The gene encoding tissue inhibitor of metalloproteinases-3 (TIMP-3) is regulated during development, mitogenic stimulation and normal cell cycle progression. The TIMP-3 gene is structurally altered or deregulated in certain diseases of the eye and in tumour cells. A detailed knowledge of the TIMP-3 gene and its regulatory elements is therefore of paramount importance to understand its role in development, cell cycle progression and disease. In this study, we present the complete structure of the human TIMP-3 gene. We show that TIMP-3 is a TATA-less gene, which initiates transcription at one major site, is composed of five exons and four introns spanning a region of approximately 30 kb, and gives rise to three distinct mRNAs, presumably due to the usage of alternative polyadenylation signals. Using somatic cell hybrids the TIMP-3 locus was mapped to chromosomal location 22q13.1 We also show that the TIMP-3 5' flanking region is sufficient to confer both high basal level expression in growing cells and cell cycle regulation in serum-stimulated cells. While the first 112 bases of the promoter, which harbour multiple Sp1 sites, were found to suffice for high basal level activity, the adjacent region spanning positions -463 and -112 was found to be a major determinant of serum inducibility. These results provide an important basis for further investigations addressing the role of TIMP-3 in physiological processes and pathological conditions.
Cyclin E is the regulatory subunit of the cdc2-related protein kinase cdk2 and is a rate limiting factor for the entry into S phase. To date, cyclin E is the only cyclin for which alternative splicing has been described. We report here the isolation of a new splice variant of cyclin E, termed cyclin ET, which has an internal deletion of 45 amino acids compared with the full-length cyclin E protein. Even though cyclin ETcontains an intact cyclin box, it is unable to complement a triple cln mutant strain of Saccharomyces cerevisiae or to interfere with rescue by cyclin E, indicating that an intact cyclin box is functionally insufficient. The expression pattern of cyclin ET during cell cycle entry, progression and differentiation differs from that of cyclin E. Thus, ET expression precedes that of the other isoforms during the G0-->S progression; it shows a sharp peak in early G1 in cells released from a mitotic block and is strongly down-regulated in terminally differentiated myeloid cells. These observations point to different functions for cyclin ET and E and show for the first time that the alternative splicing of cyclin E is a regulated mechanism governed by the cell cycle and differentiation.
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