Formalin-induced changes of NMDA receptor subunit expression in the spinal cord of the rat.

Using RT-PCR, the present study investigated the effects of formalin administration on mRNA expression coding for NMDA receptor (NR) subunits and splice variants in rat lumbar spinal cord. Subsequent to formalin injection (5%; subcutaneously) into the hind paw of Sprague-Dawley rats, the animals exhibited the typical biphasic behavioural pain response. Spinal cord (L3-6) was prepared six hours after formalin injection. In controls, NR1-b predominated over NR1-a, and NR1-2 and NR1-4 exceeded over NR1-1 and NR1-3, respectively. Regarding the NR2 subunit expression in controls, NR2B exhibited the highest expression, followed by decreasing proportions of NR2C, NR2A, and NR2D. Formalin treatment did not affect NR1 splice variant expression but significantly increased and decreased the proportion of NR2A and NR2C, respectively. In summary, the present data demonstrate adaptive changes in the NR subunit expression pattern in rat spinal cord due to formalin injection.

Cis-regulatory sequences from the first intron of the rat glutamine synthetase gene are involved in hepatocyte specific expression of the enzyme.

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.

Identification and functional characterization of regulatory elements of the glutamine synthetase gene from rat liver.

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, lambda 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 constitute 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae.

We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychia lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M(r) of 16,819. Both ends of the cloned gene have the hypotrichous telomeric C4A4 repeat. The coding region is flanked by a 158-bp 5'-leader sequence and a 3'-trailer sequence of 213 bp. S1 analysis was used to locate the transcription start point (tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca(2+)-binding protein with only one Ca(2+)-binding site. It's function and biological significance is yet unknown.