Isolation and Characterization of Cell-Specific cDNA Clones from a ...

J. Biochem. 118, 921-931 (1995)

Isolation and Characterization of Cell-Specific cDNA Clones from a Subtractive Library of the Ocular Ciliary Body of a Single Normal Human Donor: Transcription and Synthesis of Plasma Proteins1 Julio

Escribano,2

Department

of

CT

USA

06520,

Received

Javier

Ophthalmology

for publication,

A subtractive

of

significant

homologies

significant

genes

ciliary

body,

protein other confirmed

or

proteins, [35S]methionine of synthesis

the

Key

in

the

humor

mechanisms of

yet

secretory

vessels, ed

smooth

adrenergic

bilayer,

the

ciliary by

gap

(3), (4-6). epithelia

This

work

and

The

differences

barrier, (7).

The

tissues

of

was

supported

whereas

aqueous

the

the

segment

research

by

blood

forming

the (NPE) to

each

marked to

grant

and ƒÀ

and

layer

humor

anterior by

(IOP)

in comparison

NPE

carrier

establishes

PE

layer

fluid

nourishes of

from

the the

a

forms

a the

eye

and

National

Institutes of Health EY-04873. Additional support was from a Vision Core Grant EY-00785, and Research to Prevent Blindness, Inc. J. E. was a recipient of a postdoctoral fellowship from the Ministerio de Education 2 Present

y Ciencia of Spain. address: Section de

Regional, Spain. To whom

3

- 2742, Fax: mail.yale.edu

Universidad

de

correspondence +1-203-785-7694,

Vol. 118, No. 5, 1995

Biotecnologia

Castilla-La should

be e-mail:

, Instituto de Desarrollo Mancha, 02071 Albacete,

addressed: miguel

group

of

biologically

expression

cells

this

to

two

the

apolipo these and factor)

(pigmented,

that

nonpig

human

metabolically

are work

in

and

Antibodies epithelium-derived

that the in aqueous

number revealed

plasma

labeled

with

ciliary body is an organ humor. These results imported

should affecting

be the

from

outside

very

useful

ciliary

body,

of for or

C4, ƒ¿2

macroglobulin,

ocular

ciliary

exhibits immunosuppresive and antioxidant properties (8, 9). The rate of secretion and drainage of aqueous humor determines the level of IOP, which in mammalians follows a circadian rhythm influenced by a light-dark cycle (10). Previous studies have documented that elevated IOP is a high risk factor in developing primary open-angle glau coma, a severe eye disease that can lead to blindness. Very little is known about the cellular and molecular biology of the ciliary body and aqueous humor secretion. Recently, our laboratory applied several immunoscreening strategies to isolate and characterize genes highly express ed in the ocular ciliary epithelium from directional expres sion cDNA libraries (6, 11). These studies suggest that the ocular ciliary epithelium exhibits many functions other than secretory. Specifically, the high levels of expression of genes coding for anti-oxidative, and detoxification enzymes suggest an additional functional role in protecting the anterior segment of the eye from oxidative stress (6, 9). We sought to identify biologically significant genes, conferring selective phenotype to the different cell types of the ciliary body, by characterizing a subtractive cDNA library from the ciliary body of a normal human single donor. The construction of highly representative cDNA libraries is critical to determine the tissue-specific func tions. In the case of the ciliary body, which is comprised of distinct cell types, the specificity of expression might be

regulates

exhibit

receptors, the

A

evidence

abnormalities

component

subunit ƒ¿

neurotransmitter

ocular

a large

pressure.

coupled

cells

genes.

humor in

of

genes regulating

databases

transcriptional

are

aqueous

innervat

and

Na+,K+-ATPase

Furthermore,

in

sequence

epithelial

suggesting present

in

for

protein

hybridization. pigment

ciliary

of a bilayer

cells

NPE

and

reported

nonpigmented

polarized 2).

Haven,

cell-specific

selenoprotein-P;

provide

explants, proteins

containing

epithelial

physiological

blood-aqueous

avascular 1

are

including

proteins leaky

The

(1,

(CB)

postsynaptically

(PE),

cells,

and cells,

isoforms

tight

fibers.

junctions

biochemical PE

cells

pigmented

epithelial

other

the

muscle

St., New

library.

pressure

stroma

have

and ƒ¿2-macroglobulin

complement

is comprised

a

and

known

in

genes

body

CB

DNA

acid

to

We

intraocular

D,

ciliary

The

Cedar

responsible

macroglobulin;

proteins

of

intraocular

cells,

330

identifying

tissue

Partial

Northern protein

library

subtractive

the

unknown.

by

C4

candidate

regulation

and

epithelial

and

by

eye,

C4; ƒ¿2

of

a

nucleic

known

cells.

cDNA

apolipoprotein

secretion

of

not

plasma

selenoprotein-P,

mammalian

aqueous

the

words:

body,

In

that

potential

involved

of Medicine,

purpose

independently

processes of plasma

subtractive

identifying

School

body,

expression

ciliary secretion

notion

The

90

component

and the

eye.

least

the

pressure.

previously

endothelial

in

challenge

at

cell-restricted

vascular

for

searches

component

complement

University

ciliary

further demonstrated (i.e., tyrosinase-related

their

mented),

to were

complement

D, were proteins

Yale

intraocular

homology

that

Coca-Prados3

developed ocular

and

and

Science,

was

human

secretion

clones

Miguel

1, 1995

the

humor

and

Visual

library

within

cDNA

and

June

cDNA

expressed aqueous

Ortego,

Tel:

+1-203-785

coca-prados@quick

921

922

J. Escribano

determined type.

by

By

from

the

clones

genes

selecting

body

this

enriching

subtractive an

directional

a high

the

GenBank/EMBL

level

of

to

endocrine,

immune

encoded

the

proteins

(i.e.,

globulin)

was

also

processes

explants

plasma ciliary of

by

C4

labeled

in

are to

ized

genes

synthesized the protein

these

AND

Directional

cDNA

phagemid this work. the

ciliary

bodies

primary

bone,

and

cancer.

of

2

that within composition

The

of

sense

the

obtained

by

the

cDNA

eyes

enucleation. of

and

La

column

chromatography,

400-bp

were

and

titer

the

of

and

library

amplified

and

contained

the

the DNA

phagemid

procedures

(12),

cDNA

poly(A)+ ODM-2,

RNA using a human previously characterized

lower

in

than

cDNA

[as

body phage

protocol ed with

(Stratagene). VCS-M13

ity and

NH4-acetate

and

and

cells XR and

of infections significant

was

in

were ciliary

at 10:1

following

infected body

incubated

sense

tics,

NY).

and

the

found.

