The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.

We began characterization of the protein–DNA interactions necessary for UV light induced transcriptional activation of the gene encoding chalcone synthase (CHS), a key plant defense enzyme. Three light dependent in vivo footprints appear on a 90 bp stretch of the CHS promoter with a time course correlated with the onset of CHS transcription. We define a minimal light responsive promoter by functional analysis of truncated CHS promoter fusions with a reporter gene in transient expression experiments in parsley protoplasts. Two of the three footprinted sequence ‘boxes’ reside within the minimal promoter. Replacement of 10 bp within either of these ‘boxes’ leads to complete loss of light responsiveness. We conclude that these sequences define the necessary cis elements of the minimal CHS promoter's light responsive element. One of the functionally defined ‘boxes’ is homologous to an element implicated in regulation of genes involved in photosynthesis. These data represent the first example in a plant defense gene of an induced change in protein–DNA contacts necessary for transcriptional activation. Also, our data argue strongly that divergent light induced biosynthetic pathways share common regulatory units.

Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily.Michael Becker-André , Irmgard Wiesenberg, Nicole Schaeren-Wiemers, Elisabeth André , Martin Missbach, Jean-Hilaire Saurat, and Carsten Carlberg Correction submitted by Drs. Becker-André , SchaerenWiemers, and André:The article reported the identification of the pineal gland hormone melatonin as a natural ligand of the nuclear orphan receptor RZR␤. The article described transient transfection assays showing stimulation of RZR␤ transactivation by melatonin and in vitro binding studies showing a direct interaction between RZR␤ and melatonin. We were, however, not able to reproduce any of these experiments nor could we determine the reason for this failure. As the central finding of the article is based on these assays, we do not consider the drawn conclusions valid. All other data in the publication including our studies to localize RZR␤ mRNA using in situ hybridization and quantitative PCR are as published. We apologize to all investigators who have spent time and effort to reproduce parts of the work using the in vitro assays described in the article.Addition submitted by Drs. Wiesenberg, Missbach, Saurat, and Carlberg: RZR␤ belongs to the RZR/ROR family of nuclear receptors (1), where the members of this family have been shown to act as ligand-independent transcription factors that bind to monomeric binding sites mediating a high constitutive transcriptional activity (2-5). Recently, Greiner et al. (6) reported failure in observing transcriptional activity of RZR␤ on monomeric sites and its modulation by melatonin. However, in some cell lines, e.g. Drosophila SL-3 cells, the constitutive activity of RZR␤ and RZR/ROR␣ can clearly be reduced, e.g. by the omission of serum to the cell culture medium (2,7,8). Under these restricted experimental conditions RZR␤ and RZR/ROR␣ reproducibly function as ligand-dependent nuclear receptors (7-10). Ligand binding assays have been performed with cells overexpressing RZR/ROR and in vitro translated RZR/ROR (7, 8), but not with the purified receptor. This suggests but does not prove that melatonin is a ligand of RZR/ROR. However, the first report of a melatonin response element in the promoter of the human 5-lipoxygenase gene (11) has demonstrated an essential role of RZR/ROR as a mediator of nuclear melatonin signaling.

Retinoid-related orphan receptor ␣ (ROR␣) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme ␤-galactosidase. In heterozygous ROR␣ ؉/؊ mice we found ␤-galactosidase activity, indicative of ROR␣ protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the ROR␣ gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, ROR␣ is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express ROR␣. Recently, it was reported that the ataxic mouse mutant staggerer (sg͞sg) is caused by a deletion in the ROR␣ gene. The analysis of the cerebellar and the behavioral phenotype of homozygous ROR␣ ؊/؊ mice proves identity to sg͞sg mice. Although the absence of ROR␣ causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of ROR␣ ؊/؊ mice display a normal phenotype. However, the pelage hair of both sg͞sg and ROR␣ ؊/؊ is significantly less dense and when shaved shows reluctance to regrow.Nuclear receptors form a structurally related superfamily of ligand-activated transcription factors (1). They are involved in several aspects of vertebrate physiology, such as development and homeostasis. Important examples are the steroid hormone receptors that regulate, in a ligand-dependent manner, specific sets of responding genes. The retinoid-related orphan nuclear receptor (ROR) ␣ (2, 3), ROR␤ (4), and ROR␥ (5) constitute a subfamily of nuclear receptors that bind to DNA both as monomers and dimers. Distribution of ROR␣ mRNA suggests that this receptor is widely expressed and functions in several organs including brain, heart, liver, lung, and testis; highest levels were found in peripheral blood leukocytes and skin (M.B.-A., unpublished data). ROR␣ exists in four splicing isoforms: ROR␣1-4. They display different N-terminal domains causing different DNA binding site preferences (3), and they display differential expression profiles: in the thalamus there is only ROR␣1 mRNA; ROR␣4 (ϭRZR␣) (2) transcripts are predominant in leukocytes and skin; ROR␣2 and ROR␣3 transcripts are exclusively detected in testis; and in the remaining tissues including the cerebellum there is a mixture of ROR␣1 and ROR␣4 transcripts (M.B.-A., unpublished results). In the central nervous system (CNS) ROR␣ mRNA localizes to the cerebellar Purkinje cells (PCs), various thalamic nuclei, and, during development, to other brain areas (6, 7). To study the physiological role of this orphan receptor we generated ROR␣ null-mutant mice by gene targeting. In the course of this work the genetic basis of the staggerer (sg) mutation in mouse was identified by positional cloning as a deletion in the R...