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Isolation and sequence of the murine Fgf6 cDNA
Vincent Ollendorff, Olivier Rosnet, Irène Marics, Daniel Birnbaum, Odile
Delapeyrière
To cite this version:
Vincent Ollendorff, Olivier Rosnet, Irène Marics, Daniel Birnbaum, Odile Delapeyrière. Isolation and
sequence of the murine Fgf6 cDNA. Biochimie, Elsevier, 1992, 74 (11), pp.1035-1038.
�10.1016/0300-9084(92)90025-A�. �hal-02711885�
S h o r t c o m m u n k a f i o n
Isolation and sequence of the m u ~ n e Fgf6 cDNA*
V O l l e n d o r f f , O RosneK I M a f i c s , D B i m b a u m , O
deLapeyfi~**
~ ~ ~ Molecular On~logy, ~ ! 9 INSERM, 27 Bd Le~ R o u t , 13009 Marseille, France(Rec~ved 16 S ~ m ~ r 199~ ~ ~ Se~emb~ 1992)
Summary - We have ~u~ed the ~mcm~ of ~ e routine Fg/6 gene enco~ng a fibm~ast growth factor wi~ ~e purpo~ of ~oking for p~ative ~ g ~ a ~ r y sequences ~ ~e $' and 3' nomcod~g m~on~ The Fgf6 cDNA contains a yew ~ng 3' untransla~d portion of 4015
n u c l i d e s .
f l b ~ M ~ t g r o w ~ factor / o n c o ~ n e / nucleotide sequence / ~ g u M ~ ~emen~
~ t ~ d u ~ n
The fibrobla~ growth factor ( F G ~ ~ m i ~ comprises ~ date seven memb~s ~ ~ e ~ v ~ v e d ~ ~ v e m l ~ o g ~ processes [!1. We h ~ e characterized ~ e ~ n e enco~ng ~ e f i ~ h member of t ~ s ~mily ~ man [2-4] m d in mouse ~]. The transcripts of the
FGF
genes ~ general, and of Fgf6 ~ p a n ~ , comprise a ~ n g 3' tail co~esponding m an untranslated ~ o n . The m ~ of ~ s e q u e ~ present in humm as wall as in mouse ~ m ~ n s unknown. Howeve~ several ~eces ~ evidence ~ d ~ a t e that t ~ s ~ p e of ~ q u ~ m ~ ~ some role. A d d ~ o n ~ open ~ading flames (ORF~ have been ~ u n d ~ 5' or 3' reg~ns ~
FGF
genes D, 7]. M o ~ impo~anfl~ Curat~a and B a ~ o [ ~ have e~a~ished ~ e p~sence of r e g ~ m o ~ ~ e - meres w i ~ enhancer activ~y in ~ e 3' nomco~ng reg~n ~ the c ~ s d y related
Fgf4/K-Fgf
~ n e . T ~ s enhancer ~ n d s nuc~ar factors [9]. For ~ e pu~ose of stud~ng the stru~ure and posfi~e ~ g ~ a m ~ ro~ of ~ e 5' m d 3' untranflated ~ o n s of Fg/6, we c~ned and sequenced the mufine Fgf6 cDNA.M a t e r , s and m ~ h o d s cDNA cloning and screening
~o~sDtr~NcAt~nar~ gf~t~. "~h~Nc~I~ 15s~n~hd~sT~ioenm~7~c~ *The nucleotide sequence data presented in thh paper have been submitted to the EMBL-Genbank Data Libraries under the accession numbers M92415 (550 first nucleotides, of geno- ,m~x~n~edncMe92an4 ~ n ~ s ~ t of the sequence).
fink~s and l~ation m ~ ~10 arms wen performed us~g Amersham syntheMs and c ~ n g ~ts as ~commended by ~e manufacm~ The l~a~d mastiff was packaged us~g Gigapack GAd exffac~ (S~a~gene) and the phages wen ~ ~d u~ng NMSI4 as a bac~fi~ host swan. T~s I~ra~ was ~ e n e d with genomic (EcoRV-Na~ ~agme~) or cDNA (Psd-Nc~ fragment) mouse Fgf6 probes ~ h p~v~u~y de- scribed ~ ~1).
