Sarcomeric TPM3α in developing chicken

Abstract

Cloning and sequencing of various tropomyosin isoforms expressed in chickens have been described since the early 1980s. However, to the best of our knowledge, this is the first report on the molecular characterization and the expression of the sarcomeric isoform of the TPM3 gene in cardiac and skeletal muscles from developing as well as adult chickens. Expression of TPM3α was performed by conventional RT-PCR as well as qRT-PCR using relative expression (by ΔC_T as well as ΔΔC_T methods) and by determining absolute copy number. The results employing all these methods show that the expression level of TPM3α is maximum in embryonic (10-day/15-day old) skeletal muscle and can barely be detected in both cardiac and skeletal muscles from the adult chicken. Our various RT-PCR analyses suggest that the expression of high molecular weight TPM3 isoforms are regulated at the transcription level from the proximal promoter at the 5'-end of the TPM3 gene.

Keywords: TPM3α; TPM3ν; developmentally regulated; qRT-PCR.

Figure 1. Alternative splicing pattern of the TPM3 gene in chicken

Exon composition of the TPM3 gene is derived from the published documents, the recently submitted data in Gen Bank (Accession# MF347378), and the predicted sequence available in Gen Bank (Accession # XM_015298570). Exons are shown in boxes.

Figure 2. Conventional RT-PCR amplification of TPM3a with RNA from developing chicken heart and skeletal muscle

Primer-pairs were designed from the predicted TPM3 variant #7 sequence (GenBank accession # XM_015298570) Panel a. Ethidium bromide staining of the amplified products of TPM3α Panel b. Long exposure. Hybridization with TPM3α probe, the sequences of which are in Table 1. Panel c. Short exposure for the Southern hybridization. Panel d. Ethidium bromide staining of the amplified products of GAPDH cDNAs were made with oligo dT Lane 1. Skeletal muscle from 7-day old embryos; Lane 2. Skeletal muscle from 10-day old embryos; Lane 3: Skeletal muscle from 15-day old embryos; Lane 4. Skeletal muscles from 15-day post hatch chicken; Lane 5. Skeletal muscle from adult chicken; Lane 6.Heart from 7-day old embryos; Lane 7. Heart from 10-day old embryos; Lane 8. Heart from 15-day old embryo; Lane 9. Heart from 15-day post hatch chicken; Lane 10. Heart from adult chicken; Lane 11. Primer control.

Figure 3. Nucleotide and deduced amino acid sequence of TPM3α

The nucleotide sequence has been submitted in the GenBank (Submission Id. 2024828 MF347378)

Figure 4. Conventional RT-PCR amplification of TPM3β with RNA from developing chicken heart and skeletal muscle

Panel a. Ethidium bromide staining of the amplified products of TPM3β Panel b. Hybridization with TPM3β probe, the sequences of which are in Table 1. Panel c. Ethidium bromide staining of the amplified products of GAPDH. cDNAs were made with oligo dT Lane 1. Skeletal muscle from 7-day old embryos; Lane 2. Skeletal muscle from 10-day old embryos; Lane 3: Skeletal muscle from 15-day old embryos; Lane 4. Skeletal muscles from 15-day post hatch chicken; Lane 5. Skeletal muscle from adult chicken; Lane 6.Heart from 7-day old embryos; Lane 7. Heart from 10-day old embryos; Lane 8. Heart from 15-day old embryos; Lane 9. Heart from 15-day post hatch chicken; Lane 10. Heart from adult chicken; Lane 11. Primer control.

Figure 5. Relative expression of TPM3α in heart and skeletal muscle from developing, 15-day post hatch, and adult chicken

a. Relative expression using ΔC_T method b. Relative expression using ΔΔC_T method E7: 7-day old embryos; E10: 10-day old embryos; E15: 15-day old embryos; P15: 15 day post hatch; Adult. cDNA was synthesized using gene-specific oligonucleotide Chi.TPM3-9b(-).qRT-PCR was carried out in triplicate with isoform specific primer-pair (Chi.TPM3α.qRT (F)/Chi.TPM3α.qRT(R) sequences of which are given in Table 1. The primer used for18S rRNA is shown in Table 1. c. Melt curves of qRT-PCR of chicken TPM3a show a single melting point. The multiple curves represent the products from multiple replicates of the RT-PCR assay. d. PCR products separated by agarose gel electrophoresis and subsequently stained with ethidium bromide. The results demonstrate amplification of a single product forTPM3α. Lane 1. Skeletal muscle from 7-day old embryos; Lane 2. Skeletal muscle from 10-day old embryos; Lane 3: Skeletal muscle from 15-day old embryos; Lane 4. Skeletal muscles from 15-day post hatch chicken; Lane 5. Skeletal muscle from adult chicken; Lane 6. Heart from 7-day old embryos; Lane 7. Heart from 10-day old embryos; Lane 8. Heart from 15-day old embryo; Lane 9. Heart from 15-day post hatch chicken; Lane 10. Heart from adult chicken; Lane 11. Primer control.

Figure 6. Quantitative RT-PCR of chicken TPM3α by determining absolute copy number

a. Standard curve derived from amplification of chicken TPM3α plasmid clone DNA after serial dilution. Copy number of TPM3α was calculated per microgram of total RNA from heart and skeletal muscle from developing, post hatch, and adult chicken. b. Number of copies of TPM3α transcripts per μg of total RNA from heart and skeletal muscle from embryonic, post hatch, and adult chicken. E7: 7-day old embryos; E10: 10-day old embryos; E15: 15-day old embryos; P15: 15 day post hatch; Adult.

Figure 7. Conventional RT-PCR amplification of total high molecular weight TPM3 transcripts with RNA from developing chicken heart and skeletal muscle

The positive and negative primer pairs were designed from exon 1a and exon 3 respectively. The hybridization was carried out with a oligonucleotide from exon 3 of the TPM3 gene. The nucleotide sequences of the primer-pair are given in Table 1. cDNA was made with oligo dT. Panel a. Ethidium bromide staining of the amplified total high molecular weight TPM3 transcripts. Panel b. Short exposure of hybridization with total high molecular weight TPM3 probe, the sequences of which are available in Table 1. Panel c. Long exposure for the Southern hybridization. Lane 1. Skeletal muscle from 7-day old embryos; Lane 2. Skeletal muscle from 10-day old embryos; Lane 3: Skeletal muscle from 15-day old embryos; Lane 4. Skeletal muscles from 15-day post hatch chicken; Lane 5. Skeletal muscle from adult chicken; Lane 6.Heart from 7-day old embryos; Lane 7. Heart from 10-day old embryos; Lane 8. Heart from 15-day old embryos; Lane 9. Heart from 15-day post hatch chicken; Lane 10. Heart from adult chicken; Lane 11. Primer control.