1:2

4h

12•~106 and

at 37•Ž.

(helper: were

cell)

detected

a pre-hybridization

and

multiplic also yield

step

specific

resulted

fied

this

step,

of

was EcoRI

in a considerable

Diagnos

extracted the in

to

of

ssDNA

block

and

Mole

carried the

pBluescript T3

by

presence

was

of

by

PCR

48-bp

the

169-bp

out, multi

phagemid T7

reduction

promoters. in

multicloning

pBluescript using

T3/T7

were

generated

multicloning used

(Enzo

the

loss

of

clones.

For

- and

polymerase,

in

the

Xhol

RNA

Region

by

with biotinylated

hybridization

flanked

10M

inserts

vitro

kit

step

of

DNA

digested

precipitated

1

amplifica

phenol-chloro

ethanol.

T3

480

consisted 63•Ž for

volume

purified,

sequences

are

by

in

at

Cycler

After

of 0.1

was

Multicloning

polylinker)

which

non-recombinant

pfu from the 12•~107 R408

in the

the

performed

This

and ethanol.

5 min.

labeling

RNA

1.5 cycle

amplification

Thermal

of

RNA

and

polymerization

purified,

presence

Biotinylated

(or

1mM

(T7),

by

synthesize

we

12•~107

were

the

BioProbe

of

ssDNA,

and

cold

were to

subtractive

was compar ssDNA, and

Other

kb

of

cules-Before

cloning

primers. PCR of 0.25mM

denaturation

extracted

volume

(ss)

the

initial

for

modified and T7

polymerase,

presence

was

manufacturer's

Routinely

3

in

ENZO

4•Ž,

phenol-chloroform, NH4-acetate

human

using

at

templates

RNA(+)

line, of

using

primer

DNA

72•Ž

in the

0.3-1.6

as

the

(ssDNA) the

cells

phage R408 in rescuing

with library

for

differences

from

XL1-Blue

The helper its efficiency was

DNA

cooled

from used

using

Single-Stranded

SK(-)] in

and

difference

phage

no

precipitated

from

libraries

phagemid

library

R408

significant

helper

to

pBluescript

cDNA

helper

XL1-Blue ă-Uni-Zap

cDNA

Libraries

single-stranded

rescued

no

and

1.5•~1010

constructed

PCR

stan

phagemid

Cetus, Norwalk, CT). The process at 94•Ž for 1 min, annealing at at

double by

in

followed

Elmer

the cDNA cells,

The

volumes

63•Ž

was

polymerization

ranging

epithelial cell The proportion

amplified

DNA

at

This

a Perkin

form

an

excising

isolated

DNA

with

first

inserts

reverse

annealing

min.

on

Blocking of

DNA-Antisense

ciliary

ciliary (12).

1.5

tion

were

AmpTaq 1mM

inserts ODM-2

the ODM-2 and SOLR

then

cDNA

for

the

Stratagene.

was

70•Ž

dsDNA-Bio

by from phage

of

cycles,

1 min,

for

and

unamplified

contained

40

min,

was

recombinants

was

for

for

72•Ž

1%.

Conversion

was

estimated

MgCl2

of

the

dsDNA

from

DNA helper

and

and

from

accomplished

DNA

(T3),

by than

size

5U

(Perkin Elmer of denaturation

cloning

insert

mix, primer

The as

polymerase

2 min,

from

(5•Œ-GTAATACGACTCACTATAGGGCG-3•Œ) reactions were carried out in 300ƒÊl

performed

bigger Xhol

for

RNA

recommendations

kit

manufacturer's fractionated

average

library

both

synthesized

and

library

A

of

DNA

50•Ž

pBluescript SK(-) were amplified by T3 (5•Œ-GCAATTAACCCTCACTAAAGGGAA-3•Œ)

forward

cells.

phagemid

amplification

was

stranded

at 94•Ž

24h

at

phagemid ExAssist

following

mM

National

synthesis

inserts

million

pfu/ml.

non-recombinants

were

to size

an

One

amplified

second

the

This

XL1-Blue

synthesized

DNA

ssDNA

electrotransformation

AmpliTaq

was

DNA

the

(5•Œ-GCAATTAACCCTC

Biotinylated

PCR

(ds) by

ssDNA

primer

sug

was evaluated and the dsDNA synthes

coli

CT)

RNA

library.

nucleotide

and breast

within

EcoRI

The

donor liver

was

a cDNA

cDNAs the

phage.

pfu/ml.

death

strands using

into

in

through

CA) according cDNAs were

XR

4•~106

of

(NDRI)

second

subcloned

of ă-Uni-Zap

1-kb,

cause

RNA,

Jolla, The

female

metastasis

obtained

poly(A)+

(Stratagene, recommendations.

was

XR

were used in RNA from

a 34-year-old

Interchange

First

8ƒÊg

of

death

were

Vectors-Two

after

and

of

tinylated

enzymes,

previously

obtained

using

Wilton,

eliminated

Double-Stranded

ssDNA,

T3

pBluescript SK(-) library using the

in ă-Uni-ZAP

La Jolla, CA) from poly(A)+

secondary

Research

Phagemid

constructed

eyes

cause whose

Disease

from

in

libraries

the

a modified

was

above, with

as

contami

restriction as

units

synthesized

Preparation

of ciliary

METHODS

vectors (Stratagene, One was constructed

whose

sites

Libraries

cDNA

into

Escherichia

was

dard

directional

from

(Perkin-Elmer, 4 min.

humor.

MATERIALS

vitro

Xhol

as described titer obtained

electrocompetent

template,

and ƒ¿2-macro

propose

ssDNA

colony-forming

purified with the

in

preparation and

purified,

(dsDNA)

MgCl2-phenol,

ACTAAAGGGAA-3•Œ)

of

from

We

of

was

DNA

EcoRI with

15). of

titer

phagemid

phagemid

with

extraction

dsDNA

immunoprecipitation

vitro.

