeled probe was used for southern hybridization of TPM1α or TPM1κ. The sequences of various primer pairs as well as probes are shown in Table 1. We amplified TPM1α and TPM1κ by RT-PCR with cDNA from mouse heart muscle using the mus TPM1α/κ primer pair. The amplified DNA was run on a 1.5% agarose gel and the DNA band of ~850 bp was extracted using MinElute Gel Extraction kit of QIAGEN. The eluted DNA was ligated to T/A cloning vector (Invitrogen) following the manufacturer's specification as described earlier [10]. The ligation mix was used for transformation of competent one shot E. coli cells (Invitrogen) using the protocol supplied by Invitrogen. Appropriate hybridization positive colonies were picked up from the plates after filter hybridization with [32P]-labeled probe. Exon 2a specific probe was used for TPM1κ and exon 2b specific probe was used for TPM1α. Colonies were grown overnight with LB medium containing appropriate concentration of antibiotic and plasmid DNA was isolated using QIAprep Spin Miniprep kit (QIAGEN). Isolated plasmid DNA was sequenced at the Cornell University DNA sequencing facility (Figure 5).

Bottom Line: We have cloned the PCR amplified DNA and determined the nucleotide sequences.Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1 κ is significantly lower compared to TPM1 α in both mouse heart and skeletal muscle.To the best of our knowledge, this is the first report of the expression of TPM1 κ in mammalian skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

ABSTRACT
We have investigated the expression of TPM1 α and TPM1 κ in mouse striated muscles. TPM1 α and TMP1 κ were amplified from the cDNA of mouse heart by using conventional RT-PCR. We have cloned the PCR amplified DNA and determined the nucleotide sequences. Deduced amino acid sequences show that there are three amino acid changes in mouse exon 2a when compared with the human TPM1 κ . However, the deduced amino acid sequences of human TPM1 α and mouse TPM1 α are identical. Conventional RT-PCR data as well as qRT-PCR data, calculating both absolute copy number and relative expression, revealed that the expression of TPM1 κ is significantly lower compared to TPM1 α in both mouse heart and skeletal muscle. It was also found that the expression level of TPM1 κ transcripts in mouse heart is higher than it is in skeletal muscle. To the best of our knowledge, this is the first report of the expression of TPM1 κ in mammalian skeletal muscle.

No MeSH data available.