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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.


Expression analysis of TPM1α and TPM1κ RNA in mouse heart and skeletal muscle by RT-PCR. Plate (a) illustrates the ethidium bromide stain. Plate (b) illustrates hybridization with TPM1α probe. Plate (c) illustrates hybridization with TPM1κ probe. Lane 1: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 2: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 3: heart cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 4: heart cDNA amplified with TPM1α (+)/Mus TPM1α/κ-2 (−). Lane 5: heart cNDA amplified with Mus TPM1κ-1 (+)/Mus UTR (−). Lane 6: primer control for Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 7: primer control for Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 8: primer control for Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 9: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 10: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 11: skeletal cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 12: skeletal cDNA amplified with Mus TPM1α-1 (+)/Mus TPM1α/κ-2 (−).
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fig2: Expression analysis of TPM1α and TPM1κ RNA in mouse heart and skeletal muscle by RT-PCR. Plate (a) illustrates the ethidium bromide stain. Plate (b) illustrates hybridization with TPM1α probe. Plate (c) illustrates hybridization with TPM1κ probe. Lane 1: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 2: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 3: heart cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 4: heart cDNA amplified with TPM1α (+)/Mus TPM1α/κ-2 (−). Lane 5: heart cNDA amplified with Mus TPM1κ-1 (+)/Mus UTR (−). Lane 6: primer control for Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 7: primer control for Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 8: primer control for Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 9: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 10: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 11: skeletal cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 12: skeletal cDNA amplified with Mus TPM1α-1 (+)/Mus TPM1α/κ-2 (−).

Mentions: Figure 2 depicts the results of conventional RT-PCR analysis of TPM1α and TPM1κ with cDNAs synthesized from mouse heart and skeletal muscle total RNA (provided by Dr. David Wieczroek), using a variety of generic as well as isoform specific primer pairs. Panel (a) in Figure 2 represents the ethidium stained PCR products run on a 1.5% agarose gel. Lanes 1 and 2 show the PCR products amplified from heart cDNA with two different primer pairs, which amplify both TPM1α and TPM1κ. Southern hybridization with TPM1α-specific probe demonstrated stronger signals in both lanes (lanes 1 and 2, panel (b)) compared to hybridization with TPM1κ-specific probe (lanes 1 and 2, panel (c)). The weaker signals in lanes 1 and 2 in panel (c) compared to panel (b) indicate much lower expression level of TPM1κ compared to TPM1α. Lanes 3 and 4 in panel (a) represent the ethidium staining of the PCR products when amplified with TPM1κ or TPM1α-specific primer pairs, respectively. Southern hybridization with TPM1α-specific probe shows a strong hybridization signal (lane 4, panel (b)) with the TPM1α-specific primer pair. A strong hybridization signal was also detected with TPM1κ-specific probe (lane 3, panel (c)) with the TPM1κ-specific primer pair. The results indicate that both TPM1α and TPM1κ RNA are expressed in mouse heart. This was further confirmed by amplification with another TPM1κ-specific primer pair and subsequent southern hybridization with TPM1κ-specific probe (lane 5, panels (a), (b), and (c)). All primer controls were negative (lanes 6, 7, and 8). Lanes 9 and 10 represent the PCR amplicons of mouse skeletal muscle cDNA with two sets of TPM1α/κ primer pairs. Ethidium bromide stained gels indicate a strong expression of TPM1 in skeletal muscle (panel (a)). Hybridization signal with TPM1α-specific probe (panel (b)) versus the TPM1κ-specific probe (panel (c)) indicates a much stronger expression of the former. However, amplification of skeletal muscle cDNA with TPM1κ-specific primer pair (lane 11 in panel (a)) and subsequent hybridization with TPM1κ-specific probe (lane 11 in panel (c)) validate the expression of TPM1κ in mouse skeletal muscle. Similarly, the expression of TPM1α in skeletal muscle was authenticated by the PCR amplification with TPM1α-specific primer pair (lane 12, panel (a)) and by subsequent hybridization with TPM1α-specific probe (lane 12, panel (b)). A much lower expression level of TPM1κ compared to TPM1α in mouse heart and skeletal muscle was further substantiated by qRT-PCR as described below. Similar conventional RT-PCR results were obtained from the RNA procured from Stratagen and Biochain (data not shown).


