DNA and Cell Biology 1992.11:497-510. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/05/15. For personal use only.
DNA AND CELL BIOLOGY Volume 11, Number 7, 1992 Mary Ann Liebert, Inc., Publishers Pp. 497-510
Evolutionary Origins of the Transforming Growth Factor-ß Gene Family DAVID W. BURT and IAN R. PATON
ABSTRACT A molecular phylogeny for the transforming growth factor-/? (TGF-/3) gene family based on a comparison of nucleotide sequences is proposed. A phylogenetic tree constructed from these sequences shows that the family evolved from a common ancestral gene that came into existence at about the time of arthropod and chordate divergence. This model suggests that the present day TGF-/3 gene family consists of four members: TGF-/31 (= TGF-/34), TGF-/32, TGF-/33, and TGF-/35. The molecular phylogeny and Southern hybridization data also suggest that the proteins for mammalian TGF-01 and chicken TGF-/34 are the products of homologous rather than duplicated genes. If the gene duplication event that produced the ancestral gene for TGF-/31 occurred before the divergence of birds and mammals, then sufficient time would have elapsed to generate these quite distinct avian and mammalian TGF-/31 proteins. Therefore, the TGF-/3 family contains four distinct proteins, TGF-/31, 2, 3, and 5
INTRODUCTION
FACTOR-0 (TGF-/3) SUpercomposed of several closely related polypepan increasing number of more distantly related tides proteins. The TGF-/3 proteins are capable of influencing a wide range of differentiation processes (for a review, see Roberts and Sporn, 1990). Five distinct TGF-0 proteins (TGF-01 to TGF-/35) have been characterized from mammalian, avian, and amphibian sources (Roberts and Sporn, 1990). TGF-01 cDNAs have been isolated from a wide range of species, including human (Derynck et al, 1985), African green monkey (Sharpies et al, 1987), mouse (Derynck et al, 1986), pig (Derynck and Rhee, 1987), cow (Van Obberghen-Schilling et al, 1987), rat (Qian et al, 1990), and chicken (Jakowlew et al, 1988a). TGF-,32 cDNA clones have been isolated from chicken (Jakowlew et al, 1990), human (Madisen et al, 1988), African green monkey (Hanks et al, 1988), mouse (Miller et al, 1989a), and Xenopus laevis (Rebbert et al, 1990). TGF-/33 cDNAs have been cloned from chicken (Jakowlew et al, 1988b), human (Derynck et al, 1988), mouse (Miller et al, 1989b), and pig (Derynck et al, 1988). So far, a TGF-/34 cDNA has only been isolated and characterized from the chicken (Jakowlew et al, 1988c). The published TGF-/34 cDNA se-
THEfamily and
precursor protein lacking a typical hysequence. However, a recent analysis shows that this was the product of DNA sequencing errors and the native mRNA actually encodes a leader peptide (Burt and Jakowlew, 1992). The TGF-05 mRNA has only been characterized in a single species, X. laevis (Kondaiah et al, 1990). It remains to be determined whether TGF-/34 or TGF-05 exist in other species, and whether additional
predicts a drophobic signal quence
TRANSFORMING GROWTH
is
TGF-0 proteins also exist. The degree of amino acid identity between the various TGF-0 isoforms ranges from 28 to 45% in the pro region and from 64 to 82% in the mature peptide (Roberts and Sporn, 1990; Burt and Jakowlew, 1992). The individual forms of TGF-|3 are highly conserved throughout the precursor polypeptide; sequence identities are greater than 86% and 97% for TGF-/31, 82% and 95% for TGF-/32, and 84% and 99% for TGF-03, in the pro and mature regions, respectively. Conservation is also seen at the genomic level; the genes for TGF-/31, TGF-02, and TGF- /33 each have seven exons at homologous positions (Derynck et al, 1987, 1988; Burt and Paton, 1991). Chromosomal assignments of several members of the TGF-0 family (Barton et al, 1988; Dickinson et al, 1990; Tabas et al, 1991) indicate that these genes have become widely dispersed during their evolution. Therefore, it is likely that the entire
Department of Cellular and Molecular Biology, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, UK. 497
BURT AND PATÓN
DNA and Cell Biology 1992.11:497-510. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/05/15. For personal use only.
498
TGF-0 family evolved from a series of gene duplications, Estimation of evolutionary distances and these genes were separated by chromosome translocaFrom the alignment of protein sequences, the average tions. number of amino acid substitutions per site (D) between It is possible to deduce the phylogeny of gene families of sequences was estimated using the amino acid subbased on the proportion of amino acid differences between pairs stitution matrix of Dayhoff et al. (1978), as described by family members (Nei, 1987). However, the degree of Nei (1987). The rates of amino acid substitution were calamino acid divergence is frequently distorted by functional culated as D/2T, where T is the estimated time of species constraints, resulting in invalid conclusions as to gene andivergence. cestry. Phylogenetic relationships can also be deduced by To correct for possible multiple base substitution effects Within of nucleotide comparison sequences (Nei, 1987). in the nucleotide sequences the evolutionary distances per the coding region, most base substitutions at the 1st and site were estimated using the three-substitution-type (3ST) 2nd codon positions often lead to amino acid changes model of base substitutions (Kimura, 1981). evolutionary (nonsynonymous changes) and approximately parallel the The formulae were used to estimate the synonymous com-
differences observed in the amino acid sequence itself. However, a large fraction of base substitutions at the 3rd codon position are synonymous (i.e., do not lead to amino acid changes), and consequently very few functional constraints act at this position. Miyata et al. (1980) examined the rates of synonymous and nonsynonymous substitutions for various genes, and found that while the rate of nonsynonymous substitutions varied enormously, the rate of synonymous substitutions was more or less the same for all genes. This uniformity in the synonymous rate of base substitution has encouraged many investigators to use the number of synonymous substitutions as a measure of evolutionary distance in the calculation of molecular phytogenies of gene families (Nei, 1987). In this paper a phylogenetic analysis of the TGF-/3 family is presented based on the number of synonymous base substitutions found at the 3rd codon position, as determined from a comparison of all the available TGF-/3 cDNA sequences. The aim was to uncover evolutionary relationships within the TGF-/3 family and to estimate the times of gene duplication. The TGF-/3 gene phylogeny based on nucleotide sequences also provides a more reliable method of deciding whether genes isolated in one species are homologous or duplicated versions of those found in other species.
