Steroid 21-hydroxylase deficiency is caused by mutations in the CYP21B gene. This gene and a highly homologous pseudogene, CYP21A, alternate with the C4A and C4B genes encoding the fourth component of complement. Steroid 21-hydroxylase deficiency causes >90%o of cases of congenital adrenal hyperplasia (1), an inherited inability to synthesize cortisol. This deficiency is inherited as a monogenic autosomal recessive trait linked to the HLA major histocompatibility complex on chromosome 6p. A gene, CYP2JB, encoding an adrenal microsomal cytochrome P-450 enzyme specific for steroid 21-hydroxylation (P-450c21 or P-450XXI), is located between genes for the transplantation antigens HLA-B and HLA-DR. This gene (also termed 21-OHase B, OH21B, CA21HB, P450C21B, or P45OXXIA2), and a 98% similar pseudogene, CYP2JA (2, 3), are immediately adjacent to and alternate with the C4B and C4A genes, respectively, encoding the fourth component of serum complement (4,5).The rare HLA haplotype A3;Bw47;DR7 is strongly associated with 21-hydroxylase deficiency and carries a deletion of the CYP2JB gene (6) and the adjacent C4B gene, as determined by Southern blot analysis of DNA from individuals homozygous for this haplotype (7). This deletion is detected when DNA is digested with any of several restriction enzymes and hybridized to cDNA or genomic probes for either the C4 or CYP21 genes. As determined in a similar manner, the common HLA haplotype AJ;B8;DR3 carries a deletion of the CYP2JA pseudogene and the C4A gene (8).While the HLA-Bw47 haplotype is found on only =10% of 21-hydroxylase deficiency alleles (9), two studies of patients who did not carry this haplotype (10, 11) found apparent deletions of CYP21B on 7 of 30 and 9 of 40 deficiency alleles, respectively, for an allele frequency of -23%. Most deletions were found in individuals heterozygous for the deletion; heterozygous deletions were detected by decreased relative intensity of the restriction fragments that were absent in homozygous deletions.Because the majority ofdeficiency alleles are not deletions, several mutant C YP21B genes have been cloned and analyzed to identify the mutations. While one point mutation has been found thus far (12), several other CYP2JB genes carry deleterious mutations that normally occur in the CYP21A pseudogene, suggesting that they might have been transferred to CYP21B in gene conversion events (13). The combined frequency of various gene conversions might be roughly equal to the apparent frequency of deletions of CYP21B.In principle, gene conversions could also affect restriction enzyme recognition sites that have been used to distinguish the CYP21A and CYP21B genes. For example, the 3.2-and 3.7-kilobase (kb) Taq I fragments associated with the CYP21A and CYP21B genes, respectively, differ in size because the CYP21A gene carries an extra Taq I site located 200 base pairs (bp) upstream from the start of transcription.

We have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11.3-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage X vector gtlO was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450cMO is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of llfi-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.Congenital adrenal hyperplasia is a disease caused by any of several defects in the adrenal biosynthetic pathways to cortisol and other steroids. Cortisol is normally synthesized from cholesterol in the adrenal cortex in five enzymatic steps: the cholesterol side chain is cleaved to form pregnenolone, which is dehydrogenated at the 3,3 position to yield progesterone; three successive hydroxylations at the 17a, 21, and 1113 positions yield cortisol. In 90-95% of congenital adrenal hyperplasia patients, 21-hydroxylation is impaired. The molecular genetic basis of 21-hydroxylase deficiency has been extensively studied (1).In 5-8% of reported cases of congenital adrenal hyperplasia, 11p-hydroxylation is defective, so that 11-deoxycortisol cannot be converted to cortisol, and, usually, deoxycortisol is not metabolized to corticosterone (2,3). Blood levels of these precursors are elevated in the untreated state. Accumulated precursors are shunted into the androgen biosynthetic pathway, causing symptoms of androgen excess including disordered sexual differentiation and accelerated somatic growth. Elevated levels of deoxycortisol or other metabolites with mineralocorticoid activity may cause hypokalemia and hypertension.Steroid 113-hydroxylation takes place in mitochondria of cells in the adrenal cortex. It requires a substrate-specific cytochrome P-450 (P450cll), which is a heme-containing cDNA pBll -2 pBll -9 pBll -5 pBll -13

No abstract

The myomodulin-related peptides comprise a family of cotransmitters that modulate neuromuscular signaling in the feeding system of Aplysia. In this study, cDNA clones encoding a myomodulin precursor polypeptide were isolated and characterized. This precursor contains seven different myomodulin-related peptides, one of which, myomodulin A, is present in 10 contiguous copies. The sequence of a myomodulin genomic clone indicates that all of these myomodulin-related peptides are encoded on a single exon. The myomodulin gene is expressed in a tissue-specific manner and myomodulin mRNA is localized to specific neurons in the Aplysia CNS. The presence of multiple related neuropeptides can greatly increase the range and precision of signaling at synapses where they act as modulator cotransmitters.

The buccalin-related peptides, buccalin A and buccalin B, are members of a family of cotransmitters that modulate neuromuscular transmission in Aplysia. In this study, a third buccalin-related peptide, buccalin C, was purified from neuronal elements in the accessory radula closer, a muscle involved in the animal's feeding behavior. Oligonucleotide probes based upon the amino acid sequence of buccalin C were used to isolate cDNA clones that encode a buccalin precursor polypeptide. The buccalin precursor contains 19 distinct buccalin-related peptides, several of which are present in multiple copies. The buccalin gene appears to be present in a single copy, with one allele containing a small insert. Expression of this gene occurs in a tissue-specific manner and mRNA transcripts are abundant within neurons in the Aplysia CNS. This large family of neuropeptides may exert extraordinarily complex modulatory actions at synapses where they serve as cotransmitters.