BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 183, No. 3, 1992

Pages 1025-1032

March 31, 1992

THE GENOMIC ORGANIZATION OF THE RAT AT I ANGIOTENSIN RECEPTOR I

Kimberly Langford, Kristen Frenzel, Brian M. Martin* and Kenneth E. Bernstein

Department of Pathology,

Emory University,

Atlanta,

GA

30322

*Molecular Neurogenetics Section, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol Abuse and Mental Health Administration Bethesda, MD

Received January 30, 1992

SUMMARY The AT I receptor subtype modulates all of the hemodynamie effects of the vasoactive peptide, angiotensin II. In this report, we investigate the genomic organization of this important receptor. A rat genomic library was screened with fragments from the 5' region of a previously cloned eDNA, pCalSb, encoding the rat AT I receptor. Two lambda clones were isolated and the hybridizing restriction fragments were sequenced. Comparison of the genomic and cDNA sequences reveals that the rat AT I receptor has three exons. Two of the exons encode 5' untranslated sequence while the third exon encompasses the entire coding region, a small portion of the 5' untranslated region and the entire 3' untranslated sequence. Further analysis of the genomic sequence 5' to the start site of pCalSb demonstrates typical sequence motifs found in many eukaryotic promoters including a TATA box, a cap site and a potential Spl binding site. Southern analysis of genomie DNA indicates that the AT I receptor subtype represented by pCal8b is encoded by one gene within the rat genome. © 1992 -

Academic Press, Inc.

Angiotensin II is the final product of the renin-angiotensin system. small peptide physiologic

This

influences systemic blood pressure by triggering a cascade of

changes

resulting in the expansion of extracellular volume

constriction of vascular smooth muscle. I

and

A remarkable feature of angiotensin

II is the diversity of tissues and physiologic processes affected; the gut, the kidney, the adrenal, the liver, vascular smooth muscle, and the peripheral and central nervous systems all respond in a distinctive fashion to angiotensin stimulation.

Angiotensin II interacts with G protein-coupled cell membrane

receptors to initiate its actions. are at least two subtypes

Recent studies have demonstrated that there

of angiotensin II receptors. 2'3

called AT 1 and AT2, can be differentiated by peptidie ligands DuP753 and PD123177.

These

their affinities

subtypes,

for the non-

The AT I receptor subtype has

a high

affinity for the DuP753 compound and a very low affinity for PD123177.

It is

iSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. M86911 and M86912.

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0006-291X/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 183, No. 3, 1992

widely

distributed

cardiovascular

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

is

the

and hemodynamic

receptor effects

subtype

responsible

of angiotensin

11. 4

for

all

Recently,

of

the

the AT I

receptor was cloned from rat vascular smooth muscle cells and shown to be similar in structure

to other

described

G protein-coupled

have studied the genomie organization

membrane

receptors. 50&

of this receptor to determine

We

the number

of AT I receptor genes present in the rat genome, the exon-intron structure of the receptor and the genomic sequence immediately 5' to the receptor gene. here that within the rat genome, receptor subtype.

there is a single gene encoding the cloned AT I

This is composed of three exons, two encoding 5' untranslated

sequence and a third exon containing

the coding region,

a small portion of the

5' untranslated region and the entire 3' untranslated sequence. immediately preceeding an RNA polymerase

We report

The genomic DNA

the first exon contains many typical sequence motifs

II promoter

including a TATA box and a potential

of

Spl binding

site.

