Isolation and amino acid sequences of two Kunitz-type protease inhibitors from the sea anemone Anthopleura aff. xanthogrammica

We have isolated a novel retina-specific gene, retinal fascin, encoding a new member of actin-bundling protein gene family, from a bovine retina cDNA library. The cDNA encodes a 492 amino acid protein which shows 36-57% amino acid identity with three vertebrate fascins, echinoid fascin and Drosophila singed gene. Northern blot analysis revealed that retinal fascin mRNA was exclusively expressed in the eye and not seen in other tissues examined. In situ hybridization analysis indicated that retinal fascin mRNA signals were found only in the inner segment of the photoreceptor layer and outer nuclear layer, indicating that retinal fascin was specifically expressed in photoreceptor cells. As fascins are actin-bundling proteins important for constructing several intracellular structures, retinal fascin might play a pivotal role in photoreceptor cell-specific events, such as disk morphogenesis.     

Expression profile of active genes in granulocytes

A number of genes active in granulocytes have been intensively studied as to the function of their products and their expression controls. However, the intensities and relative order of these gene activities have not been studied. This report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 3'-directed cDNA library that faithfully represents the mRNA population in the source cells. A significant fraction (20.3% of the total) of the expressed genes in granulocytes consisted of nuclear proteins such as DNA binding proteins, of secretory proteins such as cytokines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing this expression profile with 11 profiles similarly obtained with unrelated human cells/tissues, we discovered 10 novel genes that are likely to act specifically in granulocytes. Comparison of this expression profile with that obtained with granulocytoids widely used as a granulocyte model by inducing a cultured promyelocytic leukemia cell line HL60 showed similarities and dissimilarities of gene expressions.     

Visual acuity and macular hole size after unsuccessful macular hole closure

We have identified and characterized a novel protein from adult zebrafish retina, which we named ES1. Database search revealed that the ES1 gene has significant similarity to two genes with unknown functions: the Escherichia coli sigma cross-reacting protein 27a (scrp27a) and the human KNP-I/GT335. In situ hybridization and immunohistochemistry experiments showed that both ES1 mRNA and protein are expressed specifically in adult photoreceptor cells. ES1 seems to be a cytoplasmic protein. An ES1-like antigen was also detected in photoreceptor cells of goldfish with anti-ES1 antibodies. The retina specific expression and the evolutionary conservation suggest that ES1 protein may be important for maintaining normal retina structure and function.     

Localization of HRG4, a photoreceptor protein homologous to Unc-119, in ribbon synapse

PURPOSE: To characterize further HRG4, a novel photoreceptor protein recently identified by subtractive cDNA cloning, by sequence analysis and immunolocalization. METHODS: The rat homolog of HRG4, RRG4 was expressed and used to prepare an antibody. The antibody was used in Western blot analysis, and immunofluorescent localization at the light and electron microscopic levels of HRG4-RRG4 protein. The HRG4-RRG4 sequence was also analyzed for homologies. RESULTS: HRG4-RRG4 showed 57% homology with unc-119, a Caenorhabditis elegans neuroprotein causing defects in locomotion, feeding, and chemosensation when mutated. By Western blot analysis, the HRG4-RRG4 protein was demonstrable only in retina and was soluble in nature. Immunofluorescence microscopic study of human and rat retinas, using the HRG4-RRG4 antibody, and other rod and cone photoreceptor-specific antibodies showed that the HRG4-RRG4 protein is localized in the outer plexiform layer of the retina in the synaptic termini of rod and cone photoreceptors. Electron microscopic immunolocalization showed the protein in the cytoplasm and on the presynaptic membranes of the photoreceptor synapses. CONCLUSIONS: The homology to unc-119 and localization to the photoreceptor synapse are suggestive of a function for HRG4-RRG4 in photoreceptor neurotransmission. HRG4 is the first photoreceptor-enriched synaptic protein to be reported, suggesting that its function may be unique to the specialized ribbon synapses formed between photoreceptors and the horizontal and bipolar cells of the retina.     

