Cloning and characterization of the 5′-flanking region of the rat neuron-specific Class III β-tubulin gene
Introduction
The large variety of cell types that compose the vertebrate nervous system are produced by the coordinated expression of numerous transcription factors that progressively restrict the fates of neural ectodermal cells. An important goal is to identify those factors necessary for specifying each cell type in order to influence neural stem cells to differentiate as cellular replacements to treat congenital and degenerative disorders. A useful approach for elucidating how different cell types are determined is the identification of putative transcription factor binding sites in the flanking regions of phenotype-specific structural genes. Examples include glial fibrillary acidic protein for astrocytes (Brenner, 1994), a myelin-specific protein (PLP) for oligodendrocytes (Berndt et al., 2001) and Tα1-tubulin (Gloster et al., 1994) for neurons.
The Class III β-tubulin gene is another important candidate for discovering factors necessary for the expression of a neuronal fate and/or initiation of neuronal differentiation. In vertebrates there are five β-tubulin genes that encode highly homologous protein classes (Nogales, 2001). These proteins share high amino acid identity, but there are two domains that diverge significantly among the classes (Sullivan et al., 1986). The ‘isotype-defining’ domain at the extreme carboxyl terminus provides functional diversity, and defines six different classes of β-tubulin proteins (Sullivan, 1988). Class I protein is ubiquitously expressed in all tissues; Class II and Class IVa proteins are prominent in brain but also expressed in several other tissues; Class IVb protein is expressed exclusively in the testes; Class V protein is detectable in all cell types except neurons. Class III β-tubulin is specifically expressed in the nervous system (Lewis et al., 1985a) only by neurons (Moody et al., 1989, Lee et al., 1990a, Lee et al., 1990b), with some expression in testis (Lee et al., 1990a). In both chick and mouse, Class III β-tubulin is expressed just prior to or at the terminal mitosis of nearly all developing neurons (Moody et al., 1989, Easter et al., 1993). This suggests that its expression is regulated by elements necessary for committing a neural progenitor cell to a neuron, and for initiating differentiation. Further, this gene is upregulated during sensory axon regeneration (Moskowitz et al., 1993). Thus, the promoter region of this gene should provide an excellent tool for studying these important processes. In addition, the expression of this gene is misregulated in a number of cancers (Katsetos et al., 2001), indicating that understanding its normal regulation could elucidate underlying causes of these malignancies. To provide the necessary background information for understanding its normal and abnormal regulation, we identified, cloned and sequenced ∼5 kb of the 5′-flanking region of the rat Class III neuron-specific β-tubulin (herein referred to as βIII-tubulin) gene. The rat gene was chosen because this animal is an important model for neurodegenerative disease, neural transplants and behavioral assessments of recovery of function (http://www.nih.gov/science/models/rat).
Section snippets
Construction of recombinant plasmids
The 350 bp-long PCR fragment BT-UTR containing the first 293 bp of the 5′-coding region and 57 bp of the 5′-UTR of βIII-tubulin was cloned into the pAMP1 vector (Life Technologies; pAMP1/BT-UTR plasmid). To analyse the 5′ genomic sequence of βIII-tubulin, a 6 kb-long DNA fragment was obtained from a positive P1 clone digested with BamHI and subsequently cloned into the pZero vector (InVitrogen; pZero/BTIII-6.0 plasmid).
For CAT assay studies, three βIII-tubulin promoter constructs were generated
Isolation of the 5′ βIII-tubulin coding region and 5′-UTR
The 5′-coding region and the 5′-UTR of the βIII-tubulin gene were isolated first to design probes that would not hybridize with other highly homologous classes of β-tubulins (Cleveland and Sullivan, 1985, Sullivan and Cleveland, 1986, Sullivan, 1988). A rat βIII-tubulin cDNA clone (isolated in the laboratory of Dr. Anthony Frankfurter, University of Virginia), which lacked the first 210 nucleotides of the coding region and the 5′-UTR, provided primer sequence for the RACE procedure. We obtained
Acknowledgements
This work was supported by NIH Grants NS23158 (S.A.M.) and NS41391 (A.E.C.).
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