A method is described that allows a general drawback of in vitro transcription assays to be overcome: RNA polymerases tend to add extra nucleotides to the RNA 3' end that are not encoded in the linearized DNA template. Furthermore, these polymerases show a considerable rate of premature termination close to the RNA's 3' end. These features lead to a decreased yield of full-length transcripts and often make it difficult to determine and isolate the correctly transcribed full-length RNA. The hammerhead ribozyme is frequently used in cis to cleave off these extra nucleotides. However, the upstream sequence requirements of this ribozyme restrict its general usability. In contrast, the hepatitis delta virus ribozyme has no such requirements and can therefore be applied to any RNA sequence in cis. Due to the catalytic activity of the ribozyme, the desired transcript is released as an RNA molecule with a homogeneous 3' end. The resulting 2',3'-cyclo-phosphate group of the released RNA can be easily and efficiently removed by T4 polynucleotide kinase treatment. The presented method can be applied for virtually any sequence to be transcribed and is therefore superior to other ribozyme strategies, suggesting possible applications in every field where transcripts with homogeneous 3' ends are required.

Inherited and isolated nail malformations are rare and heterogeneous conditions. We identified two consanguineous pedigrees in which some family members were affected by isolated nail dysplasia that suggested an autosomal-recessive inheritance pattern and was characterized by claw-shaped nails, onychauxis, and onycholysis. Genome-wide SNP array analysis of affected individuals from both families showed an overlapping and homozygous region of 800 kb on the long arm of chromosome 8. The candidate region spans eight genes, and DNA sequence analysis revealed homozygous nonsense and missense mutations in FZD(6), the gene encoding Frizzled 6. FZD(6) belongs to a family of highly conserved membrane-bound WNT receptors involved in developmental processes and differentiation through several signaling pathways. We expressed the FZD(6) missense mutation and observed a quantitative shift in subcellular distribution from the plasma membrane to the lysosomes, where the receptor is inaccessible for signaling and presumably degraded. Analysis of human fibroblasts homozygous for the nonsense mutation showed an aberrant response to both WNT-3A and WNT-5A stimulation; this response was consistent with an effect on both canonical and noncanonical WNT-FZD signaling. A detailed analysis of the Fzd(6)(-/-) mice, previously shown to have an altered hair pattern, showed malformed claws predominantly of the hind limbs. Furthermore, a transient Fdz6 mRNA expression was observed in the epidermis of the digital tips at embryonic day 16.5 during early claw morphogenesis. Thus, our combined results show that FZD6 mutations can result in severe defects in nail and claw formation through reduced or abolished membranous FZD(6) levels and several nonfunctional WNT-FZD pathways.

Premature ovarian failure (POF) is characterized by hypergonadotropic hypogonadism and amenorrhea before the age of 40. The condition has a heterogeneous background but genetic factors are demonstrated by the occurrence of familial cases. We identified a mother and daughter with POF both of whom carry an X;autosome translocation [t(X;11)(q24;q13)]. RNA expression studies of genes flanking the X-chromosome breakpoint revealed that both patients have reduced expression levels of the gene Progesterone Receptor Membrane Component-1 (PGRMC1). Mutation screening of 67 females with idiopathic POF identified a third patient with a missense mutation (H165R) located in the cytochrome b5 domain of PGRMC1. PGRMC1 mediates the anti-apoptotic action of progesterone in ovarian cells and it acts as a positive regulator of several cytochrome P450 (CYP)-catalyzed reactions. The CYPs are critical for intracellular sterol metabolism, including biosynthesis of steroid hormones. We show that the H165R mutation associated with POF abolishes the binding of cytochrome P450 7A1 (CYP7A1) to PGRMC1. In addition, the missense mutation attenuates PGRMC1's ability to mediate the anti-apoptotic action of progesterone in ovarian cells. These findings suggest that mutant or reduced levels of PGMRC1 may cause POF through impaired activation of the microsomal cytochrome P450 and increased apoptosis of ovarian cells.