Neurotrophic activity and gene analysis of a pigment epithelium-derived factor (PEDF)

Tuesday, Sep 22, 1992 Bristol

X ICER Abstracts

RETINA

CODE: R-4

SEPTEMBER

22fIXJESDAY

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YOFQCULAR

RSONS;

1

9:co

E&JS

2

9:22

v

DEBORA GERALD

of Growth

R. Adler

Factors II

FARBER (USA) I. CHADER (USA)

and

II

Retinoids

1. Tombran-Tink, A. Swaroop and 3

9:44

A T . Borras’

IO:06

&ucure

m a Crvstallln P. Gonzalez, S. Zigler

J.F. Hejtmancik. (USA)

and

Functi P.A.

Chwbeek

and

J.N.

faaton in im -e.

prw.ntion inoluailm

r..pMl...ha.b.en rotinoia treatments

of photoraoeptors immunoreactiv. for opsin, but not for th. interpbotor...ptor retinoid binding protein (Iimp). However, the response to bwp is aooompanied by .mtmnsive d.v.lopwmt of glial-like cells, which is not 0bwrv.d after retinoid treatment. Epidermal growth faotor (BW) alao .timulat.s abundant glial cell aewogtmwk, but no upregulation of opain iammor4raotivity has been doteeted with thf* faotor. The expression of photoreceptor-qmoific genes is nom b&q studied at th. mRNA law.1, using the poly~ra.. ohain nro+ion. ongoing studies suggest that th. 1.v.l~ of opein, p.riph.rin, B-antigen ana IRBP mBNAs .re differentially affaotd by growth factor treatment. Supported by USPIfS grant EY05404.

(USA.*Spain)

and

tlu

of trqbio -on.

seldom seen in them. Acmpl.xpatt.Mof observed. For u.mple, both b?fJ? ad determine increases in the frequenay

PI.2 -

I.R. Rodriguez, 4

on

(PEDF) Pawar.

I. Rodriguez. H. G.J. Chader (USA)

The potential ml0 and treataent of r.tfnriL

Xopkins

r.mainpoorlyundustoodt lsmutabties ftiuae the involvement of othu r.tinal cnlls, and the m1..u1ar cbangee triggered by the faotors. we are imvutigating theaal issues by testing effootn of rqulatory agent., inoiuding trophic factors and retinoids. urriw dissocLatad c~eallv defined cultures Of mous. r.iillal -L.lls. These oultur.s contain multipolar nwrcme , photoremptor cells and m0rph0logiaally udiffOr~tiatad lound o.lls, but glial-lik. flat cells are

of a r

Jolms USA

man, fe attraating imsrm&ii att&&bm. S&rai labmatorise have report.6 effeots of g?Meh faotors oa photorueptars in viva ana in vitro, hut the maaaripias meahanisms

(USA)

and Gent

lumllImIuo~#i~mR

CELLS IIU VlltV. IL. Idler, Wilmar Imetitut., university SOhool of n.dioino, Baltimore, IQ.

Hope

NEUROTROPHIC ACTIVITY AND GENE ANALYSIS OF A PIGMENT EPITHELHJM-DERIVEDFACTOR(PEDF) Tombran-Tmk, J.,’ Rodriguez, I.,’ II. Pawar,’ A. S~aroop,~ and Chader, G.J.’ ‘National Eye Institute, NIH, Bethesda, MD USA *Department of Ophthalmology and Human Genetics, University of Michigan Medical School, Ann Arbor, MI USA PEDF, a 50 kD protein, secreted by human fetal RPE cell cultures induces neuronal differentiation in human Y79 retinoblastoma cells and may be important to the development of the neural retina. Roth peptide and nt sequences of PRDF share significant homology with members of the Serpin gene family. A 12 Kb plasmid DNA (it 101 PEDF) isolated from a human genomic cosmid library with the PEDF cDNA probe (n FS 17, 1503 nt) contains most of the gene as shown by restriction enzyme mapping and Southern hybridization using PCR fragments from the 5’, middle (668bp) and 3’ regions of the cDNA. PCR and Southern blot analyses of a human-rodent somatic cell hybrid panel map the PEDF gene to the distal short arm of chromosome 17. Fluorescent in-s& hybridlltion confirms the physical location of the PEDF gene to 17~13.1, a region associated with several known cancer diseases. Southern and Northern blot analysis indicate the PEDF gene is conserved in evolution and is expressed in several mammalian tissues including RPE cells. RFLP analysis indicates the PEDF gene contains a polymorphism for the restriction enzyme RSAl. A population study of 20 individuals from the CEPH family repository identities several informative individuals useful for pedigree and linkage analysis. The studies indicate that PEDF is a unique extracellular neuronal-differentiating agent for retinoblastoma cells and interestingly whose chromosomal location is near those of a number of cancer genes. 3

202

GUINEA PIG HEREDITARY CATARACT CONTAINS A SPLICE-SITE NOTATION IN A CRYSTALLIN GENE Rodriuuez, I.R., Gonzalez. P.. Zialer. S. and B&s. T, National Bye Institute, NIH, Bethesda, MD, USA and *Laboratorios Cusl, Barcelona, Spain.

A

A congenital cataract present in a guinea pig animal model provided us with the unique opportunity to study a hereditary lens disease at the molecular level. Zetacrystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library, and found to contain an internal 102 bp deletion. The deletion is located in the coding region and does not interrupt the reading frame. The resulting mutant protein lacks 34 amino acids in a position very close to the co-enzyme binding site, therefore affecting the nucleotide binding. Sequencing analysis of a normal genomic zeta-crystallin clone revealedthatthemissing 102 bp fragment corresponded to an entire exon (exon 7) which is also present in the cataractous genome. More interestingly, genomic sequencing results indicated that a dinucleotide deletion (AC) occurred in the cataractous DNA at the acceptor splice-site of intron 6. The presence of this mutation will result in the skipping of exon 7 during mRNA processing which in turn will produce an altered zeta-crystallin protein. We believe this is the first time a mechanism of cataractogenesis has been directly linked to a gene that is overexpressed specifically in the lens.

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