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THE COUNCIL FOR TOBACCO RESEARCH--U.S.A., INC. 900 THIRD AVENUE NEW YORH. N.Y. 10022 Mem or andum To: Dr. H. McAllister as Staff Fran: D.H.Ford Re: Site visit with Dr. David Helfman, Cold Spring Harbor Laboratory, July 28, 1994 Grant No. 3008A entitled "Molecular basis for tissue-specific alternative RNA. splicing." Goal: Inasmuch as alternative RNA splicing is a fundamental proress whi.ch may influence different develcpmental, hormcnal, physiological and pathological states, to determine if there are diseases or abnormalities which may be aseociated with defects in the cellular splicing machinery. With the rat 13 -tropomyosin model system which Dr. Helfman is using, this would apply to the regulation of devel- opment of cardiac, skeletal and smooth muscle systems. Results: As noted in my last visit, Dr. Helfman had identified factors in ncn-muscle cells which blocked the expression of the ske let al muscle exon 7 and thus permit the n orma l develcpment of such ce lls . These factors interacted with sequences within and upstream of exon 7. He has currently identified RNA-binding proteins which interact with these factors. One of these factors which blocks the expression of exon 7 is the polypyrimidine tract-binding protein O'r3) which interacts with sequences involved in the a ltern ative sp li cing of 0-tropomyosin pre-mRNA. This protein was found to bind to two distinct functional elements within entrcri 6 of the (3-TM pre mRNA. It further appears that this PTB protein may interact with other proteins once it has bound to the pre-mRNA. The focus of his current study described in Gant No. 3008A is to define the binding sites £or PTB, the sequences in PTB involved in RNA binding and to indentify the proteins whiLh irlteract with PTB. In another series of investigations Dr. Helfman has identified two brain-specific tropomyosins (TMBr-l and TMBr-3) which were gener- ated from the rat aC-tropomyosin gene. The two isoforms were expressed differently during devlopment with TMBr-3 appearing in the embrycnic brain on day 16, while TMBr-3 appeared on day 20 postnat al ly. Further, TMBr-3 was detected in all brain areas whereas TMBr-l was detected primarily in those brain regicals derived from the prosencephalon. Immunocytochemical studies indicated that the brain-specific epitopes are restricted to neurons. Dr. Helfman believes that these tropomyosin isoforms in the CNS play a specialized role in the development and plasticity of the nervous systerg. They may have a significant role in the organizaticn of the structural proteins. However, whatever role the brain tropomyosins play is yet to be determined. Comment:- -An--exciting area of invstigation ccducted by an enthusiastic, well. informed scientist whose vision is not limited to the tropomyosins, but includes the broader range of how alternative splicing may influence - many forms of gene expressicn.in relation to development, maturation and function. DHF