- The

of

of

ssDNA

gested (14, Conversion

in

neuro

some

(ii)

ssDNA

Double-stranded

digestion

phagemid, compared

immunodetected of

extracted

proteins reported here body aaaaad they contribute

aqueous

also

proteins

(i)

of shar

registered

group

synthesis

demonstrated

round

secretory,

component

labeled

two

multifunctional

(i.e. were

a

body,

genes

genes

A prominent

complement

[35S]methionine

one

conferring

novo

in the

by:

The

(13).

nation be

of

many

known

which

De

cDNA

between

obtained.

described

will

ciliary

After

identified

epithelium

proteins

humor.

ssDNA

and

ocular

XR.

to

privilege).

plasma

aqueous

we

homology

of we

strategy

Uni-ZAP

ciliary

excess

characterization

the

databases,

properties

in

from

hybridization,

ing

cell

genes),

clones.

hybridization in ă

a certain

(driver)

the

library

libraries

subtractive

an

library

describe

efficient

to

(housekeeping

cell-specific

we

cDNA

following

unique

with

cDNA

report

be

genes

(target)

another

and

might

common

ciliary

from

In

which

subtracting

et al.

flanked restriction

fragment by

the site

SK(-) primers.

with T3

of

was

ampli

Two

fragments

after

the

digestion

XhoI.

The

largest

promoter

at the

fragment plasmid

3•Œ-end.

at The

the

of

the

fragment

5•Œ-end, smallest

121

of

and

the

fragment

J.

Biochem.

a

Subtracted

was

Human

flanked

at

Ciliary

the

5•Œ-end

Pre-hybridization by

mixing

ments

and

50mM

Subtractive

pre-hybridization under mineral

ssDNA

(250ƒÊg) subtracted

(Vector ssDNA

by

agar

E.

plates

mented

XhoI

and

amplification

ciliary

but

body

the

with

onto supple

library

body

were

probe

gel

DNA blots

of

expected

library,

generated

from

the

1%

at

Bevervely,

For

immunoprecipitation,

X-100;

for

washing

and

from

deoxycholate;

from

cipitates

with

body

the

inserts,

ODM-2

cDNA

on

4-8

0.01M

Tris

was

Culture

also

collected

microconcentrator

1%

with ice,

or

0.1%

detected

total

10

min

were

three

(Phar in

1%

SDS).

with

1

at

pre

times

1•~

sodium

Immunopre volume

2-ƒÀ-mercaptoethanol, and

the

for

pH=7.8;

95•Ž

and

A-Sepharose

washed

X-100; at

with

complexes

Tris-HCl

denatured

C4

1:100

was

protein

and

Triton

component diluted

medium

Immune

(0.01M

SDS-PAGE,

for

sodium

fluoride).

Incubation

ice.

on

containing

10%

were

1%

SDS

were

culture

incubation

buffer

buffer

0.1%

microcon

processes.

min

were

NaCl;

complement

either

4h

90

mCi/ml,

processes

MA).

ciliary

after

macia)

in

mCi/mmol;

of 0.5

proteins

a

old),

Essential (50

(0.15M

antisera

then

years

suspended

Ciliary

radiolabeled

with

(86

phenyl-sulphonyl

(Amicon,

cipitated

37•Ž.

using

processes

Minimal

buffer

Triton

containing

and

donor saline,

at a concentration

1mM

from

using

to

ciliary

a human

48h

concentrated

extract

Axioskop

taken

[35S]methionine

RIPA

pH=7.8;

PCR

to hybridize ciliary

for in

by

clones

the

Science)

incubated

37•Ž

transferred was then

subtracted

from

electro

obtained

cDNA

Specific

generated

the

and

with

Life

incubated

with

with Fluo

Immunoprecipitation-Prior

methionine-free

(MEM)

deoxycholate;

then

a Zeiss were

[35S]methionine,

from

and

homogenized

and antibody.

with

phosphate-buffered

serum-free

and

from

digestion

agarose

pair

and with

and ƒ¿2-macroglobulin insert

inserts

ciliary

Labeling

with

medium

performed Photographs

microdissected

- HC1

selected

after

in 0.8%

library.

probe

not

and

The

1:400; second

(Kodak).

labeling

washed

and D

(100 ƒÊg/ml),

duplicate Each

film

Amersham

from

mixture. electrotransform

Screening-The

radiolabeled

cDNA

radiolabeled

Vectrex-Avidin

isolated

run in filters.

from

the

of

cells,

separated

were

with

ODM-2

Blot was

TMAX-400

Medium

iso-propyl-b-thio-galactopyrano described (16).

clone

phoresis. Gels into nitrocellulose

the

the

continued hybridization, separated

ampicillin

Southern

hybridized

to

5-bromo-4-chloro-3-indolyl-ƒÀ-D-galacto

subtracted and

added

were

excess

XLl-Blue

(X-gal), and as previously

Differential

EcoRI

were

was

microscope.

were

(10-30ƒÊ

Bethesda) anti-rabbit

microscopy

Metabolic

Specific

RNA

NIH,

fluorescent

NaCl,

of

sense

to the hybridization used directly to

containing

with

pyranoside side (IPTG),

Purification

library

an

coli

40%

500mM

Becerra,

metabolic

hybrids

adding

Labs.) was

electrocompetent

and

Patricia

rescence

frag of

Dr.

a rhodamine-conjugated

3•~106

presence

=7.2,

T7.

10ƒÊl,

to

and hybridization at 52•Ž. After

RNA

unbound

the

and

cDNA

ssDNA/biotinylated

each

pH

mixture, for 26h

oil

of

digested

of biotynilated

ODM-2

3•Œ-end by

a volume

overnight.

Hybridization

the

923

(equivalent

in

52•Ž

at the in

multicloning

HEPES, at

microliters

from

LB

of

SK(-),

incubated

ssDNA-Ten

and out

ssDNA

1ƒÊg

pBluescript

then

g)

of

with

formamide,

XhoI,

carried

0.5-0.8ƒÊg

of

by

was

transformants)

Body cDNA Library

of

SDS

fractionated

by

fluorography

by

at

-70•Ž

days.

library. DNA

Sequencing-Double-stranded

[pBluescript

SK(-)],

sequenced using

using the

cent

the

dideoxy

Sequenase

RNA

gel

described (RNA)

the

ODM-2

was

method

(18),

size-fractionated

naturing

1%

agarose-formaldehyde

Nytran

filters

(Schleicher

with

prepared

of

probes,

or

rat

humor

sulfate

ferred

or

and

line

as

de to

NH).

The

conditions

previously

or

de

Filters

were

gels

as

from

previously

(19).

antibodies

at

anti-rat

selenoprotein-P F. 1:300; (21)

Burk

(20) and

Kristina

anti-rat (provided 1:200;

with

Vol. 118, No. 5, 1995

rat

ciliary

Sections the

bodies

were

following

(provided E. Hill,

generously PEDF

by (22)

incubated

generously

by

Dr.

