Molecular cloning of cDNA for a nuclear matrix protein

Hameed, Khwaja Shahid (1993) Molecular cloning of cDNA for a nuclear matrix protein. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The eukaryotic nucleus is organized around a proteinaceous structural framework termed the nuclear matrix. The precise way in which nuclear proteins are ordered to form a nuclear scaffolding remains to be established. In order to study structural and functional aspects of nuclear matrix proteins, it is necessary to clone and characterize their genes. As a first step towards achieving this goal, an immunological screening of a λgt11 cDNA library with antiserum raised against a nuclear matrix preparation was performed. Screening of the >-.gt11 library resulted in the isolation of several immunoreactive positive clones. One of these cDNA clones, λ7E, was further characterized. Immunoblot analysis, using antisera prepared against 7E β gal-fusion protein, resulted in the detection of an immunoreactive nuclear protein with an apparent molecular weight of 52 kDa. Furthermore, cell fractionation combined with immunoblotting showed that, the 7Eb protein was a component of the HeLa cell nuclear matrix cofractionated with nuclei. Subsequent to screening of another cDNA library (λZap II) with a λ7E DNA probe, two overlapping cDNA clones λ8.1 and λ17.1 were isolated. The composite nucleotide sequence deduced from λ8.1 and λ17 .1 cDNA inserts revealed that, the cDNA is capable of encoding a polypeptide of 422 amino acid residues with a molecular size of 49.8 kDa which is very close to the molecular weight of the protein identified by western blot analysis. -- Based upon the nucleotide sequence data the 7Eb protein was found to be rich in basic amino acids (22%). A computer search for similarity to the predicted amino acid sequence of the 7Eb protein did not reveal any resemblance to previously cloned nuclear matrix proteins including the nuclear lamins. -- The amino-terminal half of the 7Eb protein contains a stretch of about 80 amino acids which are very similar to the DNA binding HMG box domain of a number of proteins. The predicted amino acid of the 7Eb sequence also revealed a significant similarity to many of the cytoskeletal proteins. Although the function of the protein identified in this study is not yet known, the presence of a HMG box domain and sequence similarity to cytoskeletal proteins suggest that this protein is a novel DNA binding nuclear matrix protein.

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