Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.
OBJECTIVE
The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics.


METHODS
A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins.


RESULTS
Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping.


CONCLUSIONS
The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.
Wistow
Graeme
G
Section on Molecular Structure and Function, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-2740, USA. graeme@helix.nih.gov
Bernstein
Steven L
SL
Wyatt
M Keith
MK
Fariss
Robert N
RN
Behal
Amita
A
Touchman
Jeffrey W
JW
Bouffard
Gerald
G
Smith
Don
D
Peterson
Katherine
K
D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002829:Choroid / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis*; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D009316:National Institutes of Health (U.S.); D020411:Oligonucleotide Array Sequence Analysis; D009885:Ophthalmology; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011817:Rabbits; D021101:Radiation Hybrid Mapping; D000076106:SAM Domain and HD Domain-Containing Protein 1; D014481:United States
D018076:DNA, Complementary; D005136:Eye Proteins; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D020559:Monomeric GTP-Binding Proteins