Ruth

Halaban, generously

RNA

nating

dsDNA

the

EcoRl

and

XhoI

MgCl2

(Fig.

2,

as

previously

5)

DNA

Drs.

produced colonies on

then

number

with

Yale by

lane

4)

was units containing in

appoximately when compared results, to

mants

after

cDNA

library.

and shown

synthesized

these

with

separated

we 143,000

dsDNA

from

first with

within

the

the

2, lane

3).

presence

dsDNA

represented estimated of

in by

XL1-Blue

(Fig.

2,

with

transformants, synthesis

was

of

(Fig.

2,

(15).

in

vitro,

the

ssDNA

calculating cells

the

selected

on

Double-stranded lane

a 20-fold increase with ssDNA (Fig. started

intro

digested

(Fig. in

line

contami

from

ampicillin.

vitro

we

then

phenol

(+)

cell

material

2),

pBluescript

of transformants

plates

ssDNA

cut

dsDNA

mole

epithelial

ssDNA

2, lane

to by

sense

at eliminating

The (Fig.

two

vectors

with

antisense aimed

purified

of colony-forming agar

library a ciliary

of

of

XR

from used

was accomplished ssDNA (target)

endonucleases,

preparation

equivalent

(brown

of

region was

LB

(driver)

library

ssDNA

used

cDNA

(pBluescript).

from

titer

body

procedure

excised

1, we

in ă-Uni-ZAP

cDNA library. the purification a two-step

Library

in Fig.

library. This (-) circular

ciliary

cDNA

outlined

constructed

duced

sec

Human

Body-As

subtracted antisense a

phagemid

Univer

protein

of

The

(12).

were

a

(ODM-2) During

designat

Vanderbilt

(provided

cules

lane

trans

concentrations:

tyrosinase-related

and

and

semithin

and

described

first

Raymond

bovine

sodium

described

libraries

multicloning

aque body

on

probed

visualized

rat

ciliary

electrophoresed polyacrylamide

and

or

rat

Immunofluorescence-Cryostat

(0.5ƒÊm)

University)

human

cDNA

a Subtracted

Ciliary

biotinylated

transferred

washing

human

were

(SDS)-10%

specific

locus)

and

conducted

and

antibodies

Indirect

sity)

cell

on

Keene,

(0.5 ƒÊg/lane),

to nitrocellulose.

sera

as

plasma

(60 ƒÊg/lane)

dodecyl

tions

the

of

Ocular

prepare hybridizing

electrophoresis

Schuell,

was

as

isothiocyanate

gels,

(60 ƒÊg/lane),

extracts

ed

and

guanidinium

&

probes

completed

the

Blotting-Total

body

by

per

(12).

Human ous

the

hybridization,

designated

scribed

by

(Protein)

Construction

method Six

was

ciliary

RESULTS

were

Biochemical).

electrophoresis

from

DNA

inserts,

termination

(U.S.

(17). and Western

(20 ƒÊg/lane)

labeling

cDNA chain

system

polyacrylamide

previously Northern

plasmid

containing

ssDNA

6)

from

ssDNA

in transformant 2, lane 4). Based antisense

or

3•~106

from

the

(-) transfor

ciliary

body

924 J

Fig. 1. Scheme describing the subtractive hybridization strat egy. Antisense phagemid single-stranded (ss) DNA molecules were excised from a human ciliary body cDNA library and purified from contaminating double-stranded (ds) DNA with MgCl2-phenol. The ssDNA molecules [pBluescript SK(-)] were comprised of cDNA clones with inserts (common and specific), and without inserts (nonre combinants). The multicloning sequences (MCS) present in all the antisense ssDNA molecules were first blocked in a pre-hybridization step with complementary MCS, then hybridized with biotinylated

. Escribano

et al.

sense RNA prepared from the ODM-2 cDNA library. Hybrids (ssDNA/biotinylated RNA) were separated from ssDNA (specific clones) with an excess of Vectrex-Avidin D. Unbound material was purified and used directly to electrotransform electrocompetent E. coli X-Blue cells as described (16). Color selection of recombinants transformant colonies (white) from nonrecombinants (blue) was carried out on LB agar ampicillin plates in the presence of X-gal and IPTG. Specific clones were further characterized by differential Southern blot screening and Northern blot analysis.

J. Biochem.

Subtracted

The

ssDNA

region in

Human

(or

with

DNA

polylinker)

"MATERIALS

this

Ciliary

containing of

AND

of

ing

T3

and

EcoRI

molecule.

The

subtractive

adding

will

the

ciliary

then

competent on

LB

blue)

directly

and

Preliminary

estimated

of

to be

that

the

of

After

one

round

Southern METHODS." readily

signal

with Figure

to

3

shows

the

in

exposure

ential

expression by

DNA

in to

be by

a

low

of

Clones-Table

Human I lists

after

sequencing. from

the

differential

that

ODM-2

of

analysis

whereas required

showed

further

We 5•Œ and

sequenced 3•Œ ends

between of

each

cDNA

blot

(clone

#71),

and

AND

the

copies

subtracted

Southern

selenoprotein-P

than

#42), ƒ¿2

clones

Fig.

2.

body

in

with

vivo

EcoRI

differ

Purification

ciliary

Ciliary

Body

cDNA

clones

analysis 100-150

clone

and

DNA

basepairs (a total

of

R408

with

XhoI

100mM

303

phage

and

separated

of the

ssDNA

number

of to

dsDNA

positions

of

indicated

with

(lane

(lane

from to

induced indicated 3-kb

on

fragment

these

ssDNA. lanes

marker

and

con

markers 7.

and

On

the

R408

(arrow

conversion,

a 20-fold

XLl-Blue

with

extracted

conditions,

Enzymatic

Ladder 1 and

excised

is linearized

5).

coli

a human was

digestion

3),

6) originated

(E. by

to

(lane

(lane

(lane

transformants

are

subjected

Under

from

ssDNA

(pBluescript)

ssDNA

dsDNA

DNA

XR.

2),

4).

DNA

that

a

Uni-ZAP

endonucleases

double-stranded

vitro,

1kb

helper

(ss)

in ă

restriction

heads)

the

library

MgCl2-phenol

taminated

character

of single-stranded

cDNA

and

compared

identified

(clone

(72%)

some

type

Subtracted the

C4

hybridization

of

were

their

differential

library,

Clones

analysis

on

(higher

initial

55). This differential some clones were

number

proteins.

based

similarity

component #16),

plasma

2,700

with

subtracted

detected. this

from

such

and that

(clone

for was

we

significant

high

sequencing.