MATERIALS AND METHODS
Source of sequences and sequence
alignments
TGF-ßl: Human (Derynck et al, 1985), simian (Sharpies et al, 1987), bovine (Van Obberghen-Schilling et al, 1987), porcine (Derynck and Rhee, 1987), mouse (Derynck et al, 1986), rat (Qian et al, 1990), and chicken (Jakowlew et al, 1988a). TGF-ß2: Human (Madisen et al, 1988), simian (Hanks et al, 1988), mouse (Miller et al, 1989a), chicken (Jakowlew et al, 1990), and X. laevis (Rebbert et al., 1990). TGF-ß3: Human, porcine (Derynck et al, 1988), mouse (Miller et al, 1989b), and chicken (Jakowlew et al, 1988b). TGF-ß4: Chicken (Burt and Jakowlew, 1992). TGF-ß5: X. laevis (Kondaiah et al, 1990). Sequences were taken from the GenBank and EMBL nucleic acid data banks. Sequence alignments were performed using the University of Wisconsin Genetics Computer Group
(UWGCG)
program
(Devereux
et
al, 1984).
ponent of the number of base substitutions (#5) at the third position of codons and its standard error. The evolutionary rate, k'$, was calculated as K's/2T.
Estimated times
of species divergence
In the computation of rates of substitution, the followtimes of species divergence were used: apes and Old World monkeys, 20 million years (My); mouse and rat, 25 My; pig and cow, 55 My; diversification of eutherian mammals, 80 My; mammals, reptiles and birds, 300 My; reptiles and amphibians, 350 My; reptiles and fish, 400 My; arthropods and chordates, 700 My (Dayhoff et al,
ing
1978; Nei, 1987).
Calculation of molecular phytogenies
Sequence phylogenies were calculated using the evolutio-
nary distances deduced from either protein or DNA sequence alignments as input to KITSCH, a program within the PHYLIP33 package (Felsenstein, 1990). In this program, the branches of the phylogenetic tree are constrained so that the total length from the root of the tree to any sequence is the
same. Identical results were obtained from 20 independent trials, using the J (jumble) option to vary the order of sequences entered as input to the program.
Preparation of human TGF-ßl and chicken TGF-ß4 hybridization probes Short, homologous cDNA fragments coding for either human TGF-/31 or chicken TGF-/34 were used as hybridization probes. The TGF-/31 probe was a 243-bp Pvu II DNA fragment isolated from a human cDNA clone (position 1,736-1,978; Derynck et al, 1985). An homologous chicken TGF-04 cDNA fragment (position 872-1,114, Jakowlew et al, 1988c) was prepared from a cDNA clone using the polymerase chain reaction (PCR). The PCR reaction was performed on a Biometra TRIO-Thermoblock using Taq DNA polymerase (Boehringer Mannheim) according to the manufacturer's instructions. The denaturing, annealing, and extending conditions were 1 min at 94°C; 2 min at 37°C; and 3 min at 72°C, for 25 cycles. The TGF-04-specific primers used for PCR were: primer 1 [position 872-886] CTCTACATCGACTTC and primer 2 (po-
499
TGF-/3 GENE EVOLUTION
DNA and Cell Biology 1992.11:497-510. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/05/15. For personal use only.
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100
Xe TGF- ß5 LSTCKAVDME EVRKRRIEAI RGQILSKLKL DKTP.DVDSE KMTVPSEAIF LYNSTLEVIR EKATREEEHV Ck TGF- ß4 LSTCQRLDLE AAKKKRIEAV RGQILSKLRL TAPPPASETP PRPLPDDVRA LYNSTQELLK QRARLR^PP. Ck TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGDVP PGPLPEAVLA LYNSTRDRVA GES.VEPEP. Po TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGDVP PGPLPEAVLA LYNSTRDRVA GES.VEPEP. Bo TGF- ßl AILA LYNSTRDRVA GES.AETEP. Hu TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.AEPEP. Si TGF- ßl LSTCKTIDME LVKRKRIETI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.AEPEP. Mu TCF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.ADPEP. Ra TCF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.ADPEP. Xe TGF- ß2 LSTCSALDMD QFMRKRIEAI RGQILSKLKL NSPP.EDYPE PGEVSQDVIS IYNSTRDLLQ EKANERATSC Ck TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.DEYPE PEEVPPEVIS IYNSTRDLLQ EKANHRAATC Mu TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PDEVPPEVIS IYNSTRDLLQ EKASRRAAAC Hu TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PEEVPPEVIS IYNSTRDLLQ EKASRRAAAC Si TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PEEVPPEVIS IYNSTRDLLQ EKASRRAAAC Ck TGF- ß3 LSSCTTLDLE HIKKKRVEAI RGQILSKLRL TSPP.ES.VG PAHVPYQILA LYNSTRELLE EMEEEKEESC Po TGF- ß3 MSTCTTLDFD HIKRKRVEAI RGQILSKLRL TSPP.DP.SM LANIPTQVLD LYNSTRELLE EVHGERGDDC Hu TGF- ß3 LSTCTTLDFG HIKKKRVEAI RGQILSKLRL TSPP.EP.TV MTHVPYQVLA LYNSTRELLE EMHGEREEGC Mu TGF- ß3 LSTCTTLDFG HIKKKRVEAI RGQILSKLRL TSPP.EP.SV MTHVPYQVLA LYNSTRELLE EMHGEREEGC Xe TGF- ß5
