Vol.

189, No.,~,

December

BIOCHEMICAL

1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pa9es 1569-1577

30, 1992

CHARACTERIZATION OF THE HUMAN ISLET AMYLOID POLYPEPTIDE / AMYLIN GENE TRANSCRIPTS: IDE~ICAlYON

OF A NEW FOLY’~TIoN

SITE +

J.W.M. H’dppener1v23*,C. Oosterwijkl, H.J. Visser-Vemooyz, C.J.M. Lips2, and H.S. Janszl

’ Laboratory for Physiological Chemistry, Utrecht University, The Netherlands a Department of Internal Medicine, Utrecht University, The Netherlands Received

November

2,

1992

SUMMARY: Islet amyloid polypeptide (IAPP) or Amylin is synthesizedby the pancreatic B-cells. IAPP is the major componentof islet amyloid in the pancreasof patientswith noninsulin-dependentdiabetes mellitus. We report the composition and complete nucleotide sequenceof the two human IAPP mRNAs of 1.6 and 2.1 kb. A new polyadenylationsite was identified and shown to be used in generation of the 2.1 kb RNA. A previously identZ&l polyadenylation signal is assignedto the 1.6 kb RNA. We exactly determined the major transcription start site, which is used in generation of these mRNAs. Lower abundanceRNAs containing sequenceslocated further upstreamin the IAPP genewere also detected. % 1992 Academic Press, Inc.

INTRODUCTION:

The 37 aminoacid polypeptide IAPP (insuhnoma or islet amyloid

polypeptide) was isolated from amyloid which occurs in insulinomas and in pancreatic islets of patients with non-insulindependent diabetes mellitus (1, 2). IAPP, also called Amyliu (3), is also present in normal islet &cells in mammals and is co-secretedwith irmlin (4, 5). We have isolated the human IAPP gene and locahsed it to the short arm of chromosome12 (6,7). Northern blot analysis of human insulinoma RNA revealedIAPP RNAs of 1.6 and 2.1 kilobases (kb) (6). The aminoacid sequenceof the 89 aminoacid IAPP precursor was predicted by two IAPP cDNAs which correspondedto 3 exons in the human IAPP gene (8). From published cDNA sequencestwo polyadenylation sites, [All (8, 9) and [A21 (9), within exon 3 were inferred.

We have now determined the exact position of the “major” transcription startsite in the human IAPP gene, which is used in generation of both the 1.6 kb and the 2.1 kb IAPP +Sequencedata from this article have been deposited with the EMBL/GenBank Libraries under Accession No. X68830.

Data

To whom correspondenceshould be addressed: Institute of Molecular Biology and Medical Biotechnology, Padualaan8, 3584 CH Utrecht, The Netherlands. l

0006.291 X/92

1569

$4 00

Copyright 0 1992 by Academic Press, Iw All rights of reproduction in arty form reserwd.

Vol. 189, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMYUNICAT,!ONS

RNAs. Two new consensus polyadenylation signals [A3/A4] were discovered, one or both of which are used in generation of the 2.1 kb IAPP RNA. Polyadenylation site A2 is shown to be used in generation of the 1.6 kb IAPP RNA. Polyadenylation site Al is apparently used very

infrequently.

IAPP RNAs

beginning

upstream of the major

transcription startsite (at least up to position -801) were detected, but these RNAs are not abundant.

MATERIALS AND METHODS RNA isolation I Northern blot analysis: Total cellular RNA

was isolated from human insulinomaa by the guanidine thiocyanate method (lo), size-fractionated in 1.4% agarose gels, transferred onto Hybond-N membranes (Amersham) and hybridixed to “P-labeled probes (8). Poly(A) RNA was isolated using the PolyATract mRNA isolation kit II (Promega).