Characterization cDNA

the

42,

complement

work,

biologically

clones

sequence

this

heter

to

"MATERIALS

than of

in

were

large

seventy-two

stronger

results

clones #31, signal suggested

the

by

inserts

probe

identified

these

clones

the

since

1,940

hundred

in

a much

these

1%.

characterized

body

represented

longer

Six

of

nucleotide

GenBank/EMBL

to

blot (Fig. 4). of cDNA clones coded

nucleotide with

in

of few

(29%). subtract

registered

Due

clones expression

genes

additional

from

1kb).

of 127 result

sug a

of which

described

give

enriched

others

ized

as

ciliary

from

selectively

and

hybridization,

found the

(see inserts hybridization

(white).

the

Northern group

extensive

inserts

functional

identification

macroglobulin

hybridization,

obtained,

cDNA

any

were

I. Out once,

known

deposited

The

genes

in Table more than

to

genes

clones

EMBL).

known

176

cDNA

registered and

independent

(average

only

cDNA

occurred, in

of

examined

properties repeated

homology

The

subtracted

with

determined

or

large

90%),

result

than

90

(EST)

databases.

homology

have

without

(16).

estimated

clones less

subtractive

analysis,

28%

clones

the with

were

hybridization

was

studied

After

were

and

ed

The or

colonies

molecules

were

color

been blot

probe.

of

by

have

of we

all

(GenBank

related 21 were

total

genes by A small

(white

described

preliminary

subtractive

library

colonies

selected

clones

a

ogeneity an

grown

Subtracted

nonrecombinant

cDNA

transformant were

the

This

nonrecombinant

proportion

unsubtracted

their clones,

relatively

subtracted

color

transformant (white)

(blue).

of

with

colonies

by

of

to

bound

electro

and

previously

the

recombinants

during

enrichment

high

by

in

of

homologous

searched

exhibiting

sequences

unbound

The

selected

as

of

library RNA

from

(Stratagene) and

72%

cDNA

Labs.).

by

RNA

to electrotransform

IPTG,

nonrecombinants

gested

grouped identified ing

42% were

databases

since

out

sequences,

Characterization total

carried

separated

specific

ampicillin

X-gal

Clones-A

be

was

cells

with

with

the

clones

flank

biotinylated

biotinylated

D (Vector

used

XL1-Blue plates

in

sequenced

large

pBluescript

then

ODM-2 The

putative

of Vectrex-Avidin was

lost,

circular

(+)

epithelial

ssDNA

containing

ssDNA

any

of sense

mixture.

complementary

excess

been

sequences

was

excess

human

ssDNA,

a

Approximately

clones If

introduced,

have

to

clones).

described

pre-hybridized.

not

hybridization

prehybridization

to

multicloning (as

multicloning

anneal

925

Furthermore,

a 10-30-fold

from

was

would

containing

entire

SK(-)

were

clones

RNA

ssDNA

the

step

specific

biotinylated

the

pBluescript

METHODS")

prehybridization

number

Body cDNA Library

in

increase

colonies)

in when

(Gibco/BRL) left phage

of the

of gel

DNA

the are

arrows.

Fig. 3. Differential Southern blot analysis of eight subtracted cDNA clones. Inserts from eight cDNA clones were isolated and resolved by gel electrophoresis in 0.8% agarose gels in duplicated (panels A and B). After transferring DNA in gels into nitrocellulose filters, they were hybridized with two radio labeled probes, as described in "MATERIALS AND METHODS" (panels in B). Clone #55 shows a strong differential hybridization with the C. B. probe but not with the ODM-2 probe. Clones #31 and #42 (arrows in panel B) show low differential hybridization, and clone #41 (arrow head) does not show differential hybrid ization. Vol. 118, No. 5, 1995

926 TABLE I. Identification of subtracted human ciliary body library.

J. Escribano cDNA

clones

from

et al.

a

J. Biochem.

Subtracted

Rep.,

Human

number

of times

apolipoprotein blot

D

analysis,

restricted the they

were

(clone

C4,

and

cell

was

(Fig.

precursor agreed were

these by

5A).

The

detected

an

extracts, protein-P and 62

kDa.

lower

cell

humor,

humor

180

and

and

molecular

ciliary

weight

kDa,

generously (25) detected

in serum,

ciliary

body

The

and

as

Northern

muscle for

not

#82,

opposed

and

not

a

bands of 57 was detect and

to seleno

three samples apolipoprotein Univ. expected

in aqueous

actin

epithelial

Vol. 118, No. 5, 1995

this

clone

specific blot

#211,

subtractive

analysis

##136,

on

and

#223

homology

neurotrophic

epithelium

the is

cell

a marker

cells.

The

line.

This for

cross

probed

with

and

cDNA

clone

synthase, D

Oviedo, size, 26

humor

and

clones

was

few

additional

(Fig.

4).

Clone

is expected,

smooth

muscle

reaction,

seen

since cells in Fig.

RNA

from

is

abundant

the

following

to

a gene

ODM-2 in

an

glandin tissue

radiolabeled

recently

named

pigment

epithelium-derived

homology the

with

Rab

We

were

certain

with

their

case

in

homology

(28);

for the

this cell

only

detected

DNA

molecules

the

human

processing and

a group

corresponding

component

tissue

the may

of clones to

the

present that URF1-4

hybridi hybridization This

clone

carboxypeptidase enzyme

and

RNA.

escape

genes:

of

(27),

subtractive

molecules. of

D2 prosta

detected in #233 showed

(GGTase-II)

RNA

restrict line (Fig.

of

catalytic

during

group

also cell

synthesis

in the

that

prominent to

the

for

pigment factor

were NPE

clone were line. Clone

of

evidence

retinal

to prostaglandin

the

transferase

complementary two

neuropeptide

proteins

in

subunit

geranylgeranyl

present

zation,

cells

the ƒÀ

in

PEDF to the

homology

involved

Transcripts but not

the

#211

as coding

from

showed

Clone

identified

derived

#136

corresponded

cells.

factor

enzyme

D2. RNA,

cells

these

(PEDF) (22, 26). Transcripts for ed to the ciliary body as opposed 4).