MATERIALS AND METHODS A rat genomic DNA library in the vector Lambda Dash II was purchased from Stratagene. This was screened with the 5' Hindlll-EcoRl restriction fragment of the rat AT I receptor eDNA pCal8b (nucleotides 1-837). 5 Hybridizing plaques were plaque purified and DNA was prepared and analyzed by restriction analysis using portions of pCal8b. The first exon of pCal8b is encoded by the genomic fragment A2.EB.24. To sequence this portion of DNA, the genomic clone %RIO was cut with BamHl and EcoRI to release a 2398 bp fragment that was subcloned in the plasmid Bluescript (KS). DNA was prepared and i mg samples were sonicated in 200 ~i of i0 mM Tris, pH 8.0, i mM EDTA for two, four or six 8 second bursts using a Fisher Sonic Dismembrator at 50~ power. The three samples were placed on ice between bursts to cool and then were combined and sized-selected over an 8 ml Sephacryl S-500 (Pharmacia) column equilibrated in 25 mM Tris, pH 8.0, i00 mM NaCI, 0.05~ SDS and i mM EDTA. Column fractions containing DNA of about 400 to 2000 bp were combined. 200 ng of DNA were then treated with 2 units of T4 DNA polymerase (BRL) for 5 min at 37°C in 33 mM Tris-acetate, pH 7.9, 66 m M N a acetate, I0 m M M g acetate, i00 ~g/ml bovine serum albumin and I mM DTT. No nucleotides were present. The ends were further repaired with the Klenow fragment of DNA polymerase in the presence of i mM dNTPs. The sample was phenol/chloroform extracted, precipitated and resuspended in i0 ~I. Varying amounts of the DNA were ligated with i00 ng of Smal digested MI3 mpl0 (Amersham) and a library was constructed in the E. coli strain JMI01. The library was screened with the A2.EB.24 fragment. Positive plaques were selected and single-stranded DNA was prepared. The DNA sequence was determined using the dideoxy method of Sanger. 7 Sequenase and other reagents used for DNA sequencing were purchased from USB Corporation. DNA sequence was assembled and analyzed using the software program PC/GENE (IntelliGenetics). To sequence the coding exon of pCal8b, the genomic clone %R12 was digested with Hindlll and a 3.3 kb fragment was subcloned in Bluescript. This is called A2.H3.33. A unique internal EcoRl site was used to cut the cloned DNA into two Hindlll-EcoRl fragments of 853 and 2443 nucleotides. The DNA was blunt-ended using the Klenow fragment of DNA polymerase and cloned into the Smal site of MI3 mplOo Single stranded DNA was prepared and sequenced using oligonucleotide primers. Genomic DNA was prepared from the liver of a male Sprague Dawley rat using standard techniques. 12 ~g of DNA were digested with either Hindlll, EcoRl, BamHl or Xbal and Southern blots were performed as previously described. 8 Two DNA fragments, pCa18b/196 and pCal8b/ll6, were prepared using Vent polymerase

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(New England Biolabs) in a polymerase chain reaction to amplify pCalSb from positions 13 to 209 and 13 to 131. The primers 5'-ATGCTGTCCCGCTGGAGAG and 5'GATCACTTTCTGGGAGGGTT were used to prepare pCa18b/196. The primers 5'ATGCTGTCCCGCTGGAGAG and 5'-CAGGGAATGTGGCAGAGCTG were used to prepare pCal8b/ll6. The amplified fragments were purified by agarose gel electrophoresis. To prepare a radioactive probe, the fragments were denatured at 95°C, then cooled to 37°C and double stranded DNA was synthesized using T7 DNA polymerase (Stratagene), I00 ng of the same primers used to prepare the fragments, 50 pCi of [32p]dCTP (Amersham, 3000 Ci/mmol) and 0.i mM dATP, dGTP and dTTP. Unincorporated [32p]dCTP and oligonueleotides were removed using a BioRad 30 column. The integrity of the synthesized DNA was checked on a DNA sequencing gel. The Southern blots were hybridized overnight at 65°C and then washed with a final stringency of 0.5 x SSC, 0.i~ SDS at 65°C.

RESULTS We have previously described the cloning and characterization of pCal8b, a cDNA encoding the rat vascular AT I receptor. 5 genomic

library used

a restriction fragment

The initial screening of a rat of pCalSb

encompassing

the

5'

untranslated region, the translation start site and 564 nucleotides of coding region.

Three overlapping genomic clones were identified.

Restriction analysis

of the genomic DNA suggested that a single 3.1 kb Hindlll restriction fragment hybridized to pCalSb.

This portion of genomic DNA, referred to as A2.H3.33, was

subcloned and sequenced (Fig° I).

Comparison of this sequence to that of the

cDNA pCal8b demonstrated that the genomic Hindlll fragment contains a single exon encoding nucleotides nucleotides

of

the

220-2197 of pCalSb. 5'

This corresponds

untranslated region,

the

entire

to the final 52

coding region

(1077

nucleotides encoding 359 amino acids) and the entire 3' untranslated region of pCal8b (847 nucleotides).

The genomic sequence contains the poly(A) addition

signal identified in pCalSb.

One discrepancy was found between the cDNA and

genomic sequences; position 1194 of pCalSb sequence.

