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Nucleotide and amino acid sequence of a Cucurbita phytochrome cDNA clone: Identification of conserved features by comparison with Avena phytochrome

Authors:

Abstract

The amino acid (aa) sequence of Cucurbita phytochrome has been deduced from the nucleotide (nt) sequence of a cDNA clone which was initially identified by hybridization to an Avena phytochrome cDNA clone. Cucurbita, a dicot, and A vena, a monocot, represent evolutionarily divergent groups of plants. The Cucurbita phytochrome polypeptide is 1123 aa in length, corresponding to 125 kDa. Overall, the Cucurbita and Avena phytochrome sequences are 65 % homologous at both the nt and aa levels but this sequence conservation is not evenly distributed. Most of the N-terminal two-thirds of the aligned polypeptide chains exhibits localized regions of high conservation, while the extreme N terminus and the C-terminal one-third are less homologous. Comparison of the predicted hydropathic properties of these polypeptides also indicates conservation of domains of phytochrome structure. The possible correlation of these conserved structural features with previously identified functional domains of phytochrome is discussed.
Gene, 41 (1986) 287-295
Elsevier
287
GEN 01764
Nucleotide and amino acid sequence of a Cucurbitu phytochrome cDNA clone: identification of
conserved features by comparison with Avena phytochrome
(Plant photoreceptor structure; hydropathy analysis; molecular evolution; recombinant DNA; zucchini; oat)
Robert A. Sbarrock, James L. Lissemore and Peter H. Quail*
Department of Botany, University of Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608) 262-0476
(Received July 18th, 1986)
(Accepted August 15th, 1986)
SUMMARY
The amino acid (aa) sequence of Cucurbitu phytochrome has been deduced from the nucleotide (nt) sequence
of a cDNA clone which was initially identified by hybridization to an Avena phytochrome cDNA clone.
Cucurbitu, a dicot, and Avena, a monocot, represent evolutionarily divergent groups of plants. The Cucurbitu
phytochrome polypeptide is 1123 aa in length, corresponding to 125 kDa. Overall, the Cucurbitu and Avenu
phytochrome sequences are 65 % homologous at both the nt and aa levels but this sequence conservation is
not evenly distributed. Most of the N-terminal two-thirds of the aligned polypeptide chains exhibits localized
regions of high conservation, while the extreme N terminus and the C-terminal one-third are less homologous.
Comparison of the predicted hydropathic properties of these polypeptides also indicates conservation of
domains of phytochrome structure. The possible correlation of these conserved structural features with
previously identified functional domains of phytochrome is discussed.
INTRODUCTION
Phytochrome is the best-characterized of the regu-
latory photoreceptors that control plant growth and
* To whom correspondence and reprint requests should be
addressed.
Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilo-
base(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame;
PAGE, polyacrylamide-gel electrophoresis; Pfr, far-red light
absorbing; poly(A)’ , polyadenylated; Pr, red light absorbing;
SDS, sodium dodecyl sulfate.
development in response to light (Shropshire and
Mohr, 1983; Lagarias, 1985). The native molecule is
a dimer, with each subunit comprising a linear tetra-
pyrrole chromophore covalently attached to a poly-
peptide of 120-127 kDa (Vierstra et al., 1984).
Photoconversion of phytochrome from the Pr form
(red absorbing) to the Pfr form (far-red absorbing)
induces many morphogenic responses (Shropshire
and Mohr, 1983) and alters the expression of several
plant genes (reviewed by Tobin and Silverthome,
1985). The mechanism through which this induction
occurs is not known and the regions of the phyto-
0378-I 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
288
CP
AP3
CP
-143 ATC TACGCCTTCT CTCTCCTCAT CTCCTTTCTC
-57 CAGGAGC ATA GGG GTATA GA C nG GTG MT AC T AG CAGGCG
GCTCTAAATT CTCCTCCACC ATGCCTCTCA CCTTTCACTC TCAGCCCCTC TMTTCACCG GCGCCACTCT TGCCCCACCG GMAATCTGT TCTTCTTTTG GTTGAGAAAC
AP3