(Fig.

to smooth muscle actin, and tran to the ciliary body (likely ciliary to

were

with

which

showed

Seleno

in serum,

of

to ƒÀ-actin

transcripts

homology restricted

smooth

levels

cells

Rab geranylgeranyl transferase ƒÀ subunit, clone #223; and CPE= carboxypeptidase E, clone #294. ƒÀ-actin was used as control. C. B. = human ciliary body; ODM-2=human cell line. Arrows to the left of each probe indicate the position and size (in kb) of specific transcript. Arrowhead with probe SM actin, denotes cross reaction with ƒÀ-actin.

4,

body

5B).

two band

C. Lopez-Otin, band of the

ODM-2

probes: URF-4, clone #3; C4= Complement component C4, clone #42; ƒ¿ƒ¿ 2-M=ƒ¿2-macroglobulin, clone #16; SM actin=smooth muscle actin, clone #82; PEDF=pigment epithelium-derived factor, clone # 211; Se-P=selenoprotein P, clone #71; Apo D=apolipoprotein D, clone #158; PD2S=prostaglandin D2 synthase, clone #136; GGT=

the

5D).

by

#82 showed scripts were

ciliary

(corresponding in the purified

of

immuno

in

(Fig.

but

body and

antibodies.

also

serum

(ƒ¿, ƒÀ

and

analysis of subtracted ciliary body tissue and ODM-2 cells. Blots containing amounts of RNA from intact human ciliary

C4 antibodies these proteins

same

(24)

body,

low

of selecting

including

muscle)

in very

(Fig.

efficiency

demonstrated clones,

by

kDa

band

by Dr. a major

ciliary humor size

(Sigma)

of

to

private investiga three subunits

of C4 in in aqueous

line

in using

antibodies

molecular

protein-P) was immunodetected 5C). Finally, antibodies against (provided Spain)

by

antibodies (20) immunodetected The higher molecular weight

ed in aqueous the

band

aqueous

or

Fig. 4. Northern blot clones in ciliary body approximately equivalent body

among

analysis,

detected with (23). None of

antibodies

expected

subtractive

proteins

blot

(pro), subunits

ODM-2

that

enriched

plasma

Western

expected

in the

ƒ¿ 2-Macroglobulin

in

indicating

5%). of

provided recognized

subunits studies

were

detected

the

antibodies

generously (Sigma)

and three with previous

4),

highly

groups

verified

not

during

available

recognized

(Fig.

was

, and ƒÁ), and the precursor extracts, and only the three serum

line

Northern

clones

were

(approximately

commercially

purified proteins tors. C4 antibodies

four

they

enriched

of

By

these

in particular,

clones

humor

either

body

927

determined.

respectively. for

efficiently

identification

aqueous

N. D., not

#158),

epithelial

subtracted The

repeated;

ciliary

ciliary

hybridization. the

Body cDNA Library

transcripts

to the

NPE

Ciliary

was

#294, E

the with

(CPE),

in neuroendocrine

encoded subunits

mitochondrial of

NADH

a

928

J . Escribano

dehydrogenases, URF-A6L (ATPase 6), cytochrome b, and cytochrome c oxidases I, II, and III. URF-4, alone, was found to represent 5-8% of the entire subtracted library, and by Northern blot it was found to be very abundant in

the ciliary body when compared to the NPE cell line (Fig. 4, clone #3). One possible explanation for the less effective hybridiza tion between some of the common clones in both libraries

Fig.

5.

Western

blot

noprecipitation plasma D)

proteins.

or

rat

humor,

tionation

by and

nent

C4

Human and

ciliary

of

subunits

of

Arrow ing

in

and

(panel

the

tein-D

(panel culture - Sepharose.

A)

of

molecular

protein

markers

and ƒ¿2-macroglobulin

medium

were

Immune

(panel

is B).

immunoprecipitated complexes

were

indicated Ciliary

with resolved

in processes

complement by

SDS-PAGE

kDa.

Immunoprecipitation were

metabolically

component and

C4

antibodies

of labeled

(panel

A)

Arrows

a

compo

in

A,

and ƒÁ

component main

band Arrow

in

related

position On

the

left

protein.

of

each

blot

component

proteins

or ƒ¿2-macroglobulin

indi kDa),

of apolipopro

complement and

C, (57

a possible

kDa).

C4.

correspond

selenoprotein-P

labeled

indicate

(pro), ƒ¿, ƒÀ,

indicate

[35S]methionine

frac into

A); ƒ¿2-macro

complement

of

(26-32

[35S]methionine with

D).

B indicates

D,

were

(panel B); selenoprotein C); and apolipoprotein-D

arrowhead in

and

serum,

complement

precursor

position

Arrows

position

body

to ƒ¿2-macroglobulin.

cates

B,

from

transferred

against

(panel

positions

A,

C)

SDS-PAGE,

antibodies

antibodies

immu labeled

(panels

(panel

blotted

and

metabolically

proteins

aqueous

filter

analysis

of

globulin antibodies P antibodies (panel

the

et al.

secreted

(panel

B),

C4 into

and

the

protein

A

autoradiography.

Fig.

6.

Differential

labeling

selenoprotein-P, related

protein,

bodies

on

tion

of

and

and

rat

indirect

bovine (A-D)

sections

or

were

with

antibodies (A

sinase-related (C

antibodies

and

F).

B,

pigmented

(21)

D,

ciliary

PEDF

dilution)

and

photographs

tyro (1:200

or

(1:300 F

are

of A,

respectively.

mented

B),

D),

(22)

(20)

and

protein

dilution)

E,

by

immunofluorescence

dilution)

and

bovine

labeled

(1:300

contrast

sec ciliary

selenoprotein-P

(E

anti

frozen

and Rat

F)

PEDF

semithin

processes. (E

of

tyrosinase

NPE,

phase C,

and

nonpig

epithelium;

ciliary

PE,

epithelium;

ve,

vascular

endothelium;

bm,

basal

plasma

membrane.

Magnifica

tion•~600.

J.

Biochem.

Subtracted

could

be

Human

the

presence

synthesized

in

of

- F,

the

coded

and

indirect

rat

bovine

and

-

in

pigmented rat ciliary

ciliary processes

labeled

with

either

macroglobulin

or

subtracted

cell-specific

phenotype.

further

we

methionine.

Fig.

[35S]methionine complement

ly

a

proceses

labeled

in

proteins

secreted

of

[35S]methionine

identical

pattern

of

immunodetected serum, and

B).

These is

and

show

an

results organ

proteins with those present

were

(data the

not

and

medium

(Fig.

5,

complement component immunoprecipitated

labeled

secreted

and

proteins

mobilities Western

ciliary

body

blot (Fig.

demonstrate and

identical

molecular

from

A

ciliary plasma with

this

work,

a human

ciliary

generated summarized

in

body phagemid in Fig.