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)

Expression analysis of TPM1α and TPM1κ RNA in mouse heart and skeletal muscle by RT-PCR. Plate (a) illustrates the ethidium bromide stain. Plate (b) illustrates hybridization with TPM1α probe. Plate (c) illustrates hybridization with TPM1κ probe. Lane 1: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 2: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 3: heart cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 4: heart cDNA amplified with TPM1α (+)/Mus TPM1α/κ-2 (−). Lane 5: heart cNDA amplified with Mus TPM1κ-1 (+)/Mus UTR (−). Lane 6: primer control for Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 7: primer control for Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 8: primer control for Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 9: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 10: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 11: skeletal cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 12: skeletal cDNA amplified with Mus TPM1α-1 (+)/Mus TPM1α/κ-2 (−).
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fig2: Expression analysis of TPM1α and TPM1κ RNA in mouse heart and skeletal muscle by RT-PCR. Plate (a) illustrates the ethidium bromide stain. Plate (b) illustrates hybridization with TPM1α probe. Plate (c) illustrates hybridization with TPM1κ probe. Lane 1: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 2: heart cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 3: heart cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 4: heart cDNA amplified with TPM1α (+)/Mus TPM1α/κ-2 (−). Lane 5: heart cNDA amplified with Mus TPM1κ-1 (+)/Mus UTR (−). Lane 6: primer control for Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 7: primer control for Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 8: primer control for Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 9: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus UTR (−). Lane 10: skeletal cDNA amplified with Mus TPM1α/κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 11: skeletal cDNA amplified with Mus TPM1κ-1 (+)/Mus TPM1α/κ-2 (−). Lane 12: skeletal cDNA amplified with Mus TPM1α-1 (+)/Mus TPM1α/κ-2 (−).
Mentions: Figure 2 depicts the results of conventional RT-PCR analysis of TPM1α and TPM1κ with cDNAs synthesized from mouse heart and skeletal muscle total RNA (provided by Dr. David Wieczroek), using a variety of generic as well as isoform specific primer pairs. Panel (a) in Figure 2 represents the ethidium stained PCR products run on a 1.5% agarose gel. Lanes 1 and 2 show the PCR products amplified from heart cDNA with two different primer pairs, which amplify both TPM1α and TPM1κ. Southern hybridization with TPM1α-specific probe demonstrated stronger signals in both lanes (lanes 1 and 2, panel (b)) compared to hybridization with TPM1κ-specific probe (lanes 1 and 2, panel (c)). The weaker signals in lanes 1 and 2 in panel (c) compared to panel (b) indicate much lower expression level of TPM1κ compared to TPM1α. Lanes 3 and 4 in panel (a) represent the ethidium staining of the PCR products when amplified with TPM1κ or TPM1α-specific primer pairs, respectively. Southern hybridization with TPM1α-specific probe shows a strong hybridization signal (lane 4, panel (b)) with the TPM1α-specific primer pair. A strong hybridization signal was also detected with TPM1κ-specific probe (lane 3, panel (c)) with the TPM1κ-specific primer pair. The results indicate that both TPM1α and TPM1κ RNA are expressed in mouse heart. This was further confirmed by amplification with another TPM1κ-specific primer pair and subsequent southern hybridization with TPM1κ-specific probe (lane 5, panels (a), (b), and (c)). All primer controls were negative (lanes 6, 7, and 8). Lanes 9 and 10 represent the PCR amplicons of mouse skeletal muscle cDNA with two sets of TPM1α/κ primer pairs. Ethidium bromide stained gels indicate a strong expression of TPM1 in skeletal muscle (panel (a)). Hybridization signal with TPM1α-specific probe (panel (b)) versus the TPM1κ-specific probe (panel (c)) indicates a much stronger expression of the former. However, amplification of skeletal muscle cDNA with TPM1κ-specific primer pair (lane 11 in panel (a)) and subsequent hybridization with TPM1κ-specific probe (lane 11 in panel (c)) validate the expression of TPM1κ in mouse skeletal muscle. Similarly, the expression of TPM1α in skeletal muscle was authenticated by the PCR amplification with TPM1α-specific primer pair (lane 12, panel (a)) and by subsequent hybridization with TPM1α-specific probe (lane 12, panel (b)). A much lower expression level of TPM1κ compared to TPM1α in mouse heart and skeletal muscle was further substantiated by qRT-PCR as described below. Similar conventional RT-PCR results were obtained from the RNA procured from Stratagen and Biochain (data not shown).

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.