Metastasizing placental site trophoblastic tumor: immunohistochemical and DNA analysis. 2 case reports and a review of the literature

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.     

Expression and localization in the fish retina of a homologue of the Alzheimer's related PS1 gene

Early-onset familial Alzheimer's disease (early-onset FAD) has been linked with mutations in the presenilin gene, PS1. Mutations in PS1 may affect the processing/trafficking of b-amyloid precursor-protein (bAPP) and favour the production of toxic amyloid-b fragments that are associated with neural degeneration. This study reports the expression of a PS1-like cDNA in the carp (Cyprinus carpio) retina (the encoded protein shows 76% identity to the human PS1 protein). Carp PS1 mRNA was localized by in situ hybridization to the photoreceptor cell, inner nuclear and ganglion cell layers. Expression of the PS1 gene in the rat retina was also confirmed. The retinal expression of PS1 raises the possibility that PS1 mutations also lead to neural degeneration in the retina.     

Identification of two novel diurnal genes by screening of a rat brain cDNA library

While the hypothalamus is fundamental for sleep and circadian regulation, the molecular mechanisms involved are poorly understood. We have used a differential gene expression technique to identify hypothalamic genes which have altered expression in rat sleep periods. Complex cDNA probes from rat hypothalami removed at Zeitgeber times 4 and 15 were hybridised to rat brain cDNA library girds. From 30 differentially expressed clones, six were further analysed and two were confirmed to exhibit increased expression at Zeitgeber time 4. A Northern blot hybridization of brain, heart, kidney, lung, testis and skin mRNA showed that both clones were brain specific. Therefore, we have identified two novel brain specific diurnally expressed hypothalamic genes. Both genes may have roles in sleep or circadian regulation.     

Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.     

Physiologically based pharmacokinetic models of 2'',3''-dideoxyinosine

In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10(5) and 10(6) clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 and Alu repeat sequences, housekeeping genes, and tissue-specific genes, such as alpha-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing "tissue-specific" genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.     

Arrestin and phosducin are expressed in a small number of brain cells

Retinal photoreceptor rods and pinealocytes contain well-characterized proteins such as arrestin and phosducin whose expression is highly restricted to these cell types. Transgenic mice having a LacZ gene under the control of an arrestin promoter expressed beta-galactosidase (beta-Gal) in the photoreceptor rods and pinealocytes. In addition, it was expressed in very small numbers of discrete cells in the habenular commissura, amygdala, ventral tegmental area and superior colliculus of the brain. Immunocytochemical studies with antibody probes revealed that high level of arrestin and phosducin were also found in the same cell types. Furthermore melatonin was found in those cells of the habenula commissura. The results indicate that novel cell types are present in the brain tissues. Since high levels of arrestin and phosducin expression are generally restricted to photoreceptor rod cells and pinealocytes, these data suggest that certain brain cells may have functions similar to pinealocytes.     

Isolation of a gene product expressed by a subpopulation of human lung fibroblasts by differential display

