Two divergent types of nerve pathology in patients with different P0 mutations in Charcot-Marie-Tooth disease.

In seven unrelated patients with a demyelinating motor and sensory neuropathy, we found mutations in exons 2 and 3 of the P0 gene. Morphologic examination of sural nerve biopsy specimens showed a demyelinating process with onion bulb formation in all cases. In four patients, ultrastructural examination demonstrated uncompacted myelin in 23 to 68% of the myelinated fibers, which is in agreement with the widely accepted function of P0 as a homophilic adhesion molecule. Three patients showed normal compact myelin, but morphology was dominated by the abundant occurrence of focally folded myelin. The two divergent pathologic phenotypes exemplify that some mutations act differently on P0 protein formation or function than others, which is probably determined by site and nature of the mutation in the P0 gene.

Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth disease type 1B (CMT1B) is genetically linked to chromosome 1q21-23. The major peripheral myelin protein gene, P0, has been cloned and localized to the same chromosomal region. P0 is a 28 kDa glycoprotein involved in the compaction of the multilamellar myelin sheet and accounts for more than half of the peripheral myelin protein content. We checked whether P0 is altered in CMT1B, and show here that a 3 basepair deletion in exon 2 of the P0 gene is present in all affected individuals of a CMT1B family. The mutation results in the deletion of serine 34 in the extracellular domain of P0, suggesting that alterations of P0 cause CMT1B.

A system to study transcription by yeast RNA polymerase I within the chromosomal context: functional analysis of the ribosomal DNA enhancer and the RBP1/REB1 binding sites.

We have developed a novel system to study transcription by yeast RNA polymerase I (Pol I) of mutated rDNA units within the chromosomal context. For this, complete rDNA units carrying specific oligonucleotide tags in both the 17S and 26S rRNA genes were integrated into the chromosomal rDNA locus. Using this novel system, we analysed the action of the rDNA enhancer in stimulating transcription within the chromosomal context. We found that the enhancer acts as a stimulatory element in both directions, mainly on its two most proximal rRNA operons. Deletion of the sequences between the enhancer and the Pol I promoter in the tagged, integrated unit indicated that this part of the intergenic spacer contains no other transcriptional regulatory elements for Pol I. We also applied the system to study the function of the rDNA binding protein RBP1/REB1. For this purpose, we analysed tagged units in which either one or both of the binding sites for this protein have been inactivated. We found that mutations of both binding sites strongly diminish the transcription of the adjacent operon. The protein is hypothesized to play a crucial role in keeping the chromosomal rDNA units in an optimal spatial configuration by anchoring consecutive enhancers and promoters to the nucle(ol)ar matrix.

The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro.

Using an in vitro transcription system for Saccharomyces cerevisiae RNA polymerase I, we have analyzed Pol I promoter deletion mutants and mapped the boundaries of the promoter between positions -155 and +27. The 5'-boundary of the minimal core promoter capable of transcription initiation, however, was found to lie between -38 and -26. The 3'-deletion extending to -2 and -5 still allowed some transcription, suggesting that the positioning of Pol I is directed by upstream sequences. The results of in vitro analysis of linker scanning mutants (LSMs) combined with the deletion analysis showed that the promoter consists of three domains: two essential core domains (I: -28 to +8 and II: -76 to -51) and a transcription modulating upstream domain (III: -146 to -91). These results are in general agreement with those obtained in vivo (1). Using a template competition assay we also analyzed these mutant promoters for their ability to form a stable preinitiation complex. We found that the ability of 5'-deletion mutants to sequester an essential factor(s) correlates with their transcriptional activity. In contrast, several 3'-deletions and some LSMs in domain I and II decrease transcription activity greatly without significantly decreasing competition ability. The results indicate that the stimulatory function of domain III is achieved through its interaction with an essential transcription factor(s), although the other domains also participate in this interaction, perhaps directly or through another protein factor.

A yeast ribosomal DNA-binding protein that binds to the rDNA enhancer and also close to the site of Pol I transcription initiation is not important for enhancer functioning.

Using the gel retardation assay we have identified a protein that can specifically bind to a site within the enhancer of the 37S pre-ribosomal RNA operon in yeast, as well as to a site 210 bp upstream of the site of transcription initiation of this operon. This protein (RBP1) has been partially purified by means of heparin-agarose chromatography and protects 20 bp in the rDNA enhancer, and 25 bp in the initiation region, against DNase I in an in vitro footprinting assay. In vivo footprinting studies using methylation of intact yeast cells with dimethylsulphate, indicate that the same binding sites are occupied in vivo as well. Deletions that abolish binding of RBP1 to the enhancer in vitro, as well as linker insertions into the RBP1 binding site in the initiation region that strongly diminish in vitro binding of RBP1, have no effect whatsoever on the enhancement of rDNA transcription in vivo. This was studied by deletion/mutation of the RBP1 binding site in vitro in an artificial ribosomal minigene and measuring the effect on the minigene transcription in vivo in yeast cells, transformed with the deleted/mutated minigenes. It can therefore be concluded that binding of RBP1 is not an important parameter in the functioning of the rDNA enhancer in yeast. Using the same minigene system we also show that RBP1 is not involved in termination of RNA polymerase I (Pol I) transcription at the main terminator T2.

Termination of transcription by yeast RNA polymerase I.

Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.

Paracetamol, 3-monoalkyl- and 3,5-dialkyl derivatives. Comparison of their microsomal cytochrome P-450 dependent oxidation and toxicity in freshly isolated hepatocytes.

The effects of 3-monoalkyl- and 3,5-dialkyl-substitution on the cytotoxicity of paracetamol (PAR) in rat hepatocytes was studied. PAR is known to be bioactivated by the hepatic microsomal cytochrome P-450 containing a mixed-function oxidase system presumably to N-acetyl-para-benzoquinone imine (NAPQI), a reactive metabolite which upon overdosage of the drug causes depletion of cellular glutathione (GSH) and hepatotoxicity. The four 3-mono- and the four 3,5-di-alkyl-substituted derivatives of PAR investigated in this study (R = CH3, C2H5, C3H7, C4H9) interacted with cytochrome P-450 giving rise to reverse type I spectral changes. Like PAR, all derivatives underwent cytochrome P-450-mediated oxidation to NAPQIs. In contrast to induction by phenobarbital, induction of cytochrome P-450 by 3-methylcholanthrene enhanced the microsomal oxidation of PAR and its derivatives. The NAPQIs formed from PAR and the 3-mono-alkyl derivatives by microsomal oxidation were found to conjugate with GSH and to oxidise GSH to GSSG. The NAPQIs formed from the 3,5-dialkyl-substituted derivatives, however, only oxidized GSH to GSSG. PAR and the 3-monoalkyl derivatives were found to deplete cellular GSH to about the same extent and to be equally toxic in freshly isolated hepatocytes from 3-methylcholanthrene treated rats. In contrast, the 3,5-di-alkyl-substituted derivatives of PAR did not affect the GSH levels and were not toxic in the hepatocytes, even at higher concentrations. It is suggested that the difference between the way of reacting of 3,5-dialkyl-NAPQIs and NAPQIs from PAR and 3-monoalkyl derivatives with thiols of cellular GSH and protein could account for the observed difference between the toxicity of the 3,5-dialkyl- and the 3-monoalkyl-substituted derivatives of PAR.