Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and cAMP-dependent gene expression

Cyp-21 (the mouse steroid 21-hydroxylase gene) is expressed exclusively in cells of the adrenal cortex, is induced by ACTH and cAMP, and is required for corticosteroid synthesis. This review examines the molecular basis for the regulated expression of Cyp-21 in the ACTH-responsive, mouse adrenocortical tumor cell line, Y1. We demonstrate that 330 bp of 5′-flanking DNA from the Cyp-21 gene are sufficient for cell-selective and ACTH-induced expression of Cyp-21, and that this promoter region comprises multiple, closely spaced enhancer elements each of which is required for promoter function. Within this promoter, we define three related elements that contain variations of an AGGTCA motif and that contribute to the cell-selective expression of Cyp-21. Variations of these same AGGTCA-bearing elements are also involved in the expression of Cyp 11a and Cyp 11b in Y1 adrenocortical cells. These elements interact with the same or closely related nuclear proteins found only in steroidogenic cell lines. Taken together, these results suggest that shared elements contribute to the adrenal cell-selective expression of at least three steroidogenic cytochrome P450 genes.

The element at −170 and the related elements at −65, −140 and −210 in the Cyp-21 promoter are not active as enhancers in the mutant Y1 cell line, Kin-8. Kin-8 cells contain a mutation in the regulatory subunit of the type 1 cAMP-dependent protein kinase that renders the enzyme resistant to activation by cAMP. Therefore, these elements appear to be selectively dependent upon an intact cAMP-dependent protein kinase for enhancer function. Individually, none of these elements confer cAMP-dependence to a reporter gene driven by a heterologous promoter. On the basis of these observations, we suggest that ACTH- and cAMP-dependent expression of Cyp-21 requires the combined actions of the element at −170, and the related elements at −140, −210 and −65.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.