Renin gene expression, biosynthesis, and cellular pathways of secretion

The molecular biology of the human renin gene is reviewed. This 12.5 kb gene contains 10 exons and 9 introns. In its 5' flanking region, major control elements are present. These include promoters and enhancers as well as regulatory elements. The combined action of these elements would result in tissue specific expression and regulation of the gene. In addition to the control at the gene expression level, renin is also regulated at the posttranslational and secretory levels. The translational product of renin mRNA is preprorenin, which is cotranslationally cleaved to prorenin, an inactive precursor of renin. The majority of new synthesized human prorenin is constitutively secreted. However, prorenin is also processed intracellularly to the mature single chain active renin which is stored in secretory granules. Active renin is released by a regulated mechanism which can be stimulated by cAMP and other secretagogues. Studies are under way to examine the responses of renin gene expression, biosynthesis and secretion to various physiological conditions.     

Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons.

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-transferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 10(-11) M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5'-TGTCGC-3', which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN8AAN5.     

Promoter analysis of tumor suppressor gene PTEN: identification of minimum promoter region

Mutations of PTEN, a tumor suppressor gene located on chromosome 10, which encodes a protein-tyrosine and lipid-phosphatase, are prevalent in various human cancers, including glioblastoma. Despite extensive characterization of PTEN mutations in human cancers and a relatively good understanding of the molecular roles of PTEN in the control of cellular processes, little is known about modes of PTEN regulation. To understand the regulation of expression of the tumor suppressor gene PTEN, we isolated a 2212 bp fragment from the human BAC clone 46B12 DNA. The 3' end of this fragment starts at the Not I site of -745 relative to the first translation codon ATG (+1) and ends at the Sal I site of -2957 at the 5' end. Using classical 5'RACE and primer extension techniques, nine start sites were observed between -817 and -984 upstream of the ATG start site. We located a 137 bp fragment (-958/-821) as the minimum promoter region using promoter deletion and luciferase assays. A 704 bp fragment (-33/-737) downstream of the 2212 bp fragment was also cloned. As indicated by luciferase assays, the data show that this region possesses no promoter function. Interestingly, a p53 binding sequence is located within the 599 bp fragment (-1344/-745), although p53 expression had a minimal effect on PTEN, demonstrating its insignificant role in PTEN gene expression.     

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.