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Isolation and characterization of 5’ upstream regulatory region of survival motor neuron gene

University of British Columbia

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder characterized by loss or degeneration of lower motor neurons in the spinal cord, leading to progressive symmetrical limb and trunk paralysis and muscular atrophy. The disease has been classified into three types and mapped to chromosome 5ql3. The telomeric survival motor neuron (SMNt) gene has been identified to be the SMA determining gene. The centromeric SMN (SMNc) may be related to the SMA phenotype. Five nucleotides differences between SMNt and SMNc do not affect the encoded protein. SMN proteins are distributed widely in all tissues with highest concentration in CNS and liver. Little is known about the regulatory mechanism of the SMN gene in cells. To identify the regulatory sequence elements for the expression of the SMN gene in both neuronal and non-neuronal cells, we cloned a 3132 bp fragment of 5' upstream region of SMN gene isolated from the chromosome 5 library into the luciferase reporter gene system. Serial deletion constructs containing various lengths of 5-flanking region of SMN gene were transfected into both SY5Y and Vero cells. The results of the promoter activity assay showed that 1) The reporter gene could be expressed in both neuronal and non-neuronal cells; 2) 48 potential binding sites for transcription factors were localized in the region from -1530 to +34; 3) The region from -157 to +114 displayed the highest promoter activity while the lowest activity was shown in the region from - 28 to +114; 4) The 5' upstream region beyond - 899 showed lower promoter activity than that of more approximal region (-306 to +114); 5) The promoter was more active in the neuronal cells in general than that in the non-neuronal cells, especially when the region contained more upstream sequences. The above results suggested that the SMN gene might be well regulated by many potential transcription factors. A negative regulatory element might be located in the region between -899 to -306 and the region between -157 to +114 might be critical for the SMN gene expression.

Thesis/Dissertation

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2009-06-17

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http://hdl.handle.net/2429/9380

Surgery

University of British Columbia

UBCV

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