Vol. 118, No. 5, 1995

and

a single

related

compounds

cDNA

library human

from donor

ciliary

pBluescript, following the 1. Previous studies demonstrated

the

On

was

tein-P,

strategy that

the

epithelial

(20).

plasma,

The

in the

eye?

differ

Our

prelimi in

the

between proteins

in

aque

targeting; (24).

arachidonic

C4

it may

cells

as

roles

gene

by

actively

partici as

that

has

selenopro

extracellular in the

in

expression

glycoprotein an

(Dr.

responses, (23, 31).

suggested

selenium-rich

sub heme

properties

important

component

been

Apolipo

acid)

These

suggests

im

hydrophobic esters, or

biologically

of this

root

of ƒ¿2-macroglobulin

(or

functions

the

the

differences

peptides

30),

it has

expression

through

immunodetected

inflammation and kidney

extracellular, in

of the

different cholesteryl

play

plasma from

synthesized

small

an the

pigmented that

translocated eye

by into

modifications

foreign

cells

the

clearance;

Complement

hand,

of

communication).

proteins

other an

be

properties

in immune-mediated shown in intestinal

originally idant

are

to

Whether

indicate

proteins

inhibition;

humor.

com

are synthe contribute

cells

that

(25,

certain

chamber

plasma

of

least,

anterior

serum and plasma body (see Fig. 5).

personal these

the

of the

and transport cholesterol,

is that

outside results

the

nonpigmented

outside

processing

D binds including

pate been

normal

from ciliary

protein stances,

suggest

at

space

indicate

ciliary

ciliary body are directly by the ciliary epithelial

could

synthesized

of

humor.

studies

mobility

Lopez-Otin,

that

posttranslational

proteinase

aqueous

subtractive of

Do the

multifunctional

port;

the

ocular

those

include:

DISCUSSION In

29).

observations

The

a

explants

complement

chamber

undergo

plasma proteins ous humor or

those

mass

(7,

body

see

5, panels the

iris

electrophoretic

in

of

of the

novo C and

of these findings time assumption

aqueous

Recent humor

de

component

are synthesized based on

paracellular

anterior

the

analysis

that secretion

the

nary

with as

into

ent

the

aqueous

blood

ciliary

ciliary with

in

proteins

selonoprotein-P) and they could

the

and

plasma

in

into

from

P,

Furthermore,

in the chamber

redirected

known.

cDNA

these

suggested

of

not

proteins

separate the

is

four

(complement

indicates

humor However,

be

(i.e.,

se

which

relevance the long

composition

are

ciliary

subtractive dbEST data

on

complement

D, body

route

were

the

expressed

body and

demonstrated

can

their

evidence for

apolipoprotein the ciliary

through

cells)

300

(i.e., ƒ¿2-macroglobulin,

or they

stroma

of

it

on

in

proteins

body.

aqueous liver).

I).

clones

selenoprotein

of

of

Table

based

attention

serum

proteins synthesized into the posterior

alternative

im

in

here,

plasma secreted

with respec

from

ciliary

secretion

protein

cells,

over

present

was

C4, within

the

pattern

carried

shown)

culture

synthesis

appararent in serum.

the

the

proteins

ponent sized

com

B). Both antibodies

further of

only

We

I

search

homology

identified

ciliary GenBank

our

to

D).

proteins eye (i.e.,

plasma

im

and autoradiogra

focused

Ninety by

(see

expressed of

selection

date for

Table

the

being

in vitro. The since it challenges

plasma of the presented

to

for

character

sequenced.

genes

in of

activity

and

processes important

medium,

human

we homology

color

databases

the human entered in

ƒ¿ 2-macroglobulin

[35S]

were

to

derived

conventional

humor,

culture

antibodies

we

synthesis

ciliary

with

work

by

useful estimated

partially

identified

sequencing

from been

within

a

were

as

transcriptional

a

clones

C4, ƒ¿2-macroglobulin,

occurs

processes

48h

proteins,

electrophoretic

by

aqueous

body

for

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apolipoprotein

component

ciliary

eye

extract

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component

from

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classified

identified

are An

registered

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with

were

EMBL to

partial

this

at

with

within

In clones

together,

proteins immunoprecipitated C4 and ƒ¿2-macroglobulin

into

[35S]Met in panels C4 and ƒ¿2-macroglobulin

bases.

and

vectors (28).

isolated

clones

been

identity.

tags have

in to

followed

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vitro

the

not

proteins

complement

B,

B);

ciliary

consistent

immunoprecipitations protein

the

synthesized

A and

total

the

identified

labeled

labeled component

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with

set

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to

extract

plement component C4 munocomplexes resolved In

hown).

donor

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cells

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tively.

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labeled

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containing

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of

F). Immunocytochemistry human ciliary processes

clones

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cDNA profile

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clones

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membrane

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epithelium

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using

obtained

cDNA libraries out subtractive

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genes

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patterns

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protein

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nonpigmented

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distribution

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the

sections of paraffin

shown).

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processes analysis

on

RNA

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proteins

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secondary

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several

Body cDNA

vitro.

Cell-Specific Genes-We of

Ciliary

human

found antiox ciliary

930

J. Escribano

body,

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that

its

the

detection

ciliary

antioxidant

in aqueous

body

humor

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site

supports

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anterior

the

idea

detoxification

segment

of

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the

eye

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clones

(6,

highly

expressed

9). The

largest

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enzymes

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may

ciliary

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body

division, the

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cells.

no

in

activity

cell

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that

the

than

cell

at

occurs

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in

vivo. gene

identified E,

candidate

enzyme

was

a

and

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thank

would

and

with opens

for

hormones

being

nonpigmented The

ciliary

homology

contraction

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con

hormones. neuropeptides

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receptors

human

in

contraction ocular

synthase, which

the of

(32), ciliary

ciliary

have

in the

3.

of PGD2 there

and

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4.

of

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identified

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tissues,

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by

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body

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tissue

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subunit

blot

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exhibited

catalytic

product

transferase were found

other

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eye.

and

compared

lens

when

cornea, not

iris,

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to

and

other

(27). the

ocular

retinal

the

6.

Rab

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tissues,

pigment

such

epithelium

7.

as

(data

8.

few

cell

specificity

cases,

using

acterized.