Ck TGFCk TGFPo TGFBo TGFHu TGFSi TGFMu TGFRa TGFXe TGFCk TGFMu TGFHu TGFSi TGFCk TGFPo TGF-
....HEFKFK FNASHVRENV GMNSLLHHAE
ß4 QPQSHSIFFV FNVSRAR.RG ßl KGTPHSLYML FNTSELREAV
ßl KGTPHSLYML FNTSELREAV ßl KSSSHSIYMF FNTSELREAV ßl ßl ßl ßl ß2 ß2 ß2
ß2 ß2 ß3
ß3
Hu TGF- ß3 Mu TGF- ß3
KQSTHSIYMF FNTSELREAV KQSTHSIYMF FNTSELREAV KDISHSIYMF FNTSDIREAV KDITHSIYMF FNTSDIREAV
LRMYKKQTDK NMDQRMELFW KYQ.EN GRPTLLHRAE LRMLRQKAAA DSAGTEQRLE LYQ.GY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAD VRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAE LRLLRRLKLK EQHVE LYQ.KY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPPLLSRAE LRL.QRLKSS EQHVE LYQ.KY PEPPLLSRAE LRL.QRFKST EQHVE LYQ.KY KNASNLVKAE FRVFR.LMNT KARVSEQRIE LYQILKSKDL
PPSYTTPYFR IVRFDVSSME PPSYYSLYFR IVRFDVSAME KNASNLVKAE PPTFYRPYFR IVRFDVSTME KNASNLVKAE PPTFYRPYFR IVRFDVSAME KNASNLVKAE PPTFYRPYFR IVRFDVSAME KNASNLVKAE GICPKGVTSN VFRFNVSSAE KNSTNLFRAE AVCPKGITSK IFRFNVSSVE KNETNLFRAE AVCPKGITSK VFRFNVSSVE KNRTNLFRAE AVCPKGITSK VFRFNVSSVE KNGTNLFRAE
FRVFR.LQNS KARVSEQRIE FRVFR.LQNP KARVAEQRIE
FRVFR.LQNP KARVPEQRIE FRVFR.LQNP KARVPEQRIE FRVLR.VPNP SSKRSEQRIE FRVLR.MPNP SSKRSEQRIE FRVLR.VPNP SSKRNEQRIE
FRVLR.VPNP SSKRTEQRIE
ID.IE.GF Xe TGF-ßS EQFGLQPACK CPTPQAKD.. Ck TGF-ß4 GVFKLSVHCP CEMGPGHAEE MRIS.IE.GF Ck TGF-ßl EGFRLSAHCS SKDNT LHVE.INAGF SKDNT LHVE.IN.GF Po TGF-ßl EGFRLSAHCS SKDNT LQVD.IN.GF Bo TGF-ßl EGFRLSAHCS SRDNT LQVD.IN.GF Hu TGF-ßl EGFRLSAHCS Si TGF-ßl EGFRLSAHCS SKDNT LQVD.IN.GF Mu TGF-ßl QGFRFSAHCS SKDNK LHVE.IN.GI Ra TGF-ßl QGFRFSAHCS SKDNV LHVE.IN.GI Xe TGF-ß2 LGFKISLHCP CCTFIPSNNY IIPNKSEELE TRFAGID.DA Ck TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEEPE ARFAGID.DY Mu TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Hu TGF-62 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Si TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Ck TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENLHEVLE IKFKGID.SE Po TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENIQEVME IKFKGVD.SE Hu TGF-R3 LGLEISIHCP CHTFQPNGD. ILENIHEVME IKFKGVD.NE Mu TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENVHEVME IKFKGVD.NE .
Xe TGF-ß5 GPNCCVKPLY Ck TGF-ß4 EKNCCVRPLY Ck TGF-ßl EKNCCVRQLY Po TGF-ßl EKNCCVRQLY Bo TGF-ßl EKNCCVRQLY Hu TGF-ßl EKNCCVRQLY
Si TGF-ßl EKNCCVRQLY Mu TGF-ßl EKNCCVRQLY Ra TGF-ßl EKNCCVRQLY Xe TGF-ß2 QDNCCLRPLY Ck TGF-ß2 QDNCCLRPLY Mu TGF-A2 Hu TGF-ß2
QDNCCLRPLY QDNCCLRPLY Si TGF-ß2 QDNCCLRPLY Ck Po Hu Mu
.
INFRKDLGWK WIHEPKGYEA IDFRKDLQWK WIHEPKGYMA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFKKDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYHA IDFKRDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYNA
TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYFA
TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA
NSGRRGDLAT SSGRRGDLAT
TTGRRGDLAT TTGRRGDLAT SPKRRGDLGT SPKRRGDLGT YMYACKDSKS
TYSSGDVKA
.DGPDEYW .EPEADYY .EPEADYY .EPEADYY
AKELRRIPME TTWDGPMEHW AKEVTRVLMV ESGNQIYDKF AKEVTRVLML ESGNQIYDKF
SQENTESEYY TQENTESEYY TQENTESEYY TQETSESEYY
AKEIHKFDMI Q.GLPEHNEL AKEIYKFDMI Q.GLEEHNDL
.
.
AKEVTRVLMV EYGNKIYDKM .EPEADYY AKEVTRVLMV ETHNEIYDKF .EPEADYY AKEVTRVLMV ETHNEIYDKF .EPEADYY AKEVTRVLMV DRNNAIYEKT .EPEADYY AKEVTRVLMV DRNNAIYDKT ERERSEDEYY AKEVYKIDML P.YYTSENVI ERERSDEEYY AKEVYKIDMQ P.FYP.ENAI ERERSEQEYY AKEVYKIDMP S.HLPSENAI ERERSDEEYY AKEVYKIDMP P.FFPSENAI ERERSDEEYY AKEVYKIDMP P.FFPSENAI
GTTHSRYLES KYITPVTDDE GNASWRYLHG RSVRATADDE SNDSWGYLSN RLLAPSDSPE SNDSWRYLSN RLLAPSDSPE SNNSWRYLSN SNNSWRYLSN SNNSWRYLSN SNNSWRYLGN
ESITELED..