Fibroblasts are the major cell type responsible for synthesizing matrix constituents in lung and other connective tissues. Evidence indicates that fibroblasts are heterogeneous, and that subpopulations with some distinct properties are clonally selected and expanded in fibrotic diseases. However, few distinct markers capable of demonstrating the presence of fibroblast subpopulations in tissues have been isolated so far. With the objective of identifying proteins that could detect fibroblast subpopulations, we compared the messenger RNA (mRNA) expression of two cultured human lung fibroblast subpopulations by differential display. Total RNA was obtained, complementary DNA (cDNA) was synthesized, and the polymerase chain reaction (PCR) products obtained with several primer pairs were compared. One 724-bp product, which was strongly expressed by one human lung fibroblast subpopulation, was identified and cloned. This product was poorly expressed by the other lung fibroblast subpopulation. The mRNA for the gene encoding this product was not detectable in human smooth-muscle cells, endothelial cells, or epithelial cells, although it was present in dermal fibroblasts. The mRNA was detected in normal and fibrotic human lungs. Search of the National Center for Biotechnology (NCBI) GenBank DNA database with the sequence obtained from this clone revealed no significant matches. However, a search of the NCBI database of expressed sequence tags (dBEST) revealed five different human expressed sequence tag (EST) clones corresponding to the LR8 cDNA sequence. Six additional mouse and one pig EST clones were identified that showed significant similarity to the human fibroblast cDNA. Composites of the entire coding sequences for the human fibroblast gene product and the mouse homologue were assembled from the respective overlapping EST sequences. The open reading frame identified for each composite sequence predicted protein products of 270 and 263 amino acids for the human and mouse sequences, respectively, which were 52% identical, with three gaps. At the amino acid level, no significant sequence similarity was detected with any other sequences in exhaustive searches of the NCBI DNA and protein databases or the Blocks databases. A PCR product with predicted length and sequence was obtained by using a sense primer upstream to LR8 and an antisense primer within LR8. Our results indicate that this differentially displayed product represents a previously undescribed protein that could be useful for distinguishing fibroblasts, and possibly fibroblast subpopulations, from other cell types in lungs and other tissues.     

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.     

Spatial and temporal expression of cone opsins during monkey retinal development

The primate retina requires a coordinated series of developmental events to form its specialized photoreceptor topography. In this study, the temporal expression of cone photoreceptor opsin was determined in Macaca monkey retina. Markers for mRNA and protein that recognize short wavelength (S) and long/medium wavelength (L/M) opsin were used to determine (1) the temporal and spatial patterns of opsin expression, (2) the spatial relationship between S and L/M cones at the time of initial opsin expression, and (3) the relative time of cone and rod opsin expression (Dorn et al. [1995] Invest. Ophthalmol. Vis. Sci. 36:2634-2651). Adult cone outer segments were recognized by either L/M or S opsin antiserum. Of all adult cone inner segments, 88-90% contained L/M opsin mRNA, whereas 10-12% contained S opsin mRNA. Fetal cones initially showed cell membrane as well as outer segment labeling for opsin protein, but cell membrane labeling disappeared by birth. No cones at any age contained markers for both S and L/M opsin mRNA or protein. S and L/M opsin protein appeared in the fovea at fetal day 75. Once opsin expression progressed beyond the fovea, both mRNA and protein for S opsin were consistently detected more peripherally than L/M opsin. Cones at the peripheral edge of S opsin expression had basal telodendria that appeared to reach toward neighboring cones. Because interactions between cone populations could organize the cone mosaic, the spatial relationship between S cones and the first cones to express L/M protein was analyzed quantitatively by using double-label immunocytochemistry. No consistent relationship was found between these two cone populations. Cones are generated at least 1 week before rods across monkey retina. However, rod opsin protein appears in and around the fovea at fetal day 66, 1 week before cone opsin protein. This suggests that independent local factors control differentiation in these two photoreceptor populations.     

Selective amplification via biotin- and restriction-mediated enrichment (SABRE), a novel selective amplification procedure for detection of differentially expressed mRNAs

We present a novel subtractive enrichment protocol for the identification of differentially expressed mRNA species. This procedure, called SABRE (selective amplification via biotin- and restriction-mediated enrichment), uses selective streptavidin-biotin affinity and restriction enzyme site reconstitution to enrich for cDNA species more abundant in one population than in another. Analysis of liver cDNA from a mouse strain expressing the neomycin resistance gene demonstrated that this procedure is capable of identifying species present in one population but absent from another. Furthermore, experiments to identify genes with circadian expression patterns in mouse liver demonstrated that SABRE is capable of detecting even modest 2- to 10-fold differences in accumulation of moderately rare mRNA species, representing as little as 0.03% of total mRNA. These experiments identified the gene encoding coumarin 7-hydroxylase as displaying circadian expression in mouse liver.     

Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis

Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.