Hepatic glutamine synthetase (GS) shows a unique expression pattern limited to a few hepatocytes surrounding the terminal hepatic veins. Starting from the genomic clone of the rat GS gene, λ GS1 [Van de Zande, L. P. G. W., Labruyère, W. T., Arnberg, A. C., Wilson, R. H., Van den Bogaert, A. J. W., Das, A. T., Frijters, C., Charles, R., Moorman, A. F. M. & Lamers, W. H. (1990) Gene (Amst.) 87, 225–232] additional genomic clones containing up to 9 kb of 5′flanking region were isolated in order to characterize cis‐acting elements involved in the regulation of GS expression. Sequence analysis of the 5′flanking region up to −2520 bp revealed a putative AP2‐binding site at −223 bp and a second GC box at −2343 bp in addition to the canonical TATA, CCAAT and GC boxes found proximal to the transcription‐start site. A possible negative glucocorticoid‐responsive element (GRE) and regions with very weak similarity to a GRE and to a known silencer element were noted at −506 bp, −406 bp and at −798 bp, respectively. Within the sequenced part of the 5′flanking region no known regulatory elements associated with liver‐specific gene expression were found except for a putative HNF3‐binding site at −896 bp. Functional analysis by transient transfection assays using constructs with the pSSCAT or the pXP1 vector revealed that the elements present within the first 153 bp and particularly the first 368 bp of upstream sequence consititute an active promoter the activity of which is decreased by additional sequences up to −2148 bp. The presence of dexamethasone led to a 2–4‐fold increase in the promoter activity of all these constructs. Using the heterologous truncated thymidine‐kinase‐gene promoter of the plasmid pT81‐luc a strong enhancer element was located between −2520 bp and −2148 bp. Its activity was not affected by dexamethasone but was negatively influenced by flanking sequences in both directions. This enhancer was also effective with the homologous GS promoter (−153 to +59 bp) and the heterologous full thymidine‐kinase‐gene promoter (pT109luc). No further enhancers were found up to −6200 bp. Using the same approach, a second enhancer was found between +259 bp and +950 bp within the first intron. Deoxyribonuclease‐I hypersensitivity studies confirmed the presence of a hypersensitive site between +350 bp and +550 bp and suggested a second site between +850 bp and + 1200 bp. The ultimate GS promoter (−153 to +59 bp) as well as the two strong enhancer regions identified drove luciferase expression most efficiently or almost exclusively in HepG2 cells but not mouse embryo fibroblasts indicating that these regions might be involved in the cell‐type specificity of GS expression. In accord with known data on GS activity and localization these findings suggest that the regulation of the GS gene in the liver is different from that of liver‐specific enzymes and proteins. Although our results do not yet indicate whether the regulatory regions identified play a role in the positional regulation of GS gene expression, they provide a good ba...

In order to identify regulatory elements involved in the hepatocyte specific expression of the enzyme glutamine synthetase [GS (E.C. 6.3.1.2)] we analyzed the first intron of the rat GS gene. A sequence analysis detected clusters of potential transcription factor binding sites in regions that are hypersensitive for DNase I, including sites for Sp1, HNF3 and elements related to binding of members from the C/EBP family. By use of DNA fragments with putative regulatory elements, reporter genes have been constructed that were transfected into isolated hepatocytes in primary culture and into HepG2 hepatoblastoma cells. By these experiments we cold show that sequences from the first intron are able to enhance transcription specifically in hepatocytes but not in cells from the hepatoblastoma cell line. The existence of enhancer effects in the first intron of the GS gene and their restriction to hepatocytes demonstrates that aside from regulatory regions upstream of the transcription start point, there are also downstream regions involved in the specific expression of the gene. We conclude that intronic elements are involved in the pretranslational regulation of the expression of the GS as part of a complex interplay between different regions of the gene.

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The enforcement of rules for food labeling and quantitative ingredient declaration presupposes appropriate test systems. Additionally, central nervous system (CNS) tissue of ruminants is classified as specified risk material for the transmission of prion diseases, and its detection is needed to support the specified risk material ban. Existing antibody-based test systems are hampered by relatively high limits of detection and susceptibility to food processing conditions. For that reason we tested a broad panel of commercially available monoclonal antibodies to identify marker antigens appropriate for the development of a sensitive test system. Western blot analysis using organ-specific samples from cow, pig, and chicken and differently processed meat products containing defined amounts of CNS tissue revealed neurofilament light (NF-L) and protein gene product 9.5 (PGP 9.5) as suitable antigens for the organ-specific and sensitive detection of porcine and bovine CNS tissue. None of the tested PGP 9.5 antibodies displayed cross-reactivity to chicken tissues. Both antigens could be detected in moderately (F(10)121.1 = 0.84) and strongly (F(10)121.1 = 4.01) heated processed meat products containing 5% (NF-L) or 0.2% (PGP 9.5) CNS tissue, respectively. Further, two monoclonal antibodies (clones 13C4 and 31A3) directed against PGP 9.5 were used for the development of a sandwich enzyme-linked immunosorbent assay. The limits of detection of the enzyme-linked immunosorbent assay were approximately 2% added CNS tissue in fresh processed meat products and approximately 0.5% for strongly heated processed meat products (F(10)121.1 = 4.01). In conclusion this test system constitutes a valuable supplementation to existing procedures, which could improve enforcement of food safety regulations.