The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.

Mice express S and M opsins that form visual pigments for the detection of light and visual signaling in cones. Here, we show that S opsin transcription is higher than that of M opsin, which supports ultraviolet (UV) sensitivity greater than midwavelength sensitivity. Surprisingly, most cones coexpress both S and M opsins in a common cone cell type throughout the retina. All cones express M opsin, but the levels are graded from dorsal to ventral. The levels of S opsin are relatively constant. However, in the far dorsal retina, S opsin is repressed stochastically, such that some cones express M opsin only. These observations indicate that two different mechanisms control M and S opsin expression. We suggest that a common cone type is patterned across the retinal surface to produce phenotypic cone subtypes.

Rhes: A striatal-specific Ras homolog related to Dexras1.

We have characterized an apparently full-length cDNA corresponding to a rat mRNA, SE6C, previously identified by subtractive hybridization as being expressed predominantly in the striatal region of the brain. The SE6C mRNA encodes a 266 amino acid protein with significant similarity to members of the Ras-like GTP-binding protein family; thus, we have chosen the name Rhes, for Ras homolog enriched in striatum. The human homolog was found in a genomic sequence from human chromosome 22q13.1 and shares 95% identity with rat Rhes. Among the family of small G-proteins, Rhes shares 62% identity with Dexras1, a mouse dexamethasone-inducible Ras-like protein. Both Rhes and Dexras1 have substantially longer C-termini than other members of the Ras-like small G-protein family. Divergence between the C-terminal sequences of Rhes and Dexras1 suggests that, although their functions are probably similar, they have unique properties. Bacterially expressed Rhes binds GTP, suggesting that the protein indeed has GTPase functionality. Although Rhes was not induced by dexamethasone, its full expression is dependent upon thyroid hormone availability. Its accumulation is postnatal, consistent with the dependence upon thyroid hormone. It is noteworthy that most striatum-"specific" mRNAs characterized to date encode components of signal transduction cascades.

Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction.

We report an improved subtractive cDNA cloning procedure, named "directional tag PCR subtraction," for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5' and 3' ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.

Identification and characterization of transcribed sequences on human chromosome 9q32-34.

The gene responsible for the neuromuscular disease idiopathic torsion dystonia (DYT1) has recently been mapped to human chromosome 9q32-34. Our goal is to identify candidate genes for torsion dystonia as well as other neurologically important genes in this region. To accomplish this we have characterized the expression patterns of transcribed sequences identified within a collection of 3000 human 9q32-34-specific clones. Screening of this clone collection with cDNA probes from various brain and peripheral tissues resulted in the identification of 143 clones corresponding to 9q32-34-specific transcripts. Thirty three of these corresponded to transcripts expressed in a brain-specific manner and thus represent preferred candidates for the dystonia gene. None of these candidates were expressed specifically in the putative dystonia target tissue, basal ganglia. The 9q32-34 collection was screened with a subtracted probe enriched in striatal sequences using a directional tag PCR subtraction method. This resulted in the identification of several genes exhibiting preferential expression in the striatum as compared to cerebellum.

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Isolation and analysis of the mouse opsin gene.

We have identified three overlapping 5'-truncated mouse opsin cDNA clones by immunologically screening a lambda gt11 retina expression library. Using one of the cDNA clones as a probe, we isolated a 5 kb genomic fragment that encompassed the complete coding sequence for mouse opsin. The coding region for opsin was interrupted by four introns positioned precisely as those previously described for other mammalian opsins. In contrast to the single major opsin mRNA in the bovine and human retina, Northern analysis of mouse retina RNA demonstrated the presence of at least five distinct species of polyadenylated opsin mRNAs. Their sizes ranged from 1.7 kb to 5.1 kb.

Exploratory studies of lipid-pectin interactions.

This study was undertaken to elucidate the mechanism responsible for the hypolipidemic action of pectin. The experiments reported here were designed to test if direct molecular interactions exist between pectin and lipids. Equilibrium dialysis of pectin and taurocholate showed binding only at high nonphysiological ionic strength. When lipid microemulsions and micelles of low charge density were used, unambiguous proof of binding to pectin were obtained by NMR spectroscopy and gel exclusion chromatography. The results suggest that the interaction in mainly by hydrogen bonds involving the pectin carboxylic moieties. The quantitation of lipid binding by pectin could be established only in presence of polyvalent cations using a membrane filtration technique. Under optimum conditions, pectin can bind four times its weight in lipids. Although the techniques presented here are physical-chemical, the conclusions are highly relevant to bioavailability. These results represent the first successful demonstration of direct lipid-polysaccharide interactions in biochemistry, and they have an obvious bearing on the physiological absorption process. The intestinal binding of dietary and biliary lipids by pectin may be a major mechanism of action of this hypolipidemic polysaccharide. This paper also cells attention to techniques which could be beneficial for the in vitro evaluation of plant fibers.

Understanding children's art: an analysis of the literature.

This article attempts to answer the question: "Why is it that an intuitively obvious means of understanding children has not been documented as such?". This is done through a critical analysis of the literature dealing with the validity of the Draw-A-Person Test (DAP). Studies reviewed are analyzed along three parameters; projective test theory, subject population, and psychodiagnostic labeling. On the basis of this analysis, explanations for inconsistencies in the results of the research and suggestions for organizing future work in this area are submitted.