Date of Award

1994

Document Type

Thesis

Publisher

Edith Cowan University

Degree Name

Bachelor of Applied Sciences Honours

Faculty

Faculty of Science and Technology

First Supervisor

Chris Meredith

Second Supervisor

Dr Nigel Liang

Abstract

Tropomyosin is one of the components of the thin filaments of muscle, binding to actin, and, together with troponin, regulating contraction in a calcium-dependent manner (Cho et al.,1990). There are at least four distinct tropomyosin genes in vertebrates and each may encode at least six different isoforms of tropomyosin by alternate splicing (Novy et al, 1993; MacLeod et al., 1988). The alpha-tropomyosin gene TPM1 has recently been localised to 15q22 (Eyre et al, 1994) and has been shown to be mutated in some cases of familial hypertrophic cardiomyopathy (Thierfelder et al., 1994). The alpha-tropomyosin gene TPM3 has been recently localised to 1q22-q23 (Wilton et al, 1994) and has been shown to be mutated in a family with autosomal dominant nemaline myopathy (Laing, 1994, unpublished observations). Each muscle-specific gene is possibly associated with an inherited muscle disease, if there is a disease causing mutation in the gene. Precise mapping of muscle genes therefore becomes important in relation to mapping muscle diseases (Eyre et al., 1993). A sequence tagged site (STS) (Olson et al., 1989) was developed for the human beta tropomyosin gene (TPM2). The STS was used to amplify DNA from somatic cell hybrids to localise TPM2 to human chromosome 9. Genomic clones isolated with the STS product were in tum used in fluorescent in situ hybridisation (Callen et al., 1992) to metaphase chromosome spreads to further localise TPM2 to 9pl3.1 (Hunt et al., 1995). This project should assist those laboratories searching for candidate genes of inherited muscular diseases that are linked within the region of the TPM2 gene and may assist in the precise diagnosis of people with these diseases (Akkari, 1994). Localising TPM2 also lays a foundation for a better understanding of the role of tropornyosins in muscle and nonmuscle cells. A polymorphism was also discovered in the 3'UTR of TPM2, using single stranded conformation analysis (SSCA). The primers used were 5'-AAGTCTATGCCAGAAGATG-3' and the complementary strand 5'-CCGTGACCGAAGTAGGAAAT-3' creating a 259 bp sequence tagged site (STS). Sequencing of the STS revealed that there were two variations of the 11th base in the 3'UTR: a guanine and an adenine. Genomic DNA from 97 unrelated individuals was screened by SSCA and the allelic frequency was determined to be for the common allele (guanine) 0.91 and the rare allele (adenine) 0.09. The heterozygosity was 0.16. There is a Bgl I restriction site at the common allele polymorphic location. A Bgl I restriction digest of the 259 bp STS produces two fragments (88 & 171 bp) for homozygous individuals with the common allele, three fragments (88, 171 and 259 bp) for heterozygous individuals and a single fragment (259 bp) for homozygous individual with the rare allele. The discovery of this polymorphism will be entered on the CEPH map and is useful for researchers as a linkage and physical marker in the human genome project, especially since the TPM2 gene has now been localised to 9p13.1.

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