(A) was not found in the genomic

This nucleotide is within the 3' untranslated region of pCal8b and has

no effect on the predicted amino acid sequence of the AT I receptor° A comparison of pCal8b and A2.H3.33 reveals that the 5' 219 nucleotides of pCal8b are not found in A2.H3.33. untranslated region. the

gene,

fragments.

genomic

This sequence is the first portion of the 5'

To understand the genomic organization of this portion of Southern

analysis was

performed using

two

separate

cDNA

One probe was prepared from position 13 to position 209 of pCalSb and

is called pCa18b/196.

In each of 4 separate digestions of rat genomic DNA, this

probe hybridized to two discrete fragments of genomic DNA (Fig 2a).

The probe

hybridized to bands of 1.75 kb and 7.3 kb when DNA was digested with Hindlll, 3.8 kb and 4.7 kb when DNA was digested with EcoRl, two bands greater than i0 kb in BamHl cut DNA and bands of 3.5 kb and 5.2 kb in Xbal cut DNAo

A second probe

from position 13 to position 131 was also prepared and used in Southern analysis of rat genomic DNA (Fig. 2b).

This probe is called pCalSb/ll6, and it hybridized

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

Hlndlll

Hindlll 3' UT

ATG

TGA

500 bp

30

40

B Hindlll

i0

20

I

I

V I 61 121 181 241

aagcttgctg ggccgccgta tccaaagcgt atcggaactt AAGTGGATTT

atgtctgatt gattttatca aaagtagaac ataattttta CGAATAGTGT

I

50

I

ggccacttaa cactacgtag gcacatgetc gctcacaagg tttcttcgga aaatatggtc caetagattt gtggtggaaa atgaaatttg actttttaat gttetttgga tttttgtttt CTGAGACCAA CTCAACCCAG AAAAACAAAA M

301 CTCTTCTGCT S S A

2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241

GAAATTTTGG cttcattatt acaaaattae tagatatagg atgtagggaa ttcattgcta gttacatgca tatttagttc agagggtgac tacatgtgct ctggggatca gccataatgt tagtatacca cctgataaca ggcactagaa cagaactgag ccagaaagta agctcagtgg aacaaactag

60

I

I tagaaaagtg taaagttttt cttatttaca tttccagGTC TGGCCCTTAA A

L

N

GAAGATGGTA TCAAAAGAAT E D G I K R I

CCAAGATGAC Q D D

TGCCCCAAGG CTGGCAGGCA C P K A G R H

CAATGTATGT taaaatagaa aataatgcat ttttatatcc gatacaacat ttaacattaa tggtaattgc ttcagtgcaa ttgtaaggcc tcaccaggta agttttcata atatacaatt ttcaatgtat ttagggaatc gtcaccctga aatatggtta taacacaact agaggagtte accagaatat

ATAAAGTAAT ttttacaatg agtttgatct aaaatcatga tttagttatt ttttttactc gaagctctgg ttttagtatt tgeaaectca actaatcccc atacatacat gcttgtccct attecagtac agcatttace cacacaaate tcgtattecc aceagctatg agaaceeagg cggctgccta

TTTATTGTAA acatggatag ggttgatagt aggccagett catataacag ataagttttg aaaaeacaaa ttagcccata aagcccaccc cagtcctgcc atgtaaatta cagtgteatt tgttaaaaaa attcttctaa tgataaacte aatatgtaag tttagetgta tgctgtaggc gtcaccagaa

TTACCTCTAA gcctatctat ttttacttea cactgttgaa ttttaatttt attaatactt aggaaatata aaatactcca tcatgtctte tgtcagaett ctcactatae ctgtatatag ttcctcaaac accaaattgg gctgatcaag taaacgtttt tatatacgct acttectcta tgccatgggg

Tgtatctaat aagtaaaaat ttaatatggt gtaccccaca caaaaacatt gttacatcta gcaatagtca agtaggaagc cagtgacaga cagccaccaa tggtgttaga gcagagtact cacaagcaca aaatactttg caagaagaag agcacgtttt gctcatggca tagaacagga agctt

A Hindlll Fig. I. Sequence of A2.H3.33. A. There is a single exon composed of 5' untranslated sequence, an open reading frame of 1067 nucleotides (Coding region), a translation stop site (TGA) and 856 nucleotides of 3' untranslated sequence (3' UT). The 5' untranslated and coding regions are identical to the sequence of the AT I cDNA pCalSb. One position in the 3' UT differs between the genomic and cDNA sequences (see text). The exon is flanked by intronic sequence (solid line). B. Exonic sequence, beginning at position 238 and continuing to 2211, is shown in CAPITAL letters. Intronic DNA is in lowercase. The translation start site is at position 290. The first 24 amino acids of the AT I receptor are shown. The 1800 nueleotides not present in the figure are identical (with one exception) to the corresponding portions of pCalSb.

