Identification of an intronic regulatory element in the human protein C (PROC) gene.

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms.

Expression of human TRPC genes in the megakaryocytic cell lines MEG01, DAMI and HEL.

Store-regulated Ca2+ entry represents a major mechanism for Ca2+ influx in non-excitable cells although many details remain to be evaluated including the identification of cation entry channels. Recently human homologues of the Drosophila proteins TRP and TRPL, have been described (TRPC1, TRPC1A, HTRP1) and suggested as candidate cation channels. In this study we sought to examine if the producers of blood platelets, megakaryocytic cells (using the cell lines MEG01, DAMI, HEL), expressed these genes. RNA was prepared from the cell lines and platelets and converted to cDNA. The cDNA was then subjected to 30-35 cycles of PCR using gene specific primers for TRPC1-3. PCR products of the expected sizes were observed for all three TRPC genes in the three cell lines. Direct sequencing confirmed their identity. Additionally for TRPC1, a larger species, and for TRPC2, a smaller species was detected in all three cell lines with sequencing revealing the fragments to contain TRPC sequence, suggesting that they were either products of alternative splicing events or from closely related genes. These results suggest that TRPC genes are expressed in megakaryocytic cell lines and that the TRPC proteins may play a role in mediating cation influx in both megakaryocytes and platelets.

Novel mutations in keratin 16 gene underly focal non-epidermolytic palmoplantar keratoderma (NEPPK) in two families.

Keratins K6 and K16 are expressed in suprabasal interfollicular epidermis in wound healing and other pathological conditions associated with hyperproliferation, such as psoriasis and are induced when keratinocytes are cultured in vitro. However, these keratins are also constitutively expressed in normal suprabasal mucosal and palmoplantar keratinocytes. Mutations in keratins have been reported in the basal keratin pair K5 and K14 in epidermolysis bullosa simplex and in suprabasal epidermal keratins K1, K2 and K10 in epidermolytic ichthyoses. Two families with autosomal dominant disorder of focal non epidermolytic palmoplantar keratoderma, have oral mucosal and follicular lesions in addition to the palmoplantar hyperkeratosis. Previous studies have shown linkage in these families to the type I keratin gene cluster at 17q12-q21 and this report shows that the cDNA of affected members of both families have novel heterozygous mutations in the expressed keratin 16 gene. These mutations (R10C and N8S) lie in the helix initiation motif of the 1A domain. These mutations do not appear to cause epidermolysis on light or electron microscopy, which may reflect differences in function, assembly or interaction of the 'hyperproliferative' or 'mucoregenerative' keratins from other major types of keratins. The mutations reported here are the first to describe the molecular pathology of focal non epidermolytic palmoplantar keratoderma.