In

obtained

of

subtracted

antibodies

which

general,

with

the

these

clones were

pattern

specific

was

studied

in

extensively

of

Coca-Prados,

immunostaining cell labeling

tyrosinase-related suggests

protein,

that

these

cell-types

within

conclusion

that

The

strategy

potential

genes

precise

onset

primary

that

correlated

are

regulate

body. cDNA

genes

to

used

in

work or

mechanisms

ciliary

humor

secretion.

Stone,

R. A.

Thus,

and St.

by

a or

donor

RNA

(driver)

genes

patient, affecting

hence from

ciliary

The

the

a ciliary be

of

possible

summary,

we

generate a subtracted body of a single normal responsible

for

applied human human

regulating

subtractive cDNA donor aqueous

library patient. humor

in

the

Kumar,

N.

gap

M.

junction

epithelium.

J. M.,

Curr.

K(+)

the

nonpig

in

aqueous

184-194

(1989)

The

Glaucomas Vol.

Na+,

along

implications

149,

Y.

Martin-Vasallo,

of multiple

of isoforms

The

nervous (Ritch,

1, pp.

system R. R.,

257-279,

The

and

Shields,

C. V.

Mosby

Isolation

Eye

Cole,

D. F.

Eye

(Davson,

protein

Hernando,

N.,

Crabb,

expression

in

Martin-Alonso,

(GST)

Exp.

S.,

Differential bovine

J. W.,

of

ciliary

the

and

cellular

epithelium.

Exp.

659-669

N.,

(1992)

J. M.,

of a cDNA

class-pi

Res.

Taylor,

Ghosh,

binding 56,

from

55,

(1984)

A. W.,

the

bovine

ed.)

fluids,

Chap.

pp.

S.,

and

Coca-Prados,

a glutathione ocular

2,

269-390,

Streilein,

S-trans

ciliary

and

epithelium.

la,

Cousins,

intestinal activity

Vol.

Academic

J. W.,

vasoactive

153,

Ghosh,

encoding

711-718 Ocular

H.,

peptide

of

normal

3rd

ed.

in

Press,

New

S. W.

(1994)

Im

contributes

aqueous

The York

humor.

to

the

J.

Im

1080-1086

Martin-Alonso,

J. M., of

the

Ghosh,

bovine

(GP) of eye.

S.,

plasma

Rowland,

and

Coca-Prados,

M.

selenium-dependent

12.

the

(1993)

glutathione

13.

Hong,

Potter,

ocular

cellular

114,

ciliary

forms

epithelium:

within

the

mam

284-291

D. E.,

intraocular

T. J.,

and

pressure:

Escribano,

clones

J.,

encoding Ku

Reiter,

R. J.

A rabbit

and the

(1981)

model.

Coca-Prados,

80-kd

(p70/p8O)

of human

Escribano,

J., M.

by

eye

donors.

Hernando,

(1994)

Circadian

Curr.

M.

subunit

Eye

(1994)

protein

antisera Invest.

N.,

cDNA

to ƒÀig-h3

protein

in the

Sambrook, Cloning:

14.

Gosh,

from

Res.

1,

raised

Isolation

of the

human

against

Ophthalmol. S.,

human

Crabb, ocular

is preferentially corneal

ciliary

Vis. J. W.,

ciliary

expressed

epithelium.

15.

Fritsch,

E. F.,

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J.

D.,

Porteus, system Nucleic

Cell

Acids

Koch,

740, Brice, Usdin,

single-stranded Res.

A.,

acid-phenol

Acta

and

Maniatis,

18,

pp.

Spring

J. L. R., M. H., using

B., of

Biophys.

Rubenstein,

Cold

Hofer,

mechanism

and

Manual,

Laboratory,

Miiller, the

J., A

Harbor

body

and

the

and

Sci.

35,

and

Coca

epithelium as an

extracel

Physiol.

160,

511

- 521

the

to

from

plasma

Biochem.

J. M., in

lular

to

cDNA

the

J.

homologous

from the ciliary This tissue is secretion

958-981

4023-4030

body.

hybridization

and

ciliary

Functional

(1993)

M.

Prados,

ciliary

with

17,

of the ƒ¿1

Expression

Physiol.

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processes

of

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N. B.,

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Cell

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In

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Sci.

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M.

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it would the

10.

the

disease.

of

and

junctions

Vis.

Gilula,

Kuwayama,

Krupin,

Res.

rhythm

pathogenesis

ssDNA

normal

a glaucoma

candidate

6)

to search

prevalent

and

Burk

antibodies.

Intercellular

a gradient

J.

Martin-Alonso,

of cDNA

applied

the

R.

discus

Louis

malian

11.

during

most

subtracting of

support

(1978)

epithelium:

and

Expression

distinct

To understand this molecular mechanisms

secretion

ssDNA of

the

unknown. into the

humor

library

be

induced

glaucoma

(Fig. in

and

could

Becerra;

providing

pigmented

N., M.

pressure

Cloning

expressed.

underlining

largely is needed

library

identify

this

We

stimulating

a

cell-specificity. with PEDF,

processed

processes

assistance.

the

P.

for

S.,

reveals

mented

munol. 9.

was

well

selenoprotein-P being

differentially

repressed

complementary cDNA

are

ciliary are

aqueous

ciliary body

the they

indicates obtained

and

proteins

open-angle

glaucoma, investigation

that pattern

Drs.

cellular

Hernando,

peroxidase

pattern of The distinct

technical for

Ophthalmol.

ocular

immunosuppressive

a

char

labeling

antibodies

the

S.,

and

munoreactive

shown). The

pharmacol

113-122

P.,

Eye

(85%)

of

and

retinaldehyde

was

homology

in

to

potential

body.

excellent

E.

Ghosh,

in

11,

Coca-Prados,

were

Such

component

(GGTase-II) exclusively

and

physiology,

Swaroop

Halaban

Invest.

Ghosh,

ferase

geranylgeranyl for clone #223

the

with

R.

Raviola,

M.,

Res.

Co.,

the 5.

clones

along

Expression

intraocular

resulting

expression

and

Coca-Prados,

M. B.,

the

A.

and

G.

- ATPase

synthesis

(33),

muscle

compared

work,

epithelium.

Eye

35).

not

Raviola,

(1992)

theory muscle

activation

muscle

smooth

(34,

we

the

to prosta

supports the in smooth

Upon

this

ciliary 2.

in

showed

is involved

body.

sphincter

hypotension

Although

strongly involved

is

human

the

enzyme

#136

Dr.

biological

ciliary

for

thank

Lopez-Otin;

gene

clone

the

differentially a framework

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