to single DNA fragments

of 1.75 kb, 4.7 kb and 3.5 kb with DNA digested with

Hindlll, EeoRI and Xbal respectively. identified

A single hybridizing DNA fragment was also

in BamHl digested DNA.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

H

E

B

X

g

H

E

B

X

6-8--

4--

6 m 2 m

4--

2 h

Fig. 2. Genomic analysis of the rat AT I receptor. A. Rat genomic DNA was digested with Hind III (H), EcoRl (E), BamHl (B) or Xbal (X) and probed with pCalSb/196. Two hybridizing bands are present in each lane. Size markers in kilobases are indicated at the left of the figure. B. Rat genomic DNA was digested as described in A and probed with pCalSb/ll6. One hybridizing band is present in each lane.

To identify the first exon of the AT I receptor,

the rat genomic library was

screened using the pCalSb/196 and pCalSb/ll6 probes.

A single hybridizing plaque

was

restriction

fragment

fragment

as A2.EB.24.

identified.

sequenced

A 2.4 kb EcoRI-BamHl

(Fig.

3).

We

refer

to

this

A2.EB.24 with the sequence of pCalSb demonstrates A2.EB.24 encodes the first 130 nucleotides DNA

sequence

immediately

5'

to

this

reveals

typically found in the promoters

of other genes. 9

signal

to

(GCAGTT)

(CTATAAATA)

immediately

36

nucleotides

(AGGGCGGGGCGGGG) entire A2.EB.24

5' 5'

the to

start the

of

of the genomic

sequence

motifs

These include a putative cap

of pCal8b,

start

of

a TATA box

pCalSb

60 nucleotides upstream of the TATA sequence. sequence

and

992 to 1121 of

Analysis several

subcloned Comparison

that positions

of pCalSb.

exon

was

identified only the DNA sequence

and

a

sequence GC

box

Analysis of the

immediately upstream

of that found in pCalSb as a likely promoter region. Two additional strand from positions 3).

features of the A2.EB.24 sequence are notable.

Such DNA has been reported to bind abnormally to histones

of nucleosomes. I0 of the AT I gene

Whether

this region influences

is not known.

A second

A2.EB.24 from positions 2116 to 2147. the

dinucleotide

AG.

The coding

274 to 417 is composed of virtually all pyrimidines

This

strains

sequence

between

different

of

rats;

markers

for linkage analysis. 11

in the formation

the transcriptional

important

feature

(Fig.

activity

is the sequence

of

Here the DNA is composed of 16 repeats of probably such

1029

will

show

minisatellite

length DNA has

polymorphisms been

used

as

Vol. 183, No. 3, 1992

EcoRl

i0

20

30

40

50

I

I

I

I

I

I

gaattctact tggcagctcg gatttcaata tgcatagaag

tctgcatttt aaagccattg tctttatttt ctgttagtct

cactttaaat tattgcacag tecactggat gaagtaagct

ggaaecaatg tgtggagtaa ttgaattgct tgtacactat

ctgettgtaa ctcagagcag agtgaggact tgtctgagtt

aatacaatct ggcacatcct cgaatctcca gagtatcaga

v I 61 121 181

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

60

241 gtgattctgg ctcccgatgc agtggggaac gtattttcet ttttcgtttc tttctttcct 301 ttcctttcct ttcetttcct ttectttcct ttcctttcct ttccttcctt ccttcctttc 361 ctttectttc cttcctttcc tttcetttcc ttccttccat cctttccttt ecttttatta 421 481 541 601 661 721 781

aatctagaat taaataaatg aactctgaaa gccacagtaa tcttcttttt ccttatgccc gctgagcttg

tgtectcaaa cagctcgagc ttaaaccatg agacaaggga tttttttttc acctccctec gatctggaag

tattttgtgc aacgtgtaaa ccatctgtaa gttccctaaa tttctaagtg atcttcaaca gcgacactgg

cattttgttt ctagaaggaa tccacaacaa tcacccttaa gagcatttat cttcaagctg gtgaetggca

tggeagtctg ctgataatct aggcatcaca aagttttcca cttggagtca ggatgaggga geagggaagg

tttttatttt tttattttac ttttgagtea agcttcttct gttcatgtgg gtcaggacca cgccgtcaga

841 aactagagag ggagcgggtt ggacagaggg tctcagggcg gggcggggga ggagtgcaaa 901 gccgagcctg agggttggaa cctgcagagc agcgacgccc cctaggctat aaatatggaa 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341

