The chicken Ig lambda light chain locus is composed of a single V gene closely linked (1.8 kb) to a single J‐C unit in its natural configuration. In mice transgenic for this locus, the transgene becomes rearranged in B cells and to a much lesser extent in T cells. Modifications were introduced in the transgene in order to characterize elements which target the recombinase to the Ig loci. In the absence of either the promoter or the enhancer located 3′ of C lambda, rearrangement of the transgene is reduced 20‐ to 100‐fold. Moreover, rearrangement is increased 5‐fold when the DNA segment between V lambda and J lambda (‘Uo segment’), which is deleted during the joining process, is replaced by a neutral DNA segment of equal length. The Uo segment behaved as a strong transcriptional silencer when tested in a CAT assay in vitro. Control transgenic mice harbouring only the two 3 bp mutations that introduced restriction sites at both ends of the Uo segment to allow for its replacement were also analysed. Rearrangement was reduced 10‐ to 100‐fold in B cells from such transgenic lines. A model is proposed whereby the sites of these two mutations would function by counteracting transiently the repressing effect of the silencer, thus giving access of the chicken light chain locus to the recombinase.

Patients with congenital adrenal hyperplasia arising from mutations of 11␤-hydroxylase, the final enzyme in the glucocorticoid biosynthetic pathway, exhibit glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo-pituitary-adrenal activity, and excess mineralocorticoid activity caused by the accumulation of deoxycorticosterone. A mouse model, in which exons 3-7 of Cyp11b1 (the gene encoding 11␤-hydroxylase) were replaced with cDNA encoding enhanced cyan fluorescent protein, was generated to investigate the underlying disease mechanisms. Enhanced cyan fluorescent protein was expressed appropriately in the zona fasciculata of the adrenal gland, and targeted knock-out was confirmed by urinary steroid profiles and, immunocytochemically, by the absence of 11␤-hydroxylase. The null mice exhibited glucocorticoid deficiency, mineralocorticoid excess, adrenal hyperplasia, mild hypertension, and hypokalemia. They also displayed glucose intolerance. Because rodents do not synthesize adrenal androgens, changes in reproductive function such as genital virilization of females were not anticipated. However, adult homozygote females were infertile, their ovaries showing an absence of corpora lutea and a central proliferation of disorganized steroidogenic tissue. Null females responded normally to superovulation, suggesting that raised systemic progesterone levels also contribute to infertility problems. The model reveals previously unrecognized phenotypic subtleties of congenital adrenal hyperplasia.The final steps leading to the production of glucocorticoids and mineralocorticoids are undertaken by 11␤-hydroxylase and aldosterone synthase, encoded by two closely linked genes, Cyp11b1 and Cyp11b2, respectively, that share 95% sequence homology. In rodents, the common substrate, deoxycorticosterone, is converted into the mineralocorticoid, aldosterone, in the adrenal zona glomerulosa by aldosterone synthase and into corticosterone, the main glucocorticoid, by 11␤-hydroxylase in the zona fasiculata. Cortisol rather than corticosterone is the main glucocorticoid in humans because Cyp17 is expressed in the zona fasciculata. Unlike rodents, human adrenals produce significant amounts of the androgen dehydroepiandrosterone (DHEA). 2 Patients with congenital adrenal hyperplasia (CAH) have a markedly reduced capacity to produce glucocorticoids. In ϳ90% of cases CAH is caused by deficiency of 21-hydroxylase, the penultimate enzyme in the pathways of both aldosterone and cortisol synthesis (1). In 8% of CAH cases, point mutations in or deletion of Cyp11b1 drastically reduce or completely destroy 11␤-hydroxylase activity (2-4).Adrenal hyperplasia is a consequence of increased ACTH secretion in the absence of normal negative feedback control by glucocorticoids of the hypothalamo-pituitary-adrenal axis. In 21-hydroxylase-deficient patients, ACTH drives the production of progesterone and 17-hydroxyprogesterone and channels earlier 17-hydroxylated intermediates in the glucocorticoid pathway toward the synthesis of adr...

The genes MBD1 and MBD2 encode methyl-CpG binding proteins that suppress transcription from methylated promoters. In contrast, CGBP encodes a protein that binds promoters containing unmethylated CpG and stimulates transcription. All three are located on human chromosome 18q21, a region of frequent loss of heterozygosity in several cancers. These genes therefore represent candidate tumour suppressor genes, whose loss of function could affect the normal regulation of gene expression, whether by lack of complete suppression of genes normally silenced (via loss of MBD1 and MBD2) or by some loss of activation of genes normally expressed (via loss of CGBP), either way contributing to the tumorigenic phenotype. We have confirmed by fluorescent in situ hybridization that MBD1 and MBD2 bracket the DCC locus giving a gene order of MBD1/CGBP-DCC 5 0 -DCC 3 0 -MBD2. Mutation analyses by single-stranded conformation polymorphism in colon and lung cancer cell lines and primary tumours revealed a small number of mutations, suggesting only a limited role of these genes in human tumorigenesis.

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The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCD cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCD parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCD cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCD cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCD cells being precursor cells.