AKEIHKFDMI Q.GLAEHNEL AKEIHKFDMI Q.GLAEHNEL 200 WMSFDVTKTV NEWLKRAEEN WLSFDVTDAV HQWLSGSELL
WLSFDVTGVV RQWLTRREAI WLSFDVTGVV RQWLTRREAI RQWLTRREEI RQWLSRCKEI RLLAPSNSPE WLSFDVTGVV RQWLSRCKEI RLLAPSDSPE WLSFDVTGVV RLLAPSDSPE WLSFDVTGVV
RLLTPTDTPE WLSFDVTGVV SNNSWRYLGN RLLTPTDTPE WLSFDVTGVV ASPTQRYIDS KVVKTRAEGE WLSFDVTEAV LYQVLKSKEL SSPGQRYIDS KVVKTRAEGE WLSFDVTEAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LFQILRPDEH IAK.QRYLSG RNVQTRGSPE WLSFDVTDTV LFQILQPDEH IAK.QRYIDG KNLPTRGAAE WLSFDVTDTV LFQILRPDEH IAK.QRYICK KNLPTRGTAE WLSFDVTDTV LFQILRPDEH IAK.QRYICK KNLPTRGTAE WLSFDVTDTV
i
.PYLMITSM .PYVLAMAL IHGMNR .PFLLLMAT .PFLLLMAT IHGMNR .PFLLLMAT IHGMNR IHGMNR .PFLLLMAT .PFLLLMAT IHGMNR .PFLLLMAT IHDMNR IHDMNR .PFLLLMAT -KTGRKKHTG RTPHLLLMLL LKSNRKKYSG KTPHLLLMLL
.PALRGDLAS LSSKENTK E.QQRGDMQS IAKKHRRV
NSGRRGDLAT
GHDQNIQDYY AKQVYRF.
RQWLNQGDGI
RQWLNQGDGI NEWLHHKDRN HEWLHHRDRN
QEWLHHKDRN HEWLHHKDRN HEWLHHKDRN
REWLLHRESN REWLLRRESN REWLLRRESN REWLLRRESN
300
PAERIDT. .V TSSRKKRGVG QEYCFGN..N PAERANE. L HSARRRRDLD TDYCFGPGTD PLERAQH. L HSSRHRRALD TNYCFSS..T PLERAQH..L HSSRHRRALD TNYCFSS. .T PLERAQH..L HSSRHRRALD TNYCFSS..T PLERAQH..L QSSRHRRALD TNYCFSS..T PLERAQH..L QSSRHRRALD TNYCFSS..T .
.
PLERAQH..L HSSRHRRALD TNYCFSS.,T PLERAQH..L HSSRHRRALD TNYCFSS..T PSYRLESQ.Q SSRRKKRALD AAYCFRN..V
PSYRLESQ.Q STYASGDQKT IKSTRKKTSG KTPHLLLMLL PSYRLESQ.Q STYTSGDQKT IKSTRKKNSG KTPHLLLMLL PSYRLESQ.Q STYTSGDQKT IKSTRKKNSG KTPHLLLMLL PSYRLESQ.Q DDYGRGDLGR LK...KQKDL HNPHLILMML PPHRLESPTL .
PSRRKKRALD AAYCFRN.,V SSRRKKRALD AAYCFRN..V
TNRRKKRALD AAYCFRN..V TNRRKKRALD AAYCFRN. .V CKQRKKRALD TNYCFPN..L
.KKKEH .SPHLILMMI PPDRLDNPGL GAQRKKRALD TNYCFRN..L LK...KQKDH HNPHLILMMI PPHRLDNPGQ CKQRKKRALD TNYCFRN..L DDHGRGDLGR LK...KQKDH HNPHLILMMI PPHRLDSPGQ GSQRKKRALD TNYCFRN..L
DDPGRGDLGR LK DDHGRGDLGR
.
.
401
NYCLGNCPYI WSMDTQYSKV LSLYNQNNPG ASISPCCVPD VLEPLPIIYY VGRTAKVEQL NFCMGPCPYI WSADTQYTKV LALYNQHNPG ASAAPCCVPQ TLDPLPIIYY VGRNVRVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASASPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASASPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLDSLTILYY IGNKPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCAGACPYL WSSDTQHTKV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGNTPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL NFCSGPCPYL RSADTTHSSV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTAKVEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL
SNMVVRSCNCS SNMVVRACKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS
SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVKSCKCS SNMIVKSCKCS
SNMIVKSCKCS SNMIVKSCKCS SNMIVKSCKCS SNMVVKSCKCS
SNMVVKSCKCS SNMVVKSCKCS SNMVVKSCKCS
FIG. 1. Sequence alignment of TGF-/3 amino acid sequences. See Materials and Methods for the source of sequences and the method of alignment. The symbols used for the species are: Xe, X. laevis; Ck, chicken; Po, porcine; Bo, bovine; Hu, human; Si, African green monkey; Mu, murine; Ra, rat. Sites of proteolytic cleavage are shown with a vertical arrow. Gaps were introduced to maximize sequence alignment and are shown with a period (•). All sequences are numbered relative to the first amino acid in the pro region, immediately after the signal cleavage site.
sition 1,100-1,114] CTGCTCCACGCGCAC (Jakowlew et were used at 100 pmoles each, with 1 ng of plasmid DNA in a total volume of 100 /tl. The expected 243-bp product was isolated and verified by digestion with Sma I, which produced the expected 152-bp and
ai, 1988c). Primers
91-bp
DNA
fragments.
Genomic Southern analysis A nylon membrane containing Eco RI-digested genomic DNAs from a range of eukaryotic species (human, monkey, rat, mouse, canine, bovine, rabbit, chicken, and yeast) was obtained from Clontech (ZOO-BLOT, Palo
BURT AND PATÓN
DNA and Cell Biology 1992.11:497-510. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/05/15. For personal use only.
