Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

Abstract

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H411 hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H41I cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Motifs Resembling Hepatocyte Nuclear Factor 1 and Activator Protein 3 Mediate the Tissue Specificity of the Human Plasminogen Gene

“…The start of transcription and numbering of upstream sequences were according to Malgaretti et al [22]. The chloramphenicol acetyltransferase reporter gene (CAT) plasmid pKT has been described [23]; to this plasmid, mouse MOS oncogene sequences were inserted in the SacI site of the polylinker creating plasmid pCAT-MOS [24]. The latter is the basic plasmid to which plasminogen genomic fragments were added.…”

The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation.

“…Rat hepatoma H4II cells and the dedifferentiated derivative H5 cells were grown and transfected, and enzymatic assays were performed, as previously described (20).…”

“…Third, additional sequences sometimes prove to be required in order to obtain the correct expression pattern in transgenic mice (18). This is the case for the albumin gene (31): in transgenic mice, abundant and liver-specific albumin gene transcription requires the presence of a region located between -8.5 and -10.4 kilobases in addition to the promoter.We have previously shown that the 150 base pairs upstream of the rat albumin gene constitutes a fully functional promoter in the differentiated hepatoma cell line H4II (32), where it is as active as the sinian virus 40 early promoter (20). In striking contrast, in dedifferentiated H5 hepatoma cells (12) or in fibroblasts, this promoter is 100-fold less active.…”

“…We have previously shown that the 150 base pairs upstream of the rat albumin gene constitutes a fully functional promoter in the differentiated hepatoma cell line H4II (32), where it is as active as the sinian virus 40 early promoter (20). In striking contrast, in dedifferentiated H5 hepatoma cells (12) or in fibroblasts, this promoter is 100-fold less active.…”

Reporter constructs with low background activity utilizing the cat gene

“…We and others have attempted to identify and delete the respective prokaryotic sequences (SchOle et al, 1988) giving rise to high background of cat expression in some cell lines. Also, insertion of 'terminator' sequences has been suggested to prevent initiation of cat transcripts at cryptic promoters in the vector (Heard et al, 1987). To improve pBLCAT2 and pBLCAT3, we have combined both strategies: 467 bp of vector sequence immediately upstream from the polylinker were deleted and replaced by two fragments, each containing the polyadenylation signal of the SV40 large T encoding gene.…”