gtgcctagct AGGATTCGTG GTACCAGCGC tcccgccagc cagagtactg ggaatgagcc tgcagggtgg actctacaga attttagctg gttgtgctac gacccccacc caggtcatcg cgcagccatt tctgacttta gagctagcgg ccaatgtctc aaggcttcac tttgccctgt cagggaagtt ctgtgtcatt cagcctgcte aatgattttc aaatataagg caaacactac

getgacctcc GCTTGAGTCC TGCGGCTCTC gtggtgatca ggcacggagg tggagcgggc tccaaaggcg aatggggagg tgacctgaat tttgagctta cccgccccta catcagacag ctgattcctc actgtagtcc agggctcacc tgcaatctca ctgcccccgt tgtctttett tggcactctg ccaggagaga tcttcagtea ttgattgaag acccccaaga ctgtctagtt

aggcagttgg TGTTCCACCC TCAGCTCTGC gagtccggag acttggggga tgcccttctc tactaatgtt caagcaagcc ttaaccgtgg agttaagggt ctgccttctc tcagtcagtg agtcagaggt ccagtggatt attcttctat gaaagctgtg gtttggtcaa aaetetgggg tgccctgagg gagagagaga gtaattetta tccctgtatt agcttgtttc tgcctgtctc

gAGGGACTGG GATCACCGAT CACATTCCCT tggtcggttc cccgggggaa cggatcaggt ttgatcaaac aattttcctc cctgtgtgac gtecccagcc tccttcctct ggtgggccac ggggagaagc tcagtggtce ctcatttccc gtctccagaa gcataggagc agatggtttt tctcatttgg gagagagaga tttccctttt gceectaaea agtgtttcat ctgaggcctc

ATGATGCTGT CACCGGGGTG Ggtgagtccc cttgtcacca cgagctcatt gggagtaaag tctttcgagt cgtgctttta cgcggggeca tcacctcttt cccccaggtt acttcatcac tagtggctgc catttcagta gttccttaga aatgtaaecc atagtctacc tgtttgtttg aagttggcaa gagagagaag caaggtaett aaatacttaa ttccttggtt cggagccatt

CCCGCTGGAG GCCGAGGCCG agcccaggcg catctgaatc cccggtcagg tgcgctggtt acagggtaga aataacttgt acaatttgaa gtttgccaat ctactagcag cacaggtgtt tcctcagtgg ctgaacaacc aacgtttagc aatcctaccc ggtgtccccg tttgcttttt cattaatttc cgcagcctca tcattcctaa ccctctttac aaaccgcaat aaggatcc ^ BamHl

Fig. 3. Sequence of A2.EB.24. Positions 992 to 1121 (CAPITAL letters) are the 5' 130 nucleotides of the AT I cDNAs pCalSb and pBa23.i401. A GC box is centered at position 881 (single line above sequence). A TATA box is centered at position 952 (double line above sequence). A possible cap site is centered at position 986 (* above sequence). The coding strand from position 274 to 417 is composed of virtually all pyrimidines. 16 repeats of the dinucleotide AG are found from position 2116 to 2147 (bold).

DISCUSSION We have previously reported the eDNA sequence of two independent clones encoding the rat AT I receptor. 5 These eDNA, called pCal8b and pBa23.i401, encode

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

an identical protein of 367 amino acids, is 3141 nucleotides. both

pCalSb is 2213 nucleotides; pBa23.i401

Both eDNA begin with identical 5' untranslated sequence and

are polyadenylated.

The

5' untranslated region of pBa23.i401

nucleotides; the similar 5' region of pCalSb is 271 nucleotides.

is 182

The difference,

89 nucleotides, reflects an insertion in the 5' untranslated region of pCal8b after position 130 of the eDNA sequence. the first 130 nucleotides of sequence.

Thus both pCal8b and pBa23.i401 share

Analysis of the genomic fragment A2.EB.24

demonstrates that this DNA sequence is contained within a single exon. not mapped the start site of RNA transcription.