RACE (rapid ampljficatio of cDNA en&): We used a modification of the protocol by Frohmau et al. (11). cDNA was synthesized on 300 ng poly(A) RNA (3’ 65°C) from a human insulinoma, using 50 pmol of an IAPP gene exon 2 oliginucleotide (E2.2, oligo A in Figure l), 20 units RNA& (Promega Biotec), 20 i&i (z-~*PdCI’P (Amer.&am) and 11 units of AMV reverse transcriptase (Stratagene). Excess oligonucleotide was removed by precipitation with 0.6 volumes of 4 M (NH,)acetate and 2.0 volumes of isopropanol. The pellet was dissolved in 50 p.l H,O and 10 p.l was used for 3’ homopolymer tailing using 20 units of terminal deoxynucleotidyl transferase (Phannacia). TE (1 m M Tris, 0.1 m M EDTA, pH 7.6) was added to 500 pl and 10 p.l was used in the first polyrnerase chain reaction (PCR). Amplification of the tailed cDNA was performed with oligonucleotides E2.2 and AB650 (50 pmol each) in a volume of 50 ~.tl using 1 unit of Tuq polymerase (Perkin ElmerKetus). 30 cycles were performed (1’ 94”C, 2’ 45”C, 3’ 72‘C), followed by an incubation at 72OC for 15’. Nucleotide sequenceunalysis: Double stranded RACE products labeled at one 5’ terminus were sequenced using the chemical modification method (12). The nucleotide sequence of exon 3 downstream of the second polyadenylation site [A2] was determined by the chain termination method (13) with oligonucleotides E3.2, E3.3 aud E3.4 as primer, respectively.

RTJPCR: cDNA was synthesized using exon 3 oligonucleotide E3.1 as primer. Aliquots of the cDNA reaction mixtum were used directly as template in subsequent PCRs, using the same exon 3 oligonucleotide as 3’ amplimer and different oligonucleotides upstream of the major transcription startsite as 5’ amplirners. After 30 cycles (1’ 94’C, 2’ 58’C, 3’ 72’C) an incubation at 72’C for 15’ was performed.

Southern blot analysis: RACE- aud RT/PC!R-generated DNA &agments were sixefractionated in 1% agarose gels, blotted onto Hybond-N membranes and hybridized to 5’ end-labeled oligonucleotides or to double-stranded DNA probes labeled by random-priming. Hybridization probes and labeling: All probes for hybridization with Northern blots were generated by PCR. The size and genomic position (numbering according to Figure 4 ) of the PCR probes was as follows: immediately upstream of the major transcription startsite: 115 basepairs (bp)(-120/-5). exon 1: 88 bp (+1/+88), exon 2: 53 bp (+124/+176), exon 3A: 121 bp (+199/+319), exon 3B: 184 bp (+1277/+1460), exon 3C: 94 bp (+1497/+1590), exon 3D: 126 bp (+1944/+2069). Oligonucleotides were labeled using y_.“P ATP (Amersham) and T, polyuucleotide kinase (BRL). PCR-generated double stranded DNA fragments were labeled by random priming (14) using a-“? dCTP and the Klenow fragment of DNA polymerase I (Boehringer Mannbeim). 1570

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Vol. 189, No. 3, 1992

Oligonucleotides: The position and orientation (5’ - 3’) of IAPP gene oligonucleotides which were used as hybridization probe, in RACE reactions, in RT/PCR or for nucleotide sequence analysis, is indicated in Figure 4. The nucleotide sequence of oligonucleotide AB650 is as follows: Y-CGGATGTCGACTCGAAGCITCCCCCCCCCCCCCCCC-3’ Oligonucleotides were synthesized on a Pharmak/LKB Gene Assembler Plus. RESULTS

Determination of the transcriutionstartsite

in the human IAPP fene

A 5’ RACE reaction was performed on RNA from an insulinoma from patient ‘%I” (see Figure 1). A product of 184 bp which hybridized to an exon 1 probe (oligonucleotide El) was obtained after the second PCR. The nucleotide sequence of this product corresponds to an mRNA which starts at position 103 upstream of the 3’ end of exon 1 (which is 25 nucleotides downstream of a consensus TATA-box)

and which contains correctly spliced

exon 1 and exon 2 sequences. To determine whether this RACE product represents the only transcription startsite in the human IAPP gene, we performed RT/PCR experiments on human insulinoma RNA (from patient “S’). RT/PCR products corresponding to IAPP RNAs which extended as far upstream as -558 nucleotides relative to the 3’ end of exon 1 (= - 455 relative to the 5’ end of the RACE product) were detected (Figure 2). In subsequent experiments we even found products upto position -904 relative to the 3’ end of exon 1 (data not shown). Since these products do not hybridize to an intron 1 probe,

- human insulinoma pdy A RNA

IAPP mRNA

- cDNA syndaesis with lAPP cxon 2 oligo A

5’ 3’

Esonl I -

G G+A T+C C AX Exon 3

Eaon 2 1 B$

IAPP

m

3’

5’

- Biogcl AJm column to remove oligo A _ cDNA Iding with excess dGTP

3’ G(n)

-

5’

- fust PCR with oligo A and oligo dC(l6) (30 cycli)

3’ G(n) 5’ C(l6)

-

5’ 3’

- second PCR with nested IAPP exon 2 oligo B and oligo dC(l6) (30 cycli)

5’ C(16) 3’ .

- “hocPCR”wirh

5’ C(l6) 3’ A

5’-l&bd oligo B and oligo dC(l6) (IO cycli)

'50

3’ 0

5’

3’ 5’ 0*

. PAA 84 clectmphomsis. elution of labeled PCR product - Muam & Gilben nucleotide sequence analysis.

Figure 1. Determination of the 5’ end of human IAPP RNAs using a RACE protocol and nucleotide sequenceanalysis. Oligo A is oligonucleotide E2.2, oligo B is otigonucleotide E2.1, oligo dC(16) is otigonucleotide AB650 (also see Materials and Methods). 1571

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-546 -596 780 ‘, 853 ,912 977

603 872 1078 1353

Fieure 2. RT/PC!R on insulinoma RNA from patient “S”. An exon 3 oligonucleotide (733.1)was used for the RT reaction, followed by PCR using the same exon 3 oligonucleotide and different oligonucleotides located further and further upstream of the major transcription startsite. A) W-irradiated 1.096 agarose gel before blotting. The location (numbering according to Figure 4) of the 5’ end of the oligonucleotide which was used as 5’ amplimer in the PCR is indicated below each lane. Size-markers in lane 1 are Hae III fragments of phage 0X174 DNA. B) Autoradiograph of a Southern blot prepared from the gel shown in A, after hybridization with a probe located between positions -120 and +18 (numbering according to Figure 4). The size of the products synthesized on correctly spliced RNA is indicated at the right. Additional hybridizing products might have been produced on residual traces of genomic DNA in the RNA preparation or produced due to non-specific priming of some of the oligonucleotides in the PCR.

they

must be synthesized on spliced IAPP

RNAs.

RT/PCR

with

E2.2 and an

oligonucleotide located at position -1496 relative to the 3’ end of exon 1 did not yield specific products on insulinoma RNA. In a control experiment, the correct product (but including intron 1) was synthesized on DNA from our genomic human IAPP clone Ih201. As a negative control, the same oligonucleotide combinations as described above were also used for RT/PCR on Hela cell RNA, but did not yield products hybridizing to IAPP gene specific probes. The integrity of RNA preparations was confirmed by RT/PCR using oligonucleotides for the household enxyrne glyceraldehyde-6-phosphate dehydrogenase. Determination of the ComDosition of the maior human IAPP RhMs The 1.6 kb and 2.1 kb human IAPP RNAs were detected in a quantitative ratio of approximately

3:l

(as determined by densitometric scanning) in all six insulinomas

investigated and also at very low levels, but in a similar ratio, in normal human pancreas. As shown in Figure 3, a probe located upstream of position -108 relative to the 3’ end of exon 1 does not detect the 1.6 or 2.1 kb IAPP RNAs or any other RNA on Northern blots. A probe containing sequences downstream of position -103 relative to the 3’ end of exon 1 does detect both the 1.6 kb and the 2.1 kb IAPP RNAs. Both of these RNAs also contain exon 2 and IAPP-encoding exon 3 sequences (see Figure 3). In addition, a probe located inbetween the fist [Al] and the second [AZ] polyadenylation site recognizes both 1572

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BIOCHEMICAL

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E2

El

AND BIOPHYSICAL

E3

RESEARCH COMMUNICATIONS

ALU 1

t

AI

A2

-

tt

AN-4

z=zzzZ

2

3A 3D

Fieure 3. Northern blot analysis of 20 pg total cellular RNA from the insulinoma from patient “S”. Probes derived from different regions of the human IAPP gene were generatedby PCR and labeled by random priming (see Materials and Methods). The suitability (in terms of detection limit) for each probe was confiied by hybridization to a spot-blot containing different amounts of DNA from our human genomic L4PP clone )ih201 (6). Sizes of probes are drawn on scale underneath the gene structure, which shows the exons (numbered boxes), protein-encoding regions (shaded areas), the L4PP-encoding region, aa Alu-repetitive sequences(stippled box) and the four identified polyadenylation signals (Al-A4).

of these RNAs.

