Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.
OBJECTIVE
To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses.


METHODS
A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering.


RESULTS
The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described.


CONCLUSIONS
The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.
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
Behal
Amita
A
Touchman
Jeffrey W
JW
Bouffard
Gerald
G
Smith
Don
D
Peterson
Katherine
K
D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009316:National Institutes of Health (U.S.); D020411:Oligonucleotide Array Sequence Analysis; D009885:Ophthalmology; D016133:Polymerase Chain Reaction; D021101:Radiation Hybrid Mapping; D014481:United States
D018076:DNA, Complementary; D005136:Eye Proteins; C044539:LIM2 protein, human; D008565:Membrane Proteins; D005974:Glutamate-Ammonia Ligase