Isolation of cDNA c~nes by PCR amplification
An ~iqu~ ~ Bg) of totfl RNA from 1 5 ~ a y mouse emb~o was used to genemm a flint g~nd eDNA. The RNA was heamd to 90°C for 5 min and the cDNA w ~ syn~etized by incubating 10 m ~ ~ room ~ m p ~ u m and 1 h at 42°C ~ a 20-~ fin~
~ ' a c l t ~ t ~ g ~ atRnaNn95°C for(Pr°mega)5 rain, hNf of and l X~e cDNA PCR buffe~action After mix was succeed ~ PCR a m ~ c a t i o n ~ a fin~ vo~me of 50 ~ of I x PCR buffer containing 25 pm~ of each ~igonuc~o- tide pfim~ and 1 U of Taq p~ymera~ (CereS. The same PCR buffer ~ 0 mM KCI, 20 mM Tr~-HCI pH 8.4, 1 ~ mM MgCI>
1 mg ml -~ BSA) was used ~ both ~v~se tran~ripmse and ~n~7~tt~£h~twe~, a ~ c i ~ i t ~ l d ~ t ~ ~ i~t wg~cCh ~efOr anne~gcyde from~mperatu~65°C to 55°C. was ~ e ~ s i ~ m ~ r ~ e 1 ; ~ ; ~
where ~e eg~n~on time was I0 m~. The PCR pmdu~s wen then Num~nded uNng the K~now p N y m e ~ and subcloned into a SmaI-d~gested Bluescd~ (Stra~gene) ~asmid.
cDNA sequencing
Nuc~m~e sequenc~g was performed by ~e d~eoxy~h~n ~ r m ~ n m~hod u~ng single-s~anded and/or dou~e-swan- ded Blue~fi~ (S~agene) ~ m ~ e s and ~ e mottled T7 p ~ y m ~ e (Sequenase) protocol (US Biochemicals~
1 ~ 6
A.
/ / /; / / / / / / ; !,/
E I . / / B . ~ ! ! ! i / / / ! / i / / / / / ~ ~ ~ C ~ • 5 ~ C . 2 1Fig 1. Clo~ng of Fgf6
cooing
and untran~ated region~ Mo~ of the coding regions (black boxes) and the tot~ity of the Y untrans~ted region (open box) of the Fgf6 gene (A) were obtained by cDNA cloning (B, dashed box~ 5' Sequences (B, open box) were derived from genomk clonin~ R e s t r i ~ n enzyme sites mentioned in the ~xt are showm abbreviations are ~s fob lows: E, EcoRV; N, Nae~ Nc, Ncol; ~ PstI; S, Sphl; X, Xhol. The insets of the two cDNA dories (1 and 2) and the pmdu~ of the PCR ~oning (3) discussed in the texL are shown (C). The portions of the oligonucleotide pfime~ (a, b, and c) are shown above. The sc~e ~ the same for the three panels.R e s ~ and d ~ c u s ~ o n
In figure 1 the ~ones ~ were ~ a t e d from a mouse e m b ~ o cDNA l~rary are describe~ A fi~t eDNA c ~ n e ( c ~ n e 1) c o m ~ n e d 2.8 kb of 3' untran~ated sequence. A second cDNA ~ o n e (clone 2) contained part of ~ e 3' r e ~ o ~ ~ e wh~e of ~ e coding ~quences of exons 3 and 2, and ex~nded 5' into the ~ g g e ~ part of exon 1. In b ~ w e e n these two cDNA~ a poRion of • e 3' sequence r a i s i n g in the cDNA clones was o ~ n e d by c ~ n g of a p o ~ m e ~ s e c h i n reaction (PCR) produ~ obtained a~er two PCR s~ps. The first ~ep used a 3' primer ~ ) d e i g n e d in ~ e 5' end of ~ e fi~t ~one and a 5' primer (b) chosen in the fi~t e x o ~ T h e e prime~ span two different exons. The~ nucleo- fide sequences were 5'-GGATTGTCTTGCAGGT- CAGG-T and 5'-AACACACGAGGAGAACCCCT- 3', respe~ively. The second step was performed in the same conditions e x c e ~ for the use of a 5~ AGAGTGCTCTCTTCATTGCC-3'- nes~d 5' primer ~ ) defigned in ~ e second exon. The combin~ion of • e cDNA c ~ n e s and PCR produ~ r e s ~ d in a 4 ~ - kb cDNA, m ~ n g the fi~t 54 n u c ~ i d e s of Fgf6 coding sequence. Further scree~ng of ~ t h e r the orig- in~ or newly made cDNA fibraries did not allow ~ ~ e eDNA ~ones ex~nded f u l ~ ~ ~ e 5' e n ~ pre-
sumably because of strong secondary structure~ Further u p . r e a m sequences corresponding to the Y coding end o f the Fgf6 gene were therefore obtained from genomic ~ones. The genomic sequence was ex~nded even more 5' to include putative regulatory e ~ m e n t ~ P o t i o n s o f p r e ~ o u ~ y ~olated cosmid clones from the Fgf6 ~ c u s were subcloned. One such fragment, named XX ~ ] , conesponded to the 5' por- tion of the Fgf6 gene. A SphI-XhoI subfragment (fig IA) derived from XX was sequenced.