However,

to the 2.1 kb RNA downstream

a probe located immediately

but not to the 1.6 kb RNA

downstream

(see Figure

of A2 does hybridize

3). We sequenced further

in the human IAPP gene and found two closely linked polyadenylation

consensus sequences (AATAAA)

signal

located 439 [A31 and 469 [A41 nucleotides downstream

of A2. A probe starting 25 nucleotides downstream of A4 does not hybridize to the 2.1 kb IAPP RNA (see Figure 3), indicating that one or both of the polyadenylation signals A3IA4 is used in generation of the 2.1 kb IAPP RNA.

The calculated sizes of the major

human IAPP FUVAs are thus 1446 nucleotides (for usage of A2) and approximately 1900 nucleotides (the exact polyadenylaton site for A3/A4 excluding a poly(A)

tail. When a poly(A)

is not yet known), respectively,

tail (average length of 200 nucleotides) is

included these sizes correspond very well to the sizes of the two polyadenylated IAPP RNA species which we detected on Northern blots (6)(Figure 3). Thus, the genomic organisation of the human IAPP gene encoding the two major human IAPP mRNAs is as indicated in Figure 3. The complete exon sequences are presented in Figure 4. 1573

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DISCUSSION We have determined the composition of the two major human IAPP mRNAs of 1.6 kb and 2.1 kb, which have the same 5’ end, and demonstrated the existence of less abundant IAPP RNAs containing sequences located further upstream in the human IAPP gene. The presence of a transcription startsite within 292 nucleotides upstream of the 3’ end of exon 1 was indicated by previous studies using human IAPP gene promoter/luciferase constructs in transfection experiments (15). However, using RNAse protection analyses, Christmanson et al. (16) detected IAPP RNAs containing nucleotide sequences as far upstream as approximately - 493 relative to the 3’ end of exon 1. The “major” transcription startsite in the human IAPP gene is located at position -103 relative to the 3’ end of exon 1. This corresponds with primer extension data which indicated a transcription startsite at this position (16, 17). IAPP RNAs starting upstream from the major transcription startsite (upto position -801) and lacking intron 1 and intron 2, were detected by RT/PCR. A cDNA described by Sanke et al. (9), which extends 18 nucleotides upstream of the major transcription startsite, might be derived from such a RNA molecule. It caunot be excluded that in addition to the major transcription startsite there is a minor transcription startsite in the human IAPP gene, located upstream of position -801. Alternatively, RNAs containing nucleotide sequences upto at least position 801 are the result of transcriptional readthrough from an upstream gene, thereby generating a dicistronic RNA. Either way, the enzyme RT may not have reached the 5’ end of such a (dicistronic) RNA, which would explain why RT/PCR products do not extend beyond approximately 1 kb upstream of the IAPP gene major transcription startsite. On Northern blots of insulinomas we did not detect RNAs containing these parts of the human IAPP gene. This makes it very difficult to determine which polyadenylation signal is used by these RNAs and what their size is. We do not know whether the translation initiation codon in exon 2 of the IAPP gene is functional in such (dicistronic) RNAs or if ATG

