Andrew Grimes

Expression of the Menkes gene homologue in mouse tissues lack of effect of copper on the mRNA levels.

The expression of the homologue of the Menkes disease gene (Mnk) in mice was studied using RNA blots. The highest level of expression of the 8.0 kb mRNA was found in placenta, substantial expression was noted in lung, heart, brain, testis and kidney and gut mucosa, but very low levels were found in spleen and adult liver. In fetal liver, the amount of Mnk mRNA is similar to that found in kidney, however, it declines soon after birth. Results with copper‐loaded normal mice and mutant mice with genetic defects in copper transport suggested that Mnk mRNA levels are not regulated by tissue copper concentrations.

Animal models of Menkes disease.

There are three recognized X-linked copper deficiency disorders in humans: classical Menkes disease (MD), mild Menkes disease and occipital horn syndrome (OHS, also known as X-linked cutis laxa). Since the features of these diseases are so distinct, it was not clear until recently whether the phenotypes were due to mutations in the same gene, or whether OHS in particular is due to mutations in a gene on the X-chromosome, closely linked to the Menkes locus (Danks, 1995). Molecular analysis has now demonstrated that MD, mild Menkes and OHS are indeed due to allelic mutations of the gene affected in Menkes disease (MNK or ATP7A), however, the basis for the phenotypic differences is still not fully understood. A similar range of phenotypes is also found in the mottled mice mutants, and these are discussed below.

A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells

A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.

The effect of d-penicillamine on metallothionein mRNA levels and copper distribution in mouse hepatocytes

Penicillamine increases the levels of metallothionein (MT) mRNA in a time and concentration dependent manner without altering either the rate of copper uptake or the amount of copper within the cell. The effect is dependent on the presence of intracellular copper, however, since depletion of copper by chelators blocks the effect, and does not alter the ability of dexamethasone to stimulate mRNA production. Penicillamine did not alter the distribution of 64Cu in the hepatocytes, as measured by fast protein liquid chromatography (FPLC), although the pattern may be affected by the amount of MT present. The data indicates that penicillamine removes copper from some intermediary ligand, thereby making it available to induce metallothionein. It is possible that this is part of the therapeutic action of the chelator in the treatment of Wilsons disease.

Isolation of a human ceruloplasmin cDNA clone that includes the N-terminal leader sequence

A number of cDNA clones encoding human ceruloplasmin were identified using two mixed oligonucleotide probes. One of these clones was shown by DNA sequence analysis to span from the complete N‐terminal leader sequence to 114 amino acids short of the C‐terminus. The leader sequence consists of 19 primarily hydrophobic amino acids. Northern blot analysis of RNA from human liver showed two species of ceruloplasmin mRNA; a minor species of 3600 nucleotides and a major one of 4400 nucleotides.