As in human [3], a 624-nucleofide-long ORF ~ able to code for a protein of 208 residues (fig 2). However, there are three in-frame ATG codons and two sho~er proteins could pos~bly be made. The 5' region com~ns n ~ t h e r CAAT nor TATA boxes Putative regulatory d e m e n t s are ~ c a t e d about 300 bp upstream of the ATG (fig 2); they i n c ~ d e a decanu- ~eotide repe~ AGGGTGGTGA, and the palindromk TGACGTCA ~ n d i n g si~ for the CREB ~ e m e n t found in the 5' part of cAMP regulated genes [11]. The 3' untran~ated region, ~arting a~er the stop codon ~ position 1121, is 4015 n u c ~ o t ~ e s ~ n ~ The polyadenylation ~ ~ preceded by an unusual poly(A) ~ g n ~ sequence at position 5122. A poly(A) stre~h ~arting ~ position 1686 may represent the remnant of an arch~c polyadenylation sequence. ~ ~
1
CCC~CC~TCC~CCT~C~G&~TGCT~T~C~C~TTCCT~TAG ~ ~ ~ ~ ~ T ~ T C T ~ G121 ~ ~ ~ A ~ T ~ T ~ C ~
181 T ~ ~ ~ ~ T A ~ C T A T ~ T ~ T C
241 ~ ~ ~ ~ A ~ C ~ C ~ T G
301 ~ A T A ~ ~ ~ ~ T ~ T ~ C ~
351
~ T ~ ~ T ~ ~ T ~ C C ~ C ~ T421 ~ C ~ G ~ ~ A ~ A ~ T T C ~ C ~ C G ~
481
~ ~ T ~ G A ~ T ~ T ~ C ~ _ _ 5~ ~ ~ ~ T T ~ G T C C T ~ T ~ T _ _ ~ ~ ~ ~ C ~ T ~ C T C ~ 6~ ~ C C K ~ C T ~ T A ~ T ~ T ~ ~I ~ T ~ ~ ~ T C T ~ T ~ C ~7~
~ C ~ C T ~ T ~ C ~ C C ~ A 8~ ~ T ~ C ~ C ~ A ~ T ~ G C C ~ ~ G9~ ~ T C ~ T T ~ T ~ ~ C ~ T ~ C ~ G C T T C ~
9~ ~ T ~ G T ~ C G ~ ~ C T ~ C ~ C T A C ~
~21 G T ~ C C T ~ T T ~ T ~ C ~ T ~ G ~ G ~ T
10~ N
~
~
T
~
~
~
~
II~ C ~ ~ T G C G ~ ~ T ~ G ~ 1201 ~ T h ~ C ~ A ~ A T C ~ T ~ G T G ~ T ~ T 1~1 ~ ~ ~ ~ C ~ G ~ 1321 ~ ~ C ~ ~ C C T ~ T A ~ T T 1381 ~ ~ T ~ ~ T ~ G ~ ? ~ 1441 T A ~ ~ ~ ~ f f f f A ~ T I ~ T T 1501 ~ ~ ~ ~ G ~ C ~ 1561 I ~ ~ ~ ~ T ~ A ~ ~1621
~ A ~ A ~ ~ A ~ A T ~ T A T ~1681 A ~ ~ ~ ? ~ C ~ C ~ T T C ~ ~
1741 ~ T ~ A ~ T A ~ T ~ ~ T C C C C ~ A ~
1801 ~ ? ~ T A ~ ~ G ~ T ~ ~ T
1861 GTCCTGTAG~C~TT~C~~CTCC~ ~? ~T~G
1921 C ~ G C C ~ G T ~ C G ~ ~ ? ~
19~
~ ~ C ? ~ T ~ C C ? C C ~ ~ G T ~ T 20~ T A ~ G ~ ~ T ~ T ~ C T C T C ~ C T ~ 21~ TCU~CT~C~G~CCC~TA~C~T~CTCG~CT~G 21~ C T A ~ T C ~ T ~ T ~ ~ T G A C C T ~ A ~ 2221 C C T ~ C f A ~ G ? A ~ T ~ ~ G ~ G C ~ 22~ T C ~ ~ f ~ ~ A T ~ T ~ C ~ ? T C 23~ C ~ A ? ~ ~ A G ~ ~ T T ~ T ~2401