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DNA AND CELL BIOLOGY Volume 11, Number 7, 1992 Mary Ann Liebert, Inc., Publishers Pp. 497-510
Evolutionary Origins of the Transforming Growth Factor-ß Gene Family DAVID W. BURT and IAN R. PATON
ABSTRACT A molecular phylogeny for the transforming growth factor-/? (TGF-/3) gene family based on a comparison of nucleotide sequences is proposed. A phylogenetic tree constructed from these sequences shows that the family evolved from a common ancestral gene that came into existence at about the time of arthropod and chordate divergence. This model suggests that the present day TGF-/3 gene family consists of four members: TGF-/31 (= TGF-/34), TGF-/32, TGF-/33, and TGF-/35. The molecular phylogeny and Southern hybridization data also suggest that the proteins for mammalian TGF-01 and chicken TGF-/34 are the products of homologous rather than duplicated genes. If the gene duplication event that produced the ancestral gene for TGF-/31 occurred before the divergence of birds and mammals, then sufficient time would have elapsed to generate these quite distinct avian and mammalian TGF-/31 proteins. Therefore, the TGF-/3 family contains four distinct proteins, TGF-/31, 2, 3, and 5
INTRODUCTION
FACTOR-0 (TGF-/3) SUpercomposed of several closely related polypepan increasing number of more distantly related tides proteins. The TGF-/3 proteins are capable of influencing a wide range of differentiation processes (for a review, see Roberts and Sporn, 1990). Five distinct TGF-0 proteins (TGF-01 to TGF-/35) have been characterized from mammalian, avian, and amphibian sources (Roberts and Sporn, 1990). TGF-01 cDNAs have been isolated from a wide range of species, including human (Derynck et al, 1985), African green monkey (Sharpies et al, 1987), mouse (Derynck et al, 1986), pig (Derynck and Rhee, 1987), cow (Van Obberghen-Schilling et al, 1987), rat (Qian et al, 1990), and chicken (Jakowlew et al, 1988a). TGF-,32 cDNA clones have been isolated from chicken (Jakowlew et al, 1990), human (Madisen et al, 1988), African green monkey (Hanks et al, 1988), mouse (Miller et al, 1989a), and Xenopus laevis (Rebbert et al, 1990). TGF-/33 cDNAs have been cloned from chicken (Jakowlew et al, 1988b), human (Derynck et al, 1988), mouse (Miller et al, 1989b), and pig (Derynck et al, 1988). So far, a TGF-/34 cDNA has only been isolated and characterized from the chicken (Jakowlew et al, 1988c). The published TGF-/34 cDNA se-
THEfamily and
precursor protein lacking a typical hysequence. However, a recent analysis shows that this was the product of DNA sequencing errors and the native mRNA actually encodes a leader peptide (Burt and Jakowlew, 1992). The TGF-05 mRNA has only been characterized in a single species, X. laevis (Kondaiah et al, 1990). It remains to be determined whether TGF-/34 or TGF-05 exist in other species, and whether additional
predicts a drophobic signal quence
TRANSFORMING GROWTH
is
TGF-0 proteins also exist. The degree of amino acid identity between the various TGF-0 isoforms ranges from 28 to 45% in the pro region and from 64 to 82% in the mature peptide (Roberts and Sporn, 1990; Burt and Jakowlew, 1992). The individual forms of TGF-|3 are highly conserved throughout the precursor polypeptide; sequence identities are greater than 86% and 97% for TGF-/31, 82% and 95% for TGF-/32, and 84% and 99% for TGF-03, in the pro and mature regions, respectively. Conservation is also seen at the genomic level; the genes for TGF-/31, TGF-02, and TGF- /33 each have seven exons at homologous positions (Derynck et al, 1987, 1988; Burt and Paton, 1991). Chromosomal assignments of several members of the TGF-0 family (Barton et al, 1988; Dickinson et al, 1990; Tabas et al, 1991) indicate that these genes have become widely dispersed during their evolution. Therefore, it is likely that the entire
Department of Cellular and Molecular Biology, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, UK. 497
BURT AND PATÓN
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498
TGF-0 family evolved from a series of gene duplications, Estimation of evolutionary distances and these genes were separated by chromosome translocaFrom the alignment of protein sequences, the average tions. number of amino acid substitutions per site (D) between It is possible to deduce the phylogeny of gene families of sequences was estimated using the amino acid subbased on the proportion of amino acid differences between pairs stitution matrix of Dayhoff et al. (1978), as described by family members (Nei, 1987). However, the degree of Nei (1987). The rates of amino acid substitution were calamino acid divergence is frequently distorted by functional culated as D/2T, where T is the estimated time of species constraints, resulting in invalid conclusions as to gene andivergence. cestry. Phylogenetic relationships can also be deduced by To correct for possible multiple base substitution effects Within of nucleotide comparison sequences (Nei, 1987). in the nucleotide sequences the evolutionary distances per the coding region, most base substitutions at the 1st and site were estimated using the three-substitution-type (3ST) 2nd codon positions often lead to amino acid changes model of base substitutions (Kimura, 1981). evolutionary (nonsynonymous changes) and approximately parallel the The formulae were used to estimate the synonymous com-
differences observed in the amino acid sequence itself. However, a large fraction of base substitutions at the 3rd codon position are synonymous (i.e., do not lead to amino acid changes), and consequently very few functional constraints act at this position. Miyata et al. (1980) examined the rates of synonymous and nonsynonymous substitutions for various genes, and found that while the rate of nonsynonymous substitutions varied enormously, the rate of synonymous substitutions was more or less the same for all genes. This uniformity in the synonymous rate of base substitution has encouraged many investigators to use the number of synonymous substitutions as a measure of evolutionary distance in the calculation of molecular phytogenies of gene families (Nei, 1987). In this paper a phylogenetic analysis of the TGF-/3 family is presented based on the number of synonymous base substitutions found at the 3rd codon position, as determined from a comparison of all the available TGF-/3 cDNA sequences. The aim was to uncover evolutionary relationships within the TGF-/3 family and to estimate the times of gene duplication. The TGF-/3 gene phylogeny based on nucleotide sequences also provides a more reliable method of deciding whether genes isolated in one species are homologous or duplicated versions of those found in other species.
