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
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol. 189, No. 3, 1992
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
Vol.
189,
No.
BIOCHEMICAL
3, 1992
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
Vol. 189, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol.
-70
189,
No.
3, 1992
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
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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
Vol..l89,
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Nq. 3, 1992
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