T C C T ~ T ~ C ~ C G T A ~ T A T ~ T A T ~~61
~ T ~ C C C T C ~ T A C C C ~ C ~ ~ T C C C C ~ 2521 ? C T ~ ? A C ~ ? ~ T A C C ~ A W C ~ A T A ~ C ~ A 2581 ~CAGATCTC~CTCACGGACTCTGAGCTGCAG~CG~TC~G~TTC~CGATGC~G~G 2641 ~ C ~ T T C ~ C T T ~ C C ~ T ~ G ~ T ~ T ~ A ~2701
A ~ G ~ C C G R ~ G A ~ G A ~ T ~ T ~ T ~ T ~ G A ~ T ~ C2761 A T ~ T C ~ T T ~ A ~ ~ C C ~ T ~ A G
2821
~ ~ T T ~ T C ~ T A T T ~ T C ~ T ~ G T ~ 288~ ~ ~ T ~ C T ~ T A T C ~ T A ~ T A T ~ T ~ T A T ~ G 294~ ~T~T~CTT~A~T~TGA~T~T~CTGATGT~CT~G 300~ ~ T ~ T ~ T ~ T A T C T G ~ T A ~ G T ~ T A C ~ C 306~ T A G A T ~ T ~ C ~ T ~ C C ~ C ~ C T ~ C ~ ~ 3~2~ ~ C C ~ C C ~ T C C T ~ T C C T T T 3~8~ CTA~TC~AT~TTA~T~CTTGA~TCTAGTG~ACT~GACCCTG 324~ ~T~TG~TCTTT~T~CTTT~T~TG~GT~TC~TCC~A~G 330~ GTTC~T~CCACTC~CCCTT~T~CG~T~G~G~GCCCCGG~G 336~ CCCTGAT~CGGCCTG~GT~CCT~GA~G~C~GTTCCT 342~ ATG~TGA~TT~TTGT~~GTA~T~CC~TTTTGCT~AC 348~ TGGTT~T~ATGGRT~TACT~CT~TTTCCTGT~G~T~TCTT~GA 354~ GGTTTCTCTTTGACC~GT~A~GTA~GT~CC~TC~C 3~0~ ~ G T T ~ G ~ A T ~ G A ~ T ~ G A G ~ T C C C T T 3 ~ CTCTCCT~TACTGT~AG~TT~TCCT~C~CACCT~CTCCTT 372~ G ~ G T ~ T T C G T ~ T ~ G T G T G T T C ~ T T ~ G ~ C 3781 ~AG~T~T~GT~GA~CT~GT~TCT~GAG~TCT~TT~GTTC 384~ CCTGTA~G~T~T~T~GT~C~T~TGTCT~GTCTCTCTCT 390~ C ~ T C ~ G ~ G ~ T ~ G A T A T G ~ C T ~ T T ~ T C 396~ ~C~CCTCGTGA~C~T~CTTTTGATA~TTTGAC~GT~ 4~2~ ~T~GAT~TAC~T~TACCTT~GA~T~CC~GTTCCT 408~ C T G ~ G A ~ C C T A K C ~ ~ T C T ~ G T ~ C ~ G T A T G G 4X4~ T T T ~ C C ~ T ~ C ~ C T ~ T ~ A ~ A ~ C ~ T C ~ C T C T T 420~ A ~ T G ~ C T C T k ~ C ~ C C T G ~ T ~ T ~ T G K C ~ C C ~ C 426~ TTGTT~TGA~T~C~AC~TTGA~CCCCTTTCCTGTC~C~C 432~ A ~ T ~ C ~ T ~ T ~ C T ~ G ~ A ~ G A G G ~ A ~ G ~ G ~ T C ~ 438~ TG~TCC~TC~CCT~G~T~T~TT~G~TAC~TGT~ 444~ ~TC~T~CCC~CTC~GTATA~T~CT~GATTCC~C~CCCTT 45~ TTTGT~GCT~X~TA~C~TCTAKCTTA~T~TCCC~G 456~ A T ~ T ~ C T ~ A ~ C ~ G A ~ T C ~ C T ~ G T ~ A T ~ A 46~ TGA~TA~CTACC~CC~C~GA~GAC~C~TTG~CTT~CTG~G 4~8~ ~ C ~ T ~ ~ T ~ C ~ A ~ T A C T ~ 4741 G T ~ A ~ T C T ~ G T ~ k T ~ C T C K C C ~ G A ~ T A C ~ T ~ 480~ A ~ G A C T C T ~ T ~ G T ~ T ~ C ~ T G T ~ G T A T A C C 48~I T ~ C T A C T G ~ C ~ C ~ T ~ C T ~ G T ~ G ~ A ~ C 492~ ~ T ~ T ~ T A ~ G ~ C T ~ T C T ~ T ~ A ~ C ~ T A ~ A ~ T ~ 498~ A~TG~T~TGT~TT~G~T~T~TTCT~TG~T~TAT~ATGTG 50~ ~ C ~ T ~ T ~ C C ~ ~ T ~ T ~ A T A C G ~ T ~~01 ACT~TTCACTCCGRCCTC~TT~C~~
Figlows:genomic
sequences2"threeNUC~°fideAT
G codonsSequence(1
to 550)(bold°fand
cDNAand
the underlined),m°USesequence
Fgf6 theC°ding(55
lstoptoandcodonthe
Theuntranflated(bold)
end~
' putativeCOdingreg~n~5,sequenceTheregulatorynUCk°fideis
boxed.dementsSequenCesped~(unded~ed),
featuresis
a compoSRear
e vafiantfol.Of
polyadenylation fign~ sequence (doubly underlined) and a putative alternative po~adenylation fign~ sequence (douNy unde~
lined, position 1664). Doubly underlined and in bald ~ a block of 15 tetranuckotide repe~m
1 ~ 8
p~ceded by a pmative po~adenylation ~gn~ sequence thin may be used ~ h o a e r Fgf6 ~anscfipts of about 1 ~ kb are indeed som~imes observed in some tissues). A block of 15 ~ a n u c ~ o f i d e r e p e l s is pres- ent from position 3099 to 3159. These r e p e l s identify a putative m ~ r o s m d f i ~ and could be used to reve~ a p d y m o r p ~ s m ~ mous~ and maybe ~ s o in human w h e ~ ~ e y c o ~ d be conserved [12].
Indee& in a more general wa~
comparison
of ~ e 5' and 3' non-co~ng sequences described h e ~ w i ~ the co~espond~g human sequences c o ~ d y i d d infor- m ~ n about impo~am conserved ~ m u ~ of the 5' and 3' ~ g ~ n s . Furthermore, the c ~ n ~ g and se- quencing of the 3' un~anslmed region of Fgf6 will h e ~ in deriving specific Fgf6 probes useful in other ~ u d ~ s such as ~ situ h y b r i d i z ~ n a n ~ y ~ s of Fgf6 e x p l a i n . Indeed, ~ e 5' ~agmem of clone 1 ob- tained by E c o R V d~estion (named dream) used as ~ m ~ e for anfisense fiboprobes gives a good fign~ in h y b r i ~ z m ~ n of mouse embryo sections (deLapeyri~re ~ al, ~ p~paration).Acknow~dgments
T~s wo~ was supposed by In~rm and grants fram ARC and AFM. We are g m ~ ! to F Coffer for h~p~l advice and J Hanche for mchn~fl assistance. VO is the ~dp~m of a ~l- ~wsh~ from the 'L~ue Nm~nfle Cont~ ~ Cancer'.
