Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.     

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.     

Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression

The proteinase inhibitor II (pin2) gene family exhibits two different modes of expression. It is, on the one hand, constitutively expressed in flowers of potato and tomato plants. and in potato tubers. On the other hand, its expression is induced in the plant foliage by mechanical wounding. To define cis-regulatory elements involved in pin2 promoter activity, deletion analysis of a potato pin2 promoter has been performed in stably and transiently transformed potato and tobacco plants. Two different elements, a quantitative enhancer and a regulatory element, are required for promoter activity. While functional promoter elements required for pin2 activity in tubers and wounded leaves could not be separated, its expression in flowers is mediated by different cis-acting sequences. Induction of pin2 expression in leaves by treatment with the plant growth regulators abscisic acid and jasmonic acid, and the general metabolite sucrose, depends on the presence of the regulatory element involved in expression in tubers and wounded leaves. Thus, pin2 expression in tubers and wounded leaves apparently results from the action of similar hormonal signals on closely linked promoter elements, while a different signal pathway leads to its constitutive expression in flowers.     

Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia

Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.     

Analysis of steroid 21-hydroxylase gene in five unrelated Japanese patients with 21-hydroxylase deficiency

DNA samples from five unrelated Japanese patients with 21-hydroxylase (21-OHase) deficiency were studied by Southern analysis using human 21-OHase cDNA. Patterns seen after digestion with not only TaqI but also KpnI showed that two out of the five patients were homozygous for a deletion of the 21-OHase B gene. This result supports the report that the 21-OHase B gene is functional. In the other three, smaller mutations might be responsible for the disorder. The parents of one of the two patients with the deletion had a common ancestor. Hybridization patterns of DNA from members of the family of the patient were consistent with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. The deletion segregated with HLA-Aw 24; Bw 61; Cw 3. Heterozygous carriers of 21-OHase deficiency could be detected by comparing the patterns as well as the HLA haplotypes in this family. The application of the family study to the prenatal diagnosis is also discussed.     

Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice

Previous studies indicated that a DNA fragment comprising nucleotides (nt) ?164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt ?3 to +5) and Box II (nt ?53 to ?39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt ?98 to ?79) and a GATA motif (nt ?143 to ?135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt ?164 to ?32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the ?103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the ?164 to +45 promoter fragment indicated that the ?164 to ?103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the ?103 to ?32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.     

Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus

Steroid 21-hydroxylase (P450c21) is absent or defective in more than 90% of patients with congenital adrenal hyperplasia. This disorder of cortisol biosynthesis occurs in a wide spectrum of clinical severity; specific mutations in the 21-hydroxylase gene (CYP21) have been found in association with particular clinical phenotypes. To determine the functional effects of mutations causing amino acid substitutions, normal P450c21 and three mutagenized P450c21 enzymes were expressed at high levels in cultured COS-1 cells using recombinant vaccinia virus. A single amino acid substitution (Val281----Leu) present in patients with mild "nonclassical" 21-hydroxylase deficiency resulted in an enzyme with 20-50% of normal activity. A mutation (Ile172----Asn) identified in patients with the "simple virilizing" form (poor cortisol synthesis but adequate aldosterone synthesis) resulted in an enzyme with less than 2% of normal activity. Finally, a cluster mutation (Ile-Val-Glu-Met234-238----Asn-Glu-Glu-Lys) found in a patient with severe "salt wasting" 21-hydroxylase deficiency (inadequate aldosterone synthesis) results in an enzyme with no detectable activity. These data indicate that the severity of 21-hydroxylase deficiency correlates with the degree of enzymatic compromise.     

Regulatory elements involved in the tissue-specific expression of the yellow gene of Drosophila

Summary We have assessed the DNA sequence requirements for the correct spatial pattern and phenotypic expression of y in the late embryo/larvae. The wild-type larval phenotype requires both the regions between-294 bp and-92 bp and a portion of the intron; the sequence element(s) located within the intron can act in a position independent manner to effect the wild-type larval phenotype. The larval expression pattern was examined by tissue experiments in situ and by staining germline transformants derived from various y/lacZ fusion constructs. The larval expression of y is restricted to the mouthparts, microsetae and anal plates. While the-495 bp to+194 bp region alone cannot effect a wild-type larval expression pattern, this region in conjunction with the intron appears to be sufficient to drive -gal expression in an essentially wild-type pattern. Our data further suggest that the-294 bp to-92 bp region contains elements which specify the larval pattern and that the element(s) in the intron normally act to enhance the level of expression necessary for the wild-type larval phenotype. We also present a phenotypic analysis of the adult cuticle structures of germline transformants derived from a variety of deletion and rearrangement constructs of the y gene. This analysis has revealed several new features associated with the regulation of y expression.     

The human complement C4B/steroid 21-hydroxylase (CYP21) and complement C4A/21-hydroxylase pseudogene (CYP21P) intergenic sequences: comparison and identification of possible regulatory elements.

We determined the 1.8 kb intergenic sequences between the human complement C4B gene and the active steroid 21-hydroxylase gene in two subjects, and between the C4A gene and the steroid 21-hydroxylase pseudogene in one subject. Comparison of these sequences with each other and with published homologues revealed no differences which were unique to either intergenic region. Sequence analysis revealed two copies of an AGGTCA motif in all sequences. This motif is common to steroidogenic enzyme gene promoters and to the response elements for nuclear hormone receptors. Similarities with human enhancers were also found.