MATERIALS AND METHODS
Source of sequences and sequence
alignments
TGF-ßl: Human (Derynck et al, 1985), simian (Sharpies et al, 1987), bovine (Van Obberghen-Schilling et al, 1987), porcine (Derynck and Rhee, 1987), mouse (Derynck et al, 1986), rat (Qian et al, 1990), and chicken (Jakowlew et al, 1988a). TGF-ß2: Human (Madisen et al, 1988), simian (Hanks et al, 1988), mouse (Miller et al, 1989a), chicken (Jakowlew et al, 1990), and X. laevis (Rebbert et al., 1990). TGF-ß3: Human, porcine (Derynck et al, 1988), mouse (Miller et al, 1989b), and chicken (Jakowlew et al, 1988b). TGF-ß4: Chicken (Burt and Jakowlew, 1992). TGF-ß5: X. laevis (Kondaiah et al, 1990). Sequences were taken from the GenBank and EMBL nucleic acid data banks. Sequence alignments were performed using the University of Wisconsin Genetics Computer Group
(UWGCG)
program
(Devereux
et
al, 1984).
ponent of the number of base substitutions (#5) at the third position of codons and its standard error. The evolutionary rate, k'$, was calculated as K's/2T.
Estimated times
of species divergence
In the computation of rates of substitution, the followtimes of species divergence were used: apes and Old World monkeys, 20 million years (My); mouse and rat, 25 My; pig and cow, 55 My; diversification of eutherian mammals, 80 My; mammals, reptiles and birds, 300 My; reptiles and amphibians, 350 My; reptiles and fish, 400 My; arthropods and chordates, 700 My (Dayhoff et al,
ing
1978; Nei, 1987).
Calculation of molecular phytogenies
Sequence phylogenies were calculated using the evolutio-
nary distances deduced from either protein or DNA sequence alignments as input to KITSCH, a program within the PHYLIP33 package (Felsenstein, 1990). In this program, the branches of the phylogenetic tree are constrained so that the total length from the root of the tree to any sequence is the
same. Identical results were obtained from 20 independent trials, using the J (jumble) option to vary the order of sequences entered as input to the program.
Preparation of human TGF-ßl and chicken TGF-ß4 hybridization probes Short, homologous cDNA fragments coding for either human TGF-/31 or chicken TGF-/34 were used as hybridization probes. The TGF-/31 probe was a 243-bp Pvu II DNA fragment isolated from a human cDNA clone (position 1,736-1,978; Derynck et al, 1985). An homologous chicken TGF-04 cDNA fragment (position 872-1,114, Jakowlew et al, 1988c) was prepared from a cDNA clone using the polymerase chain reaction (PCR). The PCR reaction was performed on a Biometra TRIO-Thermoblock using Taq DNA polymerase (Boehringer Mannheim) according to the manufacturer's instructions. The denaturing, annealing, and extending conditions were 1 min at 94°C; 2 min at 37°C; and 3 min at 72°C, for 25 cycles. The TGF-04-specific primers used for PCR were: primer 1 [position 872-886] CTCTACATCGACTTC and primer 2 (po-
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TGF-/3 GENE EVOLUTION
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Xe TGF- ß5 LSTCKAVDME EVRKRRIEAI RGQILSKLKL DKTP.DVDSE KMTVPSEAIF LYNSTLEVIR EKATREEEHV Ck TGF- ß4 LSTCQRLDLE AAKKKRIEAV RGQILSKLRL TAPPPASETP PRPLPDDVRA LYNSTQELLK QRARLR^PP. Ck TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGDVP PGPLPEAVLA LYNSTRDRVA GES.VEPEP. Po TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGDVP PGPLPEAVLA LYNSTRDRVA GES.VEPEP. Bo TGF- ßl AILA LYNSTRDRVA GES.AETEP. Hu TGF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.AEPEP. Si TGF- ßl LSTCKTIDME LVKRKRIETI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.AEPEP. Mu TCF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.ADPEP. Ra TCF- ßl LSTCKTIDME LVKRKRIEAI RGQILSKLRL ASPPSQGEVP PGPLPEAVLA LYNSTRDRVA GES.ADPEP. Xe TGF- ß2 LSTCSALDMD QFMRKRIEAI RGQILSKLKL NSPP.EDYPE PGEVSQDVIS IYNSTRDLLQ EKANERATSC Ck TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.DEYPE PEEVPPEVIS IYNSTRDLLQ EKANHRAATC Mu TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PDEVPPEVIS IYNSTRDLLQ EKASRRAAAC Hu TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PEEVPPEVIS IYNSTRDLLQ EKASRRAAAC Si TGF- ß2 LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPP.EDYPE PEEVPPEVIS IYNSTRDLLQ EKASRRAAAC Ck TGF- ß3 LSSCTTLDLE HIKKKRVEAI RGQILSKLRL TSPP.ES.VG PAHVPYQILA LYNSTRELLE EMEEEKEESC Po TGF- ß3 MSTCTTLDFD HIKRKRVEAI RGQILSKLRL TSPP.DP.SM LANIPTQVLD LYNSTRELLE EVHGERGDDC Hu TGF- ß3 LSTCTTLDFG HIKKKRVEAI RGQILSKLRL TSPP.EP.TV MTHVPYQVLA LYNSTRELLE EMHGEREEGC Mu TGF- ß3 LSTCTTLDFG HIKKKRVEAI RGQILSKLRL TSPP.EP.SV MTHVPYQVLA LYNSTRELLE EMHGEREEGC Xe TGF- ß5
Ck TGFCk TGFPo TGFBo TGFHu TGFSi TGFMu TGFRa TGFXe TGFCk TGFMu TGFHu TGFSi TGFCk TGFPo TGF-
....HEFKFK FNASHVRENV GMNSLLHHAE
ß4 QPQSHSIFFV FNVSRAR.RG ßl KGTPHSLYML FNTSELREAV
ßl KGTPHSLYML FNTSELREAV ßl KSSSHSIYMF FNTSELREAV ßl ßl ßl ßl ß2 ß2 ß2
ß2 ß2 ß3
ß3
Hu TGF- ß3 Mu TGF- ß3
KQSTHSIYMF FNTSELREAV KQSTHSIYMF FNTSELREAV KDISHSIYMF FNTSDIREAV KDITHSIYMF FNTSDIREAV
LRMYKKQTDK NMDQRMELFW KYQ.EN GRPTLLHRAE LRMLRQKAAA DSAGTEQRLE LYQ.GY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAD VRLLR.LKLK EQHVE LYQ.KY PEPVLLSRAE LRLLRRLKLK EQHVE LYQ.KY PEPVLLSRAE LRLLR.LKLK EQHVE LYQ.KY PEPPLLSRAE LRL.QRLKSS EQHVE LYQ.KY PEPPLLSRAE LRL.QRFKST EQHVE LYQ.KY KNASNLVKAE FRVFR.LMNT KARVSEQRIE LYQILKSKDL
PPSYTTPYFR IVRFDVSSME PPSYYSLYFR IVRFDVSAME KNASNLVKAE PPTFYRPYFR IVRFDVSTME KNASNLVKAE PPTFYRPYFR IVRFDVSAME KNASNLVKAE PPTFYRPYFR IVRFDVSAME KNASNLVKAE GICPKGVTSN VFRFNVSSAE KNSTNLFRAE AVCPKGITSK IFRFNVSSVE KNETNLFRAE AVCPKGITSK VFRFNVSSVE KNRTNLFRAE AVCPKGITSK VFRFNVSSVE KNGTNLFRAE
FRVFR.LQNS KARVSEQRIE FRVFR.LQNP KARVAEQRIE
FRVFR.LQNP KARVPEQRIE FRVFR.LQNP KARVPEQRIE FRVLR.VPNP SSKRSEQRIE FRVLR.MPNP SSKRSEQRIE FRVLR.VPNP SSKRNEQRIE
FRVLR.VPNP SSKRTEQRIE
ID.IE.GF Xe TGF-ßS EQFGLQPACK CPTPQAKD.. Ck TGF-ß4 GVFKLSVHCP CEMGPGHAEE MRIS.IE.GF Ck TGF-ßl EGFRLSAHCS SKDNT LHVE.INAGF SKDNT LHVE.IN.GF Po TGF-ßl EGFRLSAHCS SKDNT LQVD.IN.GF Bo TGF-ßl EGFRLSAHCS SRDNT LQVD.IN.GF Hu TGF-ßl EGFRLSAHCS Si TGF-ßl EGFRLSAHCS SKDNT LQVD.IN.GF Mu TGF-ßl QGFRFSAHCS SKDNK LHVE.IN.GI Ra TGF-ßl QGFRFSAHCS SKDNV LHVE.IN.GI Xe TGF-ß2 LGFKISLHCP CCTFIPSNNY IIPNKSEELE TRFAGID.DA Ck TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEEPE ARFAGID.DY Mu TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Hu TGF-62 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Si TGF-ß2 LGFKISLHCP CCTFVPSNNY IIPNKSEELE ARFAGID.GT Ck TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENLHEVLE IKFKGID.SE Po TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENIQEVME IKFKGVD.SE Hu TGF-R3 LGLEISIHCP CHTFQPNGD. ILENIHEVME IKFKGVD.NE Mu TGF-ß3 LGLEISIHCP CHTFQPNGD. ILENVHEVME IKFKGVD.NE .
Xe TGF-ß5 GPNCCVKPLY Ck TGF-ß4 EKNCCVRPLY Ck TGF-ßl EKNCCVRQLY Po TGF-ßl EKNCCVRQLY Bo TGF-ßl EKNCCVRQLY Hu TGF-ßl EKNCCVRQLY
Si TGF-ßl EKNCCVRQLY Mu TGF-ßl EKNCCVRQLY Ra TGF-ßl EKNCCVRQLY Xe TGF-ß2 QDNCCLRPLY Ck TGF-ß2 QDNCCLRPLY Mu TGF-A2 Hu TGF-ß2
QDNCCLRPLY QDNCCLRPLY Si TGF-ß2 QDNCCLRPLY Ck Po Hu Mu
.
INFRKDLGWK WIHEPKGYEA IDFRKDLQWK WIHEPKGYMA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFRKDLGWK WIHEPKGYHA IDFKKDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYHA IDFKRDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYNA IDFKRDLGWK WIHEPKGYNA
TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYFA
TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA TGF-ß3 EENCCVRPLY IDFRQDLGWK WVHEPKGYYA
NSGRRGDLAT SSGRRGDLAT
TTGRRGDLAT TTGRRGDLAT SPKRRGDLGT SPKRRGDLGT YMYACKDSKS
TYSSGDVKA
.DGPDEYW .EPEADYY .EPEADYY .EPEADYY
AKELRRIPME TTWDGPMEHW AKEVTRVLMV ESGNQIYDKF AKEVTRVLML ESGNQIYDKF
SQENTESEYY TQENTESEYY TQENTESEYY TQETSESEYY
AKEIHKFDMI Q.GLPEHNEL AKEIYKFDMI Q.GLEEHNDL
.
.
AKEVTRVLMV EYGNKIYDKM .EPEADYY AKEVTRVLMV ETHNEIYDKF .EPEADYY AKEVTRVLMV ETHNEIYDKF .EPEADYY AKEVTRVLMV DRNNAIYEKT .EPEADYY AKEVTRVLMV DRNNAIYDKT ERERSEDEYY AKEVYKIDML P.YYTSENVI ERERSDEEYY AKEVYKIDMQ P.FYP.ENAI ERERSEQEYY AKEVYKIDMP S.HLPSENAI ERERSDEEYY AKEVYKIDMP P.FFPSENAI ERERSDEEYY AKEVYKIDMP P.FFPSENAI
GTTHSRYLES KYITPVTDDE GNASWRYLHG RSVRATADDE SNDSWGYLSN RLLAPSDSPE SNDSWRYLSN RLLAPSDSPE SNNSWRYLSN SNNSWRYLSN SNNSWRYLSN SNNSWRYLGN
ESITELED..