References
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2 Mafics I, A d ~ d e J, Raybaud E Mauei MG, C o ~ r E ~anche J, deLapeyfi~ O, Birnbaum D (1989) Chamc- ~fizm~n of ~e Hst-related FGF-6 gen~ a new member of
t ~ f i b ~ g ~ h ~ gene ~ f l ~ O ~ e ~ ~ 5 ~ 3 C o ~ r E Bat~ ~ M~cs L ~ ~ ~ ~ m - baum D (1991) ~ t ~ e s ~ ~ ~ ~ ~ p~- duct ~ d ~ of ~e fign~ ~ d e . O ~ e ~ 1 ~ 4 ~ r E ~ n d ~ ~ M~cs L ~ s ~ t ~ B ~ M, ~ c h e L M ~ t o ~ ~ ~ , d ~ ~ ~ B ~ u m D (1991) ~ e FGF6 ~ n e ~ n the FGF multigene ~mily. Ann ~ A c a d Sci 638, 53~1
5 d e L a p e y ~ O, Rosn~ ~ B e n h ~ h ~ R~baud ~ M a ~ o S, ~ a n c ~ J, G ~ a ~ E M ~ M ~ Cop~ I~d ~ ~n~ns ~ Co~i~ E ~ m ~ u m D (19~) S ~ u r e , chromosome m ~ n g ~ d expre~ion of the routine F~-6 gene. Oncogene 5, 823-831
6 ~ffa ~ ~ s ~ ~ ~ g a w a ~ S~amo~ ~ ~rada ~ Su~mura T (1987) cDNA s~uence ~ a human t ~ n s ~ ~ ~ r ~ ~o ~ a ~ ~ f i c ~ t ~ f ~ t y ~ g ~ e ~ USA 8~ 298~2984
7 Zhan ~ Ba~s B, Hu X, G ~ d ~ b M 0988) ~ e hum~ FGF-5 gene encodes a novd pm~n ~ a ~ d to ~roblast growth factor~ Mol ~ H B~l ~ M ~ 5
8 Curato~ AM, Basfl~o C (199~ E x p l a i n of the K-f~ proto-onco~ne is co~ml~d by Y r e g ~ o ~ e~me~s wh~h ~e s ~ ~r emb~onal c~cinoma cel~ Mol ~ H B ~ l !~ 2475-24~
9 Schoodemm~ L ~ e r W (1~1) ~me~dependent ~g~m~n of ~e kFGF gene ~ e m ~ o n ~ c~inoma and emb~on~ s~m ceils. M e ~ Dev ~ ~ 6
10 Don ~ Co~ ~ W a ~ w r i ~ B, B~er K, M~6ck J (1991) '~ucMown' ~ R m c~umvem ~ o u s ~ d ~ ~ne am~cation. N u c ~ Acids Res 1~ 4 ~ 8
I1 Hyman S~ Comb M, Mn YS, ~ L G~en M ~ Goodman HM 0988) A common trans-ac~g ~ctor ~ ~v~ved ~ tr~scfi~on~ wgu|ation of neumtr~smitter ~nes ~ ~ d ~ AM~ Mol ~ H B ~ ~ ~ 2 5 ~ 3
12 Moo~ S, S~geant ~ ~ n g Z Matt~k J, Georges ~ He~el D (1991) ~ e c o n ~ n ~ d ~ u c ~ ~cro- s a ~ l ~ s among mamm~an genomes ~ w s ~e use ~ h ~ e ~ o u s PCR ~ m e r ~ ~ ~osdy ~ated s ~ e s . G e n o m ~ 1~ 6 5 ~ 6 0
