Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Prom7?+ Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-speciffc RNA probe. (13). The two strains employed, a transgenic mouse line hemizygous for a Pro347 -_+Ser mutation in the pig rhodopsin gene and a normal strain, shared the same genetic background, C57BL/6. The transgenic strain was constructed by using C57BL/6 mice and maintained by crossing hemizygous individuals with C57BL/6 mice. Potentially transgenic embryos were generated by mating hemizygous transgenic male mice to C57BL/6 female mice. As a result, half of the embryos should be transgenic. The normal mice, C57BL/6J -c2J/c2J (The Jackson Laboratory), were homozygous for albino, allowing identification of chimeras by coat color mosaicism and by observation of mosaic fundi with indirect ophthalmoscopy. Chimeras expressing the transgene were identified by standard tail-DNA analysis.Histology. Three chimeras expressing the transgene were sacrificed at 7 weeks of age, and the one lacking the transgene, at 14 weeks. Enucleated eyes were immersed in 0.1 M cacodylate buffer, pH 7.3/3% glutaraldehyde. Following overnight fixation at 4°C, eyes were bisected through the optic nerve head along either a superior-inferior or nasaltemporal axis. The eyes were postfixed in 2% osmium tetroxide and embedded in low-viscosity Spurr resin (14). Sections included the entire hemisphere that passed near the optic nerve head. Sections 0.5-1 ,um thick were taken and counterstained with toluidine blue dye.Construction and Synthesis of Probes for in Situ Hybridization. Two RNA probes were used for in situ hybridization; one specific for mouse rhodopsin and the other, pig rhodopsin. These probes were derived from DNA sequences in the 3' untranslated portions of the respective rhodopsin genes. The DNA fragments were cloned in the Novagen T-vector

The tumor suppressor gene p53 plays an important role in guarding genomic integrity. When induced in response to environmental results, the gene product of p53 functions as a transcription factor to transactivate genes involved in arresting the cell cycle and as a facilitator of DNA repair. In contrast, the status of p53 in Chinese hamster ovary (CHO) cells, commonly used as a model system for various studies including those involving the cell cycle and transformation, remains an enigma. In this study, the function and sequence of p53 in CHO.K1 cells were investigated. The level of p53 proteins was elevated on ultraviolet (UV) irradiation of the cells, and the proteins formed specific complexes as probed with DNA containing p53-binding sequences. Its activities toward responsive promoters were inducible by UV in a dose-dependent manner. Although p53 in CHO.K1 contained a single missense mutation at codon 211, the mutation apparently had no effect on the functional properties of the protein. The CHO.K1 cells on X-ray irradiation failed to arrest at G1 phase even when the cells were transfected with a wildtype human p53 gene, indicating that the failure probably was not caused by dysfunction of its p53, but by some other mechanism. This result is consistent with the finding that p21(Waf1/Cip1) is undetectable in UV-treated CHO.K1 cells, whereas Gadd45 is induced by UV light in the cells.

An indirect immunoperoxidase staining technique was employed to localize the heavy metal binding protein, metallothionein, in rat liver and kidney. Immunostaining for metallothionein was observed in all hepatocytes and most renal tubular cells. This protein was not detectable, however, in cell types such as endothelial or connective tissue cells, indicating that metallothionein synthesis or accumulation is tissue- and cell-type specific. Hepatocytes from cadmium-exposed animals showed increased staining for metallothionein. The presence of metallothionein was also seen extracellularly within the liver sinusoids and renal tubules in both normal and cadmium-exposed animals, suggesting transport and excretion, respectively, of this protein.

As reported in a preceding paper,' the analysis of the sedimentation behavior and base composition of the RNA recovered from hybrids between E. coli 16S and 238 RNA and homologous DNA, after RNase digestion of non-base-paired segments, has given results which suggest a regular and complete hydrogen bonding of the hybridized RNA with specific sites in DNA. A similar analysis has now been applied to the investigation of ribosomal RNA sites in HeLa and other human DNA's and has made it possible to resolve the ambiguities in specificity pattern and base composition presented by the raw hybridization data, and to identify among the RNA-DNA complexes a fraction which appears to involve specific sites in DNA. Materials and Methods.-Methods of growth in suspension of HeLa cells have been described previously.2 Ribosomal RNA labeled to a high specific activity with p32 was prepared by growing the cells for 48 hr in a modified Eagle's medium3 with 4.5 X 10-5 M phosphate and 5% dialyzed horse serum, in the presence of 20-25 ,.C/ml p32 carrier-free orthophosphate. In order to reduce the specific activity of the unstable RNA fraction,4 the cells were subjected to a chase for 16-20 hr in the presence of 10-2 M unlabeled phosphate. Ribosomal RNA was extracted from the ribosomal fraction2 with redistilled, water-saturated phenol containing 0.1% hydroxyquinoline,5 in the presence of 0.5% sodium dodecylsulfate, 5 mg/ml naphthalene disulfonate,2 and 1.3 mg/ml bentonite.6 Following 3 ethanol precipitations, the final product was dissolved in acetate buffer 0.01 AT, pH 5.0, NaCl 0.1 M, and resolved into the 28S and 18S components by two cycles of sucrose gradient (linear, 5-20%) centrifugation. Several P32-labeled preparations made in this way had at the time of isolation specific activities varying between 1.0 and 1.5 X 106 dpm/igg RNA. While the 288 preparations were always found to be free of any detectable P32 contaminant (less than 10-6), determined as RNase-resistant counts excluded by Sephadex G-100 or alkali stable counts, the 18S preparations usually showed between 0.1 and 1% RNase-resistant radioactivity associated with macromolecular material, which could be reduced 'to less than 10-4 by two or more passages of the 18S preparation through nitrocellulose membranes.' Reference is made to the preceding paper' for the procedures followed for 1)NA preparation and denaturation, RNA-DNA hybridization (incubation here was carried out at 70'C for 2'/2-8 hr), isolation of hybrids, and analysis of sedimentation properties and base composition of hybridized RNA after RNase digestion.

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of norma! and pathologmc human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited