Functional analysis of the promoters of the human red and green visual pigment genes.

PURPOSE: To delineate cis-acting DNA elements involved in the expression of the human red and green visual pigment genes and to correlate these with transcription factor binding sites. METHODS: Assays of promoter activity were accomplished by transient transfection into WERI cells. Nested deletion and block mutagenesis were undertaken to delineate critical elements. Transcription factor binding sites were determined by DNase I footprinting and electrophoretic mobility shift (EMSA) analyses. RESULTS: The human retinoblastoma cell line WERI, but not Y-79, was found to express the red and green pigment genes. Transfection assays in WERI cells revealed that the proximal region of the red pigment gene promoter had positive (-130 to -113 and -96 to -23) and negative (-190 to -130 and - 113 to -96) regulatory elements. The green pigment gene promoter was found to be 2 to 4 times more active than was that of the red pigment. This difference in activity was attributable mainly to a T to C substitution at position -3. DNase I protection and EMSA studies demonstrated the binding of several ubiquitous and WERI-enriched proteins to DNA sequences between - 130 and the TATA box. The locus control region (LCR) did not have any enhancer activity in transient transfection. CONCLUSIONS: The WERI cell line is a good model system for the analysis of gene expression of the human cone visual pigment genes. The expression of these genes in a cell-specific fashion seems to be controlled mainly by positive-acting elements in the region between - 130 and the TATA box. The higher activity of the green pigment gene promoter could have evolved to compensate for its longer distance from the activating LCR than that of the red pigment gene promoter (approximately 34 versus 3.5 kb). Although the LCR does not enhance transcription in transient transfection, it binds factors that also recognize the proximal promoter region. These interactions may be important for the establishment of a transcriptionally active domain in a chromatin context.

Transgenic mice expressing a functional human photopigment.

PURPOSE: Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction. METHODS: Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG). RESULTS: The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG. CONCLUSIONS: The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.

In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

Effect of temperature and ageing on the mechanical properties of dental polymeric composite materials.

Evaluation of the mechanical properties of some dental composite materials, Compact, Finesse and Prisma-Fil based on bisphenol glycidyl methacrylate resin was undertaken by applying compression, tension and hardness tests. The effects of temperature and ageing times on these properties were studied. There was a marked increase in the mechanical properties (compressive strength, diametral tensile strength, compressive elastic modulus and hardness) for all the investigated composites with increase of both temperature and time. This was explained in terms of the influence of temperature on the polymerization rate of the materials. The improvement in the mechanical properties of the samples, kept at 37 degrees C, was attributed to further and continued polymerization of the polymer content of their resin system. Such mechanical improvement was verified by the regression equation of linearity versus both temperature and time.

pH-induced difference spectrophotometric methods for drug analysis. Part 2: Determination of mefenamic and flufenamic acid.

On the basis of the spectral changes induced by changing the solvent medium from HCl (0.01 mol . l-1) to NaOH (0.01 mol . l-1), coefficient-difference spectrophotometric methods using the six-points quadratic order of the orthogonal polynomials have been developed. The optimum wavelength ranges were 326 to 386 nm for mefenamic acid and 322 to 382 nm for flufenamic acid, both measured at 12 nm intervals. The respective mean percentage recoveries were 100.1 +/- 0.89 over a concentration range of 0.6-2.6 mg/100 ml of mefenamic acid and 100.7 +/- 0.61 over a range of 0.2-2.4 mg/100 ml for flufenamic acid (p = 0.05). Both methods gave precise results with relative standard deviation less than 1%. Trials to adapt single wavelength difference technique as well as the cubic order of the orthogonal polynomials were also performed; their results were discussed on a statistical basis. The developed procedures have been applied to the analysis of some randomly collected market preparations and the results obtained proved suitability for application in routine analysis.