AKEIHKFDMI Q.GLAEHNEL AKEIHKFDMI Q.GLAEHNEL 200 WMSFDVTKTV NEWLKRAEEN WLSFDVTDAV HQWLSGSELL
WLSFDVTGVV RQWLTRREAI WLSFDVTGVV RQWLTRREAI RQWLTRREEI RQWLSRCKEI RLLAPSNSPE WLSFDVTGVV RQWLSRCKEI RLLAPSDSPE WLSFDVTGVV RLLAPSDSPE WLSFDVTGVV
RLLTPTDTPE WLSFDVTGVV SNNSWRYLGN RLLTPTDTPE WLSFDVTGVV ASPTQRYIDS KVVKTRAEGE WLSFDVTEAV LYQVLKSKEL SSPGQRYIDS KVVKTRAEGE WLSFDVTEAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LYQILKSKDL TSPTQRYIDS KVVKTRAEGE WLSFDVTDAV LFQILRPDEH IAK.QRYLSG RNVQTRGSPE WLSFDVTDTV LFQILQPDEH IAK.QRYIDG KNLPTRGAAE WLSFDVTDTV LFQILRPDEH IAK.QRYICK KNLPTRGTAE WLSFDVTDTV LFQILRPDEH IAK.QRYICK KNLPTRGTAE WLSFDVTDTV
i
.PYLMITSM .PYVLAMAL IHGMNR .PFLLLMAT .PFLLLMAT IHGMNR .PFLLLMAT IHGMNR IHGMNR .PFLLLMAT .PFLLLMAT IHGMNR .PFLLLMAT IHDMNR IHDMNR .PFLLLMAT -KTGRKKHTG RTPHLLLMLL LKSNRKKYSG KTPHLLLMLL
.PALRGDLAS LSSKENTK E.QQRGDMQS IAKKHRRV
NSGRRGDLAT
GHDQNIQDYY AKQVYRF.
RQWLNQGDGI
RQWLNQGDGI NEWLHHKDRN HEWLHHRDRN
QEWLHHKDRN HEWLHHKDRN HEWLHHKDRN
REWLLHRESN REWLLRRESN REWLLRRESN REWLLRRESN
300
PAERIDT. .V TSSRKKRGVG QEYCFGN..N PAERANE. L HSARRRRDLD TDYCFGPGTD PLERAQH. L HSSRHRRALD TNYCFSS..T PLERAQH..L HSSRHRRALD TNYCFSS. .T PLERAQH..L HSSRHRRALD TNYCFSS..T PLERAQH..L QSSRHRRALD TNYCFSS..T PLERAQH..L QSSRHRRALD TNYCFSS..T .
.
PLERAQH..L HSSRHRRALD TNYCFSS.,T PLERAQH..L HSSRHRRALD TNYCFSS..T PSYRLESQ.Q SSRRKKRALD AAYCFRN..V
PSYRLESQ.Q STYASGDQKT IKSTRKKTSG KTPHLLLMLL PSYRLESQ.Q STYTSGDQKT IKSTRKKNSG KTPHLLLMLL PSYRLESQ.Q STYTSGDQKT IKSTRKKNSG KTPHLLLMLL PSYRLESQ.Q DDYGRGDLGR LK...KQKDL HNPHLILMML PPHRLESPTL .
PSRRKKRALD AAYCFRN.,V SSRRKKRALD AAYCFRN..V
TNRRKKRALD AAYCFRN..V TNRRKKRALD AAYCFRN. .V CKQRKKRALD TNYCFPN..L
.KKKEH .SPHLILMMI PPDRLDNPGL GAQRKKRALD TNYCFRN..L LK...KQKDH HNPHLILMMI PPHRLDNPGQ CKQRKKRALD TNYCFRN..L DDHGRGDLGR LK...KQKDH HNPHLILMMI PPHRLDSPGQ GSQRKKRALD TNYCFRN..L
DDPGRGDLGR LK DDHGRGDLGR
.
.
401
NYCLGNCPYI WSMDTQYSKV LSLYNQNNPG ASISPCCVPD VLEPLPIIYY VGRTAKVEQL NFCMGPCPYI WSADTQYTKV LALYNQHNPG ASAAPCCVPQ TLDPLPIIYY VGRNVRVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASAAPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASASPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCLGPCPYI WSLDTQYSKV LALYNQHNPG ASASPCCVPQ ALEPLPIVYY VGRKPKVEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLDSLTILYY IGNKPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCAGACPYL WSSDTQHTKV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGNTPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCAGACPYL WSSDTQHSRV LSLYNTINPE ASASPCCVSQ DLEPLTILYY IGKTPKIEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL NFCSGPCPYL RSADTTHSSV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTAKVEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL NFCSGPCPYL RSADTTHSTV LGLYNTLNPE ASASPCCVPQ DLEPLTILYY VGRTPKVEQL
SNMVVRSCNCS SNMVVRACKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS
SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVRSCKCS SNMIVKSCKCS SNMIVKSCKCS
SNMIVKSCKCS SNMIVKSCKCS SNMIVKSCKCS SNMVVKSCKCS
SNMVVKSCKCS SNMVVKSCKCS SNMVVKSCKCS
FIG. 1. Sequence alignment of TGF-/3 amino acid sequences. See Materials and Methods for the source of sequences and the method of alignment. The symbols used for the species are: Xe, X. laevis; Ck, chicken; Po, porcine; Bo, bovine; Hu, human; Si, African green monkey; Mu, murine; Ra, rat. Sites of proteolytic cleavage are shown with a vertical arrow. Gaps were introduced to maximize sequence alignment and are shown with a period (•). All sequences are numbered relative to the first amino acid in the pro region, immediately after the signal cleavage site.
sition 1,100-1,114] CTGCTCCACGCGCAC (Jakowlew et were used at 100 pmoles each, with 1 ng of plasmid DNA in a total volume of 100 /tl. The expected 243-bp product was isolated and verified by digestion with Sma I, which produced the expected 152-bp and
ai, 1988c). Primers
91-bp
DNA
fragments.
Genomic Southern analysis A nylon membrane containing Eco RI-digested genomic DNAs from a range of eukaryotic species (human, monkey, rat, mouse, canine, bovine, rabbit, chicken, and yeast) was obtained from Clontech (ZOO-BLOT, Palo
BURT AND PATÓN
DNA and Cell Biology 1992.11:497-510. Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 01/05/15. For personal use only.
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