To define the cis-acting elements important for rat insulin II gene expression, we analyzed the effects of 5' deletions and linker-scanning mutations on the expression of a rat insulin II reporter gene in an insulinoma cell line (HIT). The reporter gene contained 448 base pairs of 5'-flanking sequence joined to the bacterial chloramphenicol acetyltransferase gene. Expression of the 5' deletion mutations indicated that the minimal sequence requirement for efficient expression was 218 base pairs of 5'-flanking sequence, and at least three regions downstream from -218 were important for transcription. A more precise localization of these elements and the cis-acting sequences in the promoter was achieved by analysis of the expression of 18 linker-scanning mutations. In these studies at least four other regions important for expression of the rat insulin II gene were identified. These findings suggest that the sequences important for rat insulin H and rat insulin I expression may differ significantly despite the high degree of sequence similarity in their 5'-flanking regions.To better understand the molecular basis of cell-specific gene expression, we have studied the rat insulin II gene, which is expressed exclusively in pancreatic gB cells. Unlike most vertebrates, rats have two nonallelic insulin genes (4, 19), and there is an -85% sequence similarity in the 5'-flanking regions extending 500 base pairs (bp) upstream from the mRNA cap site (4, 27). This 5'-flanking region contains a cell-specific enhancer and promoter (5, 10), and site-directed mutagenesis of these sequences in the rat insulin I gene identified several regions in the enhancer that are important for expression (16). Both genes are expressed, and the steady-state levels of both mRNA and protein are similar (3, 7). Cell-specific transcription of the insulin gene may also be achieved by repression of insulin gene expression in inappropriate cell types by negative regulatory elements in the 5'-flanking region (13,17,24).To define sequences within the rat insulin II 5'-flanking region essential for expression, we introduced a series of 5' deletions and linker-scanning (LS) mutations into a rat insulin II-chloramphenicol acetyltransferase (CAT) fusion gene. A reporter vector (pBRN/S-CAT) was constructed by introducing the coding sequences of the CAT gene and the simian virus 40 (SV40) splice and poly(A) addition sites into plasmid pBRN/S (9). This was done by subcloning the BamHI-HindIII fragment of pSV2CAT (8) The plasmid carrying the rat insulin II-CAT fusion gene, pBRN/S Ins CAT (Fig. 1), was constructed by subcloning the -448 (EcoRI) to +49 (RsaI) rat insulin II genomic fragment into the BamHI-HindIII sites in pBRN/S-CAT after appropriate end modification of the fragment by linker ligation. The 5' deletions were made according to existing protocols (1) after linearization of pBRN/S Ins CAT with BamHI (-448). Plasmid pBRN/S Ins II, constructed for the purpose of generating the 3' deletions, was made by subcloning an EcoRI (-448)-BamHI (+191) rat insul...
The recirculation of lymphocytes throughout the body, from blood through lymphoid organs and back into the bloodstream (1), plays a critical role in the normal function of the immune system by maximizing interactions of lymphocytes with antigen and by increasing collaborative interactions between many disparate cell types. The organ-specific interactions of lymphocytes with specialized high endothelial venules (HEVs) in lymphoid organs (2) are believed to be controlled by homing receptors on lymphocytes and vascular addressins on HEVs (3, 4). Thus far, at least three functionally distinct lymphocyte-HEV recognition systems governing the homing of lymphocytes to peripheral lymph nodes (PLNs), mucosal lymphoid organs [Peyer's patches (PPs) and appendix], and inflamed synovium have been identified (5, 6).Murine lymphocyte PP HEV adhesion molecule 1 (LPAM-1) involved in the organ-specific adhesion of lymphocytes to PP HEVs has been identified as an integrin receptor (7,8). The integrin receptors are engaged in cell-cell adhesion and interactions with extracellular matrix components (9). The functional role of LPAM-1 as a lymphocyte homing receptor for PP is confirmed by in vivo migration studies in the rat (10). LPAM-1 consists of a murine integrin a4 homologous to human VLA4a and an integrin (
Band-shifting and DNase I-footprinting assays have been used to study the trans-acting factor(s) binding to an important promoter element (-53 to -46 relative to the transcription start) of the rat insulin II gene. A binding activity which footprints a region between -60 and -40 was found in both HIT, a hamster insulinoma cell line, and HeLa cells. A mutation within this region which drastically decreases promoter activity in vivo also greatly reduces binding activity in vitro. This binding activity was purified from HeLa cells and identified by competition and renaturation analyses as being the same as the COUP (chicken ovalbumin upstream promoter) transcription factor, a DNA-binding protein required for efficient transcription of the ovalbumin gene in vitro. Interestingly, the binding sequences of the COUP transcription factor in the ovalbumin and the insulin promoters have only limited similarities.Accumulating evidence suggests that the regulation of gene expression is mediated by the interaction of cis elements and trans-acting factors (1,6,10,14,19,21,23,26). Many sequence elements, including the TATA box, upstream promoter elements, enhancers, and silencers, have been identified as important for the expression of a large number of eucaryotic genes transcribed by RNA polymerase II. Similarly, a number of trans-acting factors which bind to these regulatory elements have been identified. Nevertheless, a critical question remains as to the molecular mechanism by which these trans-acting factors regulate transcription. A clear understanding requires the purification and characterization of these factors.The insulin gene is expressed only in pancreatic ,B cells, thus providing a good model system for the study of tissuespecific gene expression. In rats, there are two nonallelic insulin genes which are highly homologous in flanking as well as coding regions (17) and are expressed at similar levels (2, 3). It is therefore likely that these two genes are controlled by similar transcriptional regulatory mechanisms.The DNA sequences important for the expression of the rat insulin genes have been approximately determined. With insulin-CAT (chloramphenicol acetyltransferase) fusion genes, Walker et al. (32) showed that the -300 to +51 fragment of the rat insulin I gene is sufficient for tissuespecific expression in transfected insulinoma cells. Subsequently, Edlund et al. (7) demonstrated that this fragment can be divided into two distinct sequence elements, the promoter and the enhancer, each of which can direct tissuespecific expression. For the rat insulin II gene, Hanahan (13) demonstrated that 520 base pairs of 5'-flanking sequences linked to the simian virus 40 (SV40) T-antigen gene induced P-cell-specific tumor formation in transgenic mice. A similar fragment is sufficient for tissue-specific expression in cultured cells, as shown by Episkopou et al. (8). In addition to the positive elements, sequences at 2 to 4 kilobases (15) and 250 base pairs (22) upstream from the cap site inhibit expression of the rat insu...
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