We have

However there are several

reasons to believe that A2.EB.24 contains the first exon of the AT I receptor and that transcription begins close to the 5' end of pCal8b.

Northern analysis has

demonstrated that the major species of mRNA encoding the rat AT I receptor is about

2.3 kb;

pCalSb

is 2213 nucleotides,

pCalSb

and pBa23.i401

independent eDNA yet they begin at the same position within genomic DNA.

are

two

A third

rat AT I eDNA isolated by lwai et al. begins one nucleotide 5' to the start of pCalSb. 6

Finally, the sequence of A2.EB.24 clearly exhibits features of an RNA

polymerase II promoter. and cap signal.

These include a potential Spl binding site, a TATA box

The predicted cap signal is found 35 bp 3' of the TATA box and

8 bp upstream of the 5' end of pCal8b. As indicated, pCalSb contains 89 bp of 5' untranslated sequence not found in pBa23.i401.

Both Southern analysis (Fig. 2) and genomic sequencing indicates

that the insert DNA is encoded by at least one exon separate from the exon found withinA2.EB.24.

Digestion of genomic D N A w i t h four separate restriction enzymes

failed to identify digestion conditions that generate a single DNA fragment when probed with pCa18b/196.

Southern analysis of rat genomic DNA digested with EcoRl

is informative and together with the sequence of A2.EB.24 indicates at least a 3 kb intron between these two portions of the gene. The vascular AT I receptor coding region is contained within a single exon. This genomic pattern has been described for several other G protein-coupled receptors. 12 We have not determined the distance between the first exon of the AT I receptor and the coding region.

None of the % isolates containing the coding

exon overlap the % clone containing the first exon. The AT I receptor mediates all the hemodynamic manifestations of angiotensin II.

Studies

suggest

that

receptor

levels

are

regulated

in

part

by

the

concentration of ligand. 13 While more than one mechanism will probably regulate this process, we postulate that transcription will be regulated both by the status of the renin-angiotensin system and in a tissue specific fashion.

The

isolation of genomic sequence encoding the first exon and upstream sequence of the

AT I receptor

provides

tools

to

investigate

controlling transcription of the receptor.

1031

the

regulatory

mechanisms

Vol. 183, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This work was supported by NIH grants DK39777

and DK44280.

KEB is an

Established Investigatory of the American Heart Association.

REFERENCES i. Peach, M.J., and Dostal, D.E. (1990) J. Cardiovascular Pharm. 16 (Suppl 4), $25-$30. 2. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T., and Herblin, W.F. (1991) TIPS 12, 55-62. 3. Blankley, C.J., Hodges, J.C., Kelly, J.S., and Klutchko, S.R. (1988) European Patent No. 0 245 637. 4. Wong, P.C., Hart, S.D., Zaspel, A.M., Chiu, A.T., Ardecky, R.J., Smith, R.D., and Timmermans, P.B. (1990) J. Pharm. Exp. Therap. 255, 584-592. 5. Murphy, T.J., Alexander, R.W., Griendling, K.K., Runge, M.S., and Bernstein, K.E. (1991) Nature 351, 233-236. 6. lwai, N., Yamano, Y., Chaki, S., Konishi, F., Bardhan, S., Tibbetts, C., Sasaki, K., Hasegawa, M., Matsuda, Y., and Inagami, T. (1991) Biochem. Biophys. Res. Commun. 177, 299-304. 7. Sanger, F., Nicklen, N., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 8. Bernstein, K.E., Martin, B.M., Biol. Chem. 264, 11945-11951.

Edwards, A.S.,

and Bernstein,

E. (1989) J.

9. Bucher, P. (1990) J. Mol. Biol. 212, 563-578. i0. Watson, J., Hopkins, N., Roberts, J., Steitz, J.S., and Weiner, A. (1988) Molecular Biology of the Gene, 4 th edition, p. 249, Benjamin/Cummings Publishing, Menlo Park. ii. Weber, J.L., and May, P.E. (1989) Am. J. Hum. Genet. 44, 388-396. 12. Strosberg, A.D. (1991) Eur. J. Biochem. 196, I-I0. 13. Wilkes, B.M. (1991) In Contemporary Issues in Nephrology, (Goldfarb, S., Ziyadeh, F. and Stein, J., Ed.) Vol. 23, pp. 141-160, Churchill Livingstone, New York.

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