Figure 4. Nucleotidesequenceof the exons(capitalletters)and bordering 5’ and 3’ regions (small

letters) of the human lAPP gene. Jntron sequencesare not numbered.The numberingof exon sequences, sequences upstream of the major transcription startsite and downstream of the last polyadenylation signal (A4),is relative to the position of the major transcription startsite (indicated by an open arrow). Also indicated are the TATA box starting at position -29, the translation initiation codon ATG in exon 2 (starting at position +119). the IAPP-encodiig sequence (position +218/+328), the translational termination codon TAG (starting at position +386) and an Alu repetitive sequence (position +756/+1035, underlined) in exon 3 as well as the identified polyadenylation signals Al - A4 (starting at positions +562, +1422, +1884 and +1914, respectively). Polyadenylation sites Al and A2 are indicated by an arrow. Since the exact site of addition of the poly(A)-tail for polyadenylation signals A3 and/or A4 is unknown, we have assigned “exon sequences”until the 5’ end of PCR probe “exon 3D” which does not hybridize. to the 2.1 kb RNA (also see Materials aud Methods). The position and orientation (5’-3’) of several oligonucleotides which were used as hybridization probe or in RAGE reactions, RT/PCR or nucleotide sequence analysis are also indicated underneath the nucleotide sequence:c= El, B= E2.1, A= E2.2, d= E3.1, e= E3.2, f=E3.3 and g= E3.4. The nucleotide sequenceup to position +1507 was published previously (18). However, a few corrections have now been made in tbe region between +1462 and +1507. 1574

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tqaqctqcct

+51

qttaqcaaat 4 TTTCTTTCTA

f104

aactqtaaqa

+11t3

AATGGGCATC

-10

+

AND BIOPHYSICAL

BIOCHEMICAL

qatqtcaqaq

ctqaqaaaqq

GAGGGGGTAA

ATATTCCAGT

RESEARCH COMMUNICATIONS

tqtqaqqqqt

atataaqaqc

tqqattacta

GGATACAAGC

TTGGACTCTT

TTCTTGAAGC

TCAGAAGCAT

TTGCTGATA?

TGCTGACATT

GAAACATTAA

AAGqtaaaqa

aatctcttqa

tttcaqtqct

qqattattct

ttqcaqA&AA

TTTGAGAAGC

CTGAAGCTGC

AAGTATTTCT

CATTGTGCTC

TCTGTTGCAT

TGAACCATCT

*

Be

+178

GAAAGCTACA

CCCATTGAAA

Gqttqqtaac

tttaaaatcc

tqtttctttq

taacttttqt

t199

tqttccatqt

taccaqTCAT

CAGGTGGALP.

AGCGGAAATG

CAACACTGCC

ACATGTGCAA

t243

CGCAGCGCCT

GGCAAAl!Tl-T

TTAGTTCAlT

CCAGCAA~

CTTTGGTGCC

ATTCTCTCAT

t303

CTACCAACGT

GGGATCCAAT

ACATATGGCA

AGAGGAATGC

AGTAGAGGTT

TTAAAGAGAG

+3f53

AGCCACTGAA

TTACTTGCCC

CTTTAGAGGA

CAATGTAACT

CTATAGTTAT

TGTTTTATGT

+423

TCTAGTGATT

TCCTGTATAA

TTTAACAGTG

CCCTTTTCAT

CTCCAGTGTG

AATATATGGT

t403

CTGTGTGTCT

GATGTTTGTT

1

EXON

2

EXON

3

AAAAGATTGT

TTTATATGTA GTTTTTGCTA

ATACCTTCTC

A

t543

GTACTAACTA

GCTAGGACAT 4 AGGTCCCA+j==+AGAT

+603

TAGATTTGTA

TTTTAAAACA

TAAGAACGTC

ATTTTGGGAC

AAAATGAAAT t CTATATCTCA

+663

TTTAAGAACG

AAGGAGAIQA

AGGTAGTTTG

AACCTTGGTA

AATTGTAAAC

AGCTAATAAT

+723

GAAGTTATTC

TTGACATGAG

AAiUTCAGTA

ATTGGACCAG

GCGCGGTGGC

TCTTGCCTGT

t783

AATCCCAGCA

CTTTGGGAGG

CCGAGGCAGG

CAGATCACAA

GGTCAGGAGT

TCGAGACCAG

+843

CCTGACCAAC

ATGGTGAAAC

CCTGTCTCTA

CTAAAAATAC

AAAAATTAGC

CGGGGGTGGT

+903

GACATGTGCC

TGTAATCCCA

GCTACTCAGG

AGGCTAAGGC

AGGAGAATCG

CTTAAACCCA

+963

GGAGGCGGAG

GTTGCAGTGA

GCCCAGATTG

CACCACTGCA

CTCCAGCCTG

GGTGGCAGAG

TTAGTAATTG

TAAGTACCCC

TGATAAGCAA

AGTATCTTTT

d

GTGGCACAGG

+1023

TGAGACTCGT

CTCAAAAAAA

AGAAAGAARA

+1083

ATTAGTAATT

GTCAATACCC

CTGTTAAGCA

ATTCCTTTTT

GCAGTATATT

TCTGAAATGA

t1143

CAGAATGCTG

TTTTAAAAAC

AAAGAAATAA

AATCCTGCTC

CTGACTCGGT

CAAAATATTT

t1203

TTTAAAGTCT

ATTGTTTGTT

GTGCTTGCTG

GTACTAAGAG

GCTATTTAAA

AGTATAAAAC

+1263

TGCTTTGTAT

TTCATTGTGT

GTTAGCAGCA

GTGAGCTTCT

ATTAAATGTA

+1323

TATGTCATTT

CCATGAGGGT P ATTTTGTTTA

AGTGGCTTTC

AGCAAACCTC

AGTCATATTC

TTATGCAGGG

t1383

TATTGCGAAA

CAACTTGTGT

TCTATTAATC

GTGTCTTC +TT+GACC

ACAGACTTCT

+1443

GGAAACTCTT t GAGGTTATGT

TGCTGTATAq

GAATTATTTC

TTTTGTTTRA

CAAATTAGAC

ATTTCTGGCA

ATATGATACA

CTTTTTTTGA

TAGCAGCTGC

AATGTTGGAC

AGAAGATGM

+1563

ATGCTTTGCT I

TTGAGTCAGA

TTCTTATGAA b

TATCTGCTTT

TCCCTGACTT

TGAGTTAGGT

+1623

AGCTTTGGAA

GTAGCATTAA

TTCAGATAAA

CTGCCATCAT

GCTGCGTTAT

GCCATTTCTA

t1683

AAGACCACTC

AACTTGTACT

TTTMAAAAA

TAGAMiAAAT

AGCATTTCAA

TCTAAGTGGA

+1743

AATTTGA~TC

ATTGACTTAC

ATTTCTAAGT

TRAAATTTCC

CTTTATGAAG

TGTGCCTTAG

t1803

GTTACCAAAT

TGTAGAGGCT

TTCGTTGGTG

GTGGTAATTG

GTAGCGGTAG

TGAGTGTATA

+1863

GAGGCAGGGA

AATATATTTA

$&ii+TC

TATGTCATGA

ATTAkkATTG

&j===AG

t1923

TGAATATACA

AATTTATATT

Tqtqatqctc

aattqttqqt

ccttctttca

aaqtqqcctc

+19a3

aaacccatat

ttcaaattqa

ataaqqtaca

attaaattat

aatctctcaa

acttatttqa

t2043

aaaatttccc

acaataqtct

acaqttttat

tqcatatcac

acctatatat

qatataaata

+1503

EXON

c

1575

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Vol. 189, No. 3, 1992

sequences upstream of the IAPP gene major transcription startsite are used to produce other proteins in addition to or instead of the IAPP precursor. Our results demonstrate that polyadenylation

signal A2 is used for the 1.6 kb RNA,

whereas the 2.1 kb RNA extends further downstream (polyadenylation signal A3/A4). The functional significance of two IAPP mRNAs which only differ in their 3’ non-coding sequences is unknown, but might be related to a different intracellular localization, stability or translatability. On Northern blots of several human insulinomas we did not detect RNAs for which polyadenylation

signal Al

is used. That this nonetheless is a functional

polyadenylation signal is indicated by two cDNAs which are polyadenylated at a position 22 nucleotides further downstream (8,9). Apparently polyadenylation at Al occurs very infrequently in human insulinomas. Based on transient expression studies with human IAPP promoter constructs and on RNAse protection assays it was proposed that human IAPP RNAs start at a position approximately 493 nucleotides upstream of the 3’ end of exon 1 (which is 390 nucleotides upstream of the major transcription startsite) (16). Polyadenylationsignal A2 was assigned to the 2.1 kb RNA aud an AATti

sequence inbetween Al and A2 (at position 1168-1173 in Figure

4) was proposed to be used as polyadenylation signal in generation of a more abundant 1.8 kb human IAPP RNA (16). This 1.8 kb RNA probably corresponds to the 1.6 kb RNA we described. However, we have shown that the 1.6 and 2.1 kb IAPP RNAs are generated using the major transcription startsite and that RNAs starting further upstream are much less abundant. In addition, polyadenylation site A2 is used for the 1.6 kb RNA whereas the 2.1 kb RNA uses (a) newly identified polyadenylation signal(s) A3/A4.

Acknowledjpnents: We thank Dr. A.D.M. Van Mansfeld and H.A.A.M. Vau Teeffelen for providing some of the insulinoma RNA preparations, Dr. R.J.C. Slebos for providing oligonucleotide AB650, glyceraldehyde-6-phosphate dehydrogenase oligonucleotides aud advice on the RACE reactions and G. Peek and I. Janssen for assistance in preparation of the figures. This research was supported by the Royal Dutch Academy of Sciences (KNAW) (J.W.M.H.) and the Netherlands Organization for Chemical Research (SON) with financial aid from the Netherlands Organ&ion for Scientific Research (NWO) (C.O.).

REFERENCES 1. 2. 3. 4. 5. 6.

Westermark, P., Wemstedt, C., Wilander, E., Hayden, D.W., O’Brien, T.D. and Johnson, K.H. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3881-3885. Cooper, G.J.S., Willis, A.C., Clark,.A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8628-8632. Leighton, B. and Cooper, G.J.S. (1988) Nature 335, 632-635. Westermark, P., Wilander, E., Westermadc, G,T. and Johnson, K.H. (1987) Diabetologia 30, 887-892. Van Jaarsveld, B.C., Hackeng, W.H.L., Nieuwenhuis, M.G., Erkelens, D. W., Geerdink, R.A. and Lips, C.J.M. (1990) Lancet i:60. Mosselmau, S., Hijppener, J.W.M., Zandberg, J., Van Mansfeld, A.D.M., Geurts van Kessel, A.H.M., Lips, C.J.M. and Jansz, H.S. (1988) FEBS Lett. 239, 227-232. 1576

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Buckle, V.J., Marsland, A.M., Mosselman, M. and Hoppener, J.W.M. (1989) Cytogenet. Cell. Genet. 51, 972. Mosselman, S., Hopperter, J.W.M., Lips, CJ.M. and Jansz, H.S. (1989) FEBS I&t. 247, 154-158. Sanke, T., Bell; G.I., Sample, C., Rubenstein, A.H. and Steiner, D.F. (1988) J. Biol. Chem. 263, 17243-17246. Chirgwin, J.M., Przybyla, A.E., McDonald, R.J. and Rutten, W.J. (1979) Biochemistry 18, 5294-5299. Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc. Natl. Acad. sci. U.S.A. 85, 8998-9002. Maxam, A.M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560-564. Sanger, F., Nicklen, S. and Cot&on, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. Feinberg, A. and Vogelstein, B. (1983) Anal. B&hem. 132, 6-13. Mossehnan, S., Hoppener, J.W.M., De Wit, L., Soeller, W., Lips, C.J.M. and Jansz, H.S. (1990) FEBS Lett. 271, 33-36. Christmanson, L., Rorsman, F., Stenman, G., Westermark, P. and Betsholtz, C. (1990) FEBS Len. 267, 160-166. Nishi, M., Sanke, T., Seino, S., Eddy, R.L., Fan, Y.-S., Byers, M.g., Shows, T.B., Bell, G.I. and Steiner, D.F. (1989) Mol. Endocrin. 3, 1775-1781. Van Mansfeld, A.D.M., Mossehnan, S., Hiippener, J.W.M., Zandberg, J., Van Teeffelen, H.A.A.M., Baas, P.D., Lips, C.J.M. and Jansz, H.S. (1990) Biochim. Biophys. Acta 1087, 235-240.

1577

amylin gene transcripts: identification of a new polyadenylation site.

Islet amyloid polypeptide (IAPP) or Amylin is synthesized by the pancreatic beta-cells. IAPP is the major component of islet amyloid in the pancreas o...
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