Functional variation of MC1R alleles from red-haired individuals.

Red hair in humans is associated with variant alleles of the alphaMSH receptor gene, MC1R. Loss of MC1R function in other mammals results in red or yellow hair pigmentation. We show that a mouse bacterial artificial chromosome (BAC) which contains Mc1r will efficiently rescue loss of Mc1r in transgenic mice, and that overexpression of the receptor suppresses the effect of the endogenous antagonist, agouti protein. We engineered the BAC to replace the mouse coding region with the human MC1R sequence and used this in the transgenic assay. The human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely resistant to the effects of agouti, suggesting agouti protein may not play a role in human pigmentary variation. Three human variant alleles account for 60% of all cases of red hair. We engineered each of these in turn into the BAC and find that they have reduced, but not completely absent, function in transgenic mice. Comparison of the phenotypes of alphaMSH-deficient mice and humans in conjunction with this data suggests that red hair may not be the null phenotype of MC1R.

Sooty foot, a novel mouse mutation that affects the pigmentation of exposed skin, but not hair, maps to chromosome 2.

We have characterized a novel recessive mouse mutation, named sooty foot, that increases the pigmentation of the exposed skin on the foot pads, the genital region, around the snout and muzzle, the ears, and the tail. By contrast, the pigmentation of the hair is unaffected. We have localized the mutation to Chromosome 2 by polymerase chain reaction (PCR) amplification of simple sequence repeats from pooled DNA from backcross progeny. In an extended backcross we have generated a detailed map of the region around sooty foot.

Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo.

The development of neural crest-derived melanocytes, as well as haematopoietic and germ cells, is affected by mutations of the Kit and Mgf genes, which lead to dominant spotting (W) or steel (Sl) phenotypes. Mgf codes for the ligand of the receptor tyrosine kinase encoded by the Kit locus. KitW-v, a point mutation exerting a dominant negative effect, causes a substantial reduction in tyrosine kinase activity of the Kit receptor and leads to a characteristic pigmentation phenotype, namely dilute coat colour and a white ventral and head spot with reduced pigmentation of the feet and tail in the heterozygous animal, as well as slight anaemia. Homozygous animals lack coat pigmentation and are severely anaemic and infertile. Dct is a marker for cells of the melanoblast lineage. In order to study these cells in detail we have generated transgenic mouse lines carrying the lacZ reporter under the control of the Dct promoter and have used the embryonic expression of the reporter to identify early melanoblasts before they begin to produce pigment. Our transgenic lines have simplified the study of melanoblasts in the mouse embryo, and by crossing our mice with KitW-v mutants we have been able to identify the midgestation stages at which melanoblasts rely critically on Mgf/Kit interactions. We conclude that the survival of immature melanoblasts depends crucially upon Kit signalling up until E11, and later in development Kit plays a vital role in melanoblast proliferation. Our data do not describe a dependence upon Kit for melanoblast migration or differentiation.

Structure of the mouse tyrosinase-related protein-2/dopachrome tautomerase (Tyrp2/Dct) gene and sequence of two novel slaty alleles.

We have isolated the eight exons and 5' and 3' flanking regions of the mouse tyrosinase-related protein-2 (dopachrome tautomerase) gene (Tyrp2/Dct), which is mutated in slaty mice. The gene has a structure that is considerably different from those of other tyrosinase family members in both the number and the position of introns, consistent with the suggestion that the divergence of the family represents an ancient gene duplication. We also identify in the 5' flanking DNA an 11-bp element, the M-box, conserved in other tyrosinase family genes. We have characterized point mutations in two slaty alleles recently identified at the Jackson Laboratory: slaty-2J (slt2J) has a similar phenotype to the original slaty (slt) mutation, and slaty light (Sltlt), which has a more severe effect and is semidominant. We suggest that the slaty-light phenotype is a result of the failure of the enzyme to be correctly targeted to its normal location on the inner face of the melanosomal membrane.

The origin and significance of 18-hydroxycortisol: studies in hyperaldosteronism and in bovine adrenocortical cells in vitro.

18-Hydroxycortisol has been suggested as a marker compound for a transitional zone between the adrenocortical zonae glomerulosa and fasciculata. The control of secretion of 18-hydroxycortisol has been compared with those of cortisol and aldosterone in normal subjects and patients with primary hyperaldosteronism. Comparisons were also made in isolated bovine zona glomerulosa and zona fasciculata cell preparations. Although there was considerable cross-contamination between fractions, 18-hydroxycortisol secretion occurred with equal facility in both fractions but depended on the availability of cortisol as substrate. Changes in secretion during stimulation following those of cortisol. It is concluded that, in vivo, 18-hydroxycortisol derives mainly from the zona fasciculata. The relevance of these findings to primary hyperaldosteronism and to the nature of the transition is discussed.

Production of mouse Hox-2.1 protein in Escherichia coli: characterisation of in vitro binding to DNA.

The developmentally regulated mouse Hox-2.1 gene encodes a homeodomain-containing (Hox) protein which is likely to function as a transcription factor. We expressed the DNA coding for full-length Hox-2.1 protein in a T7 promoter-containing vector in bacteria, which produced low levels of protein showing weak DNA-binding activity. Synthesis of a truncated polypeptide lacking all the sequence upstream from the homeodomain enabled us to produce greater amounts of protein and demonstrate its sequence-dependent DNA binding. The tetranucleotide ATTA is necessary for binding, but a single copy is not by itself sufficient. Flanking sequences are important; in particular a cytosine immediately 5' to the ATTA enhances binding.

The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase.

We have determined the exon structure of the mouse tyrosinase-related protein-1 (TRP-1) gene. The gene is only 15kb in length, but contains seven introns, in contrast to the tyrosinase gene which is almost 100kb long with only four introns. Only two introns are located in homologous positions in both genes. Intron I of TRP-1 has three alternative 5' splice sites clustered within 21bp, which all splice to the same 3' site. Intron V has a very unusual 5' splice site, which has the dinucleotide GC rather than the conventional GT. We show that as little as 370bp of 5'-flanking DNA is sufficient to direct cell-specific expression of the chloramphenicol acetyl transferase gene. The flanking DNA of TRP-1, unlike tyrosinase, does not contain a TATA box or a CCAAT box. Both mouse genes, however, share an 11bp sequence, also found in human tyrosinase, which we suggest may be a melanocyte-specific promoter element.

Factors affecting the secretion of 18-hydroxycortisol, a novel steroid of relevance to Conn's syndrome.

A recently developed radioimmunoassay for direct measurement of 18-hydroxycortisol (18-OH-F) in plasma and urine has been used to study the physiology of this newly described steroid in normal subjects. Plasma levels of 18-OH-F show a circadian variation similar to that of cortisol and are increased and suppressed by administration of ACTH and dexamethasone respectively. A clear increase was observed in response to sodium restriction but despite this, angiotensin II infusion failed to cause a rise in 18-OH-F levels and a possible explanation is discussed. The results are interpreted in terms of a proposed biosynthetic pathway involving 18-hydroxylation of cortisol during a second passage through the adrenal gland.

A radioimmunoassay for 18-hydroxycortisol in plasma and urine.

Increased excretion of 18-hydroxycortisol has been proposed as a specific biochemical marker for differential diagnosis of primary aldosteronism. We describe the development of a direct RIA with an 125I label that permits measurement of the steroid in less than or equal to 0.5 microL of urine or less than or equal to 25 microL of plasma. For control subjects, the mean concentrations of 18-hydroxycortisol in urine and plasma were 310 (SD 178) nmol/24 h (n = 32) and 2.27 (SD 0.80) nmol/L (n = 37), respectively; patients with Conn's adenoma or glucocorticoid-remediable aldosteronism had values for urine in the range 1084 to 6534 nmol/24 h and concentrations in plasma ranging from 6.49 to 31.20 nmol/L. Patients with idiopathic zona glomerulosa hyperplasia had values for urine and plasma ranging from 353 to 734 nmol/24 h and from 0.26 to 6.60 nmol/L, respectively. Concentrations of 18-hydroxycortisol in urine clearly discriminate patients with idiopathic hyperplasia from those with other forms of primary aldosteronism, but further work is required to assess the diagnostic accuracy of determinations in plasma.