ABSTRACT
PURPOSE: We recently used microarray and reverse transcriptase PCR (RT-PCR) analysis to show an upregulation of cathepsin S (CatS) and glutathione peroxidase 3 (GPX3) in the aging mouse RPE/choroid. To evaluate the mRNA distribution and levels in the RPE and choroid, in situ hybridizations were performed. METHODS: Eye sections from 2-month-old and 24-month-old C57BL/6 mice were probed for CatS or GPX3 mRNA by in situ hybridization. The ratio of mRNA labeled cells to total cells counted per section was compared between the two age groups for the RPE and choroid separately. RESULTS: The CatS labeled RPE cell ratio increased significantly with age. The GPX3 labeled RPE cell ratio did not increase with age. CONCLUSIONS: The increases in mRNA levels for CatS and GPX3 found in the aging C57BL/6 RPE/choroid appear to represent an increase in both the numbers of cells expressing these messages and an increase in the level of expression in individual cells.
Subject(s)
Aging/genetics , Cathepsins/genetics , Choroid/metabolism , Gene Expression Regulation/physiology , Glutathione Peroxidase/genetics , Pigment Epithelium of Eye/metabolism , Animals , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolismABSTRACT
PURPOSE: cDNA libraries from the mouse retina have recently been reported, but no well characterized library from the retinal pigment epithelium (RPE) or choroid of the mouse has yet appeared in the literature. To complement these libraries and to provide the first mouse RPE/choroid library, we used freshly dissected tissue from adult C57BL/6J mice to construct new retina and RPE/choroid libraries. METHODS: Eyes from 100 six to eight week old C57BL/6J mice were dissected in groups of 10. The whole retina and RPE/choroid were isolated individually and then homogenized before RNA isolation. Over 5000 clones each were sequenced from the unamplified and un-normalized retina and RPE/choroid libraries. All sequences were analyzed using GRIST (GRouping and Identification of Sequence Tags), a bioinformatics program for gene identification and clustering. RESULTS: The RPE/choroid library contained 3145 clusters with 76% of the clusters representing single clones. Nearly 87% of the clusters corresponded to named genes in GenBank, and 8% of the RPE clusters remain unidentified. The retina library contained 3190 clusters of which 78% represented only one clone. Approximately 85% of the clusters matched sequences in GenBank, and 9% of the clusters remain unidentified. The clones most abundant in each library were all well-known sequences and both libraries contained a number of tissue specific or tissue-enhanced genes. CONCLUSIONS: These new libraries should provide a valuable resource for gene discovery and cDNAs for expression analysis and functional studies.
Subject(s)
Choroid/metabolism , DNA, Complementary/analysis , Expressed Sequence Tags , Eye Proteins/genetics , Gene Library , Pigment Epithelium of Eye/metabolism , Retina/metabolism , Animals , Eye Proteins/metabolism , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , RNA/isolation & purification , RNA, Messenger/metabolismABSTRACT
To evaluate the age-related changes in gene expression occurring in the complex of retinal pigmented epithelium, Bruch's membrane, and choroid (RPE/choroid), we examined the gene expression profiles of young adult (2 mo) and old (24 mo) male C57BL/6 mice. cDNA probe sets from individual animals were synthesized using total RNA isolated from the RPE/choroid of each animal. Probes were amplified using the Clontech SMART system, radioactively labeled, and hybridized to two different Clontech Atlas mouse cDNA arrays. From each age group, three independent triplicates were hybridized to the arrays. Statistical analyses were performed using the Significance Analysis of Microarrays program (SAM version 1.13; Stanford University). Selected array results were confirmed by semi-quantitative RT-PCR analysis. Of 2,340 genes represented on the arrays, approximately 60% were expressed in young and/or old mouse RPE/choroid. A moderate fraction (12%) of all expressed genes exhibited a statistically significant change in expression with age. Of these 150 genes, all but two, HMG14 and carboxypeptidase E, were upregulated with age. Many of these upregulated genes can be grouped into several broad functional categories: immune response, proteases and protease inhibitors, stress response, and neovascularization. RT-PCR results from six of six genes examined confirmed the differential change in expression with age of these genes. Our study provides likely candidate genes to further study their role in the development of age-related macular degeneration and other aging diseases affecting the RPE/choroid.
Subject(s)
Aging/genetics , Choroid/metabolism , Gene Expression Profiling/methods , Pigment Epithelium of Eye/metabolism , Retina/metabolism , Transcription, Genetic , Animals , Bruch Membrane/chemistry , Bruch Membrane/metabolism , Choroid/chemistry , DNA, Complementary/genetics , Gene Expression Profiling/statistics & numerical data , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Pigment Epithelium of Eye/chemistry , Retina/chemistry , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical dataABSTRACT
Oxidative stress has been studied in the retinal pigmented epithelium (RPE) in vitro but not in vivo. Our purpose, therefore, was to develop an in vivo model of acute oxidative stress in the C57BL/6J mouse. Mice were exposed to > or = 98% oxygen for 0, 2, or 6 h, and amplified total RNA from the RPE/choroid was applied to microarrays examining about 2200 unique genes. Statistical analysis determined that 642 genes, out of a total of 1349 expressed, were significantly downregulated at only 2 h, only 6 h, or both 2 and 6 h, and a single gene, ubiquitin, was upregulated. These genes are involved in all aspects of cellular functions, and there are no major differences among the three groups. The effect of hyperoxia on the RPE/choroid in vivo appears to be very similar to oxidative stress studies performed with an RPE cell line in vitro. All 11 genes identified as being regulated by all three oxidants in our previous study, and were expressed by mouse, were also differentially regulated by hyperoxia. At least for the initial response to an oxidative challenge, the in vitro ARPE-19 cell line is a reasonable model for in vivo studies.
Subject(s)
Choroid/metabolism , Gene Expression Regulation/physiology , Hyperoxia/metabolism , Oxidative Stress , Pigment Epithelium of Eye/metabolism , Transcription, Genetic , Animals , DNA, Complementary/genetics , Eye Proteins/metabolism , Gene Expression Profiling/methods , Male , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The general stress response of Bacillus subtilis is triggered by a variety of environmental and metabolic stresses which activate the sigmaB transcription factor. Among the more than 100 genes controlled by sigmaB (the csb genes), the functions identified thus far include resistance to oxidative stress, resistance to protein denaturation and resistance to osmotic stress. To understand the breadth of functions in which csb genes participate, the transcriptional organization and predicted products of two such genes previously identified in a screen for sigmaB-dependent lacZ fusions were analysed. The csb-22::Tn917lacZ and csb-34::Tn917lacZ fusions are unusual among csb genes in that their expression appears to be completely dependent upon sigmaB. By plasmid-integration experiments, fusion analyses and site-directed mutagenesis, stress-inducible, sigmaB-dependent promoters for both these fusions were identified. The csb-34 fusion marked an ORF (yxcC or csbC) which by sequence analysis lay in a monocistronic transcriptional unit. This ORF encoded a predicted 461-residue product which had high identity with Class I sugar transporters of the major facilitator superfamily. It was speculated that the csbC product could serve either a nutritional or an osmotic protection function. In contrast, the csb-22 fusion identified an ORF (ywmG or csbD) which appeared to be the second gene of a two-gene operon. This ORF encoded a predicted 62-residue product which resembled a small Escherichia coli protein of unknown function. The sigmaB. dependent promoter lay immediately upstream from csbD and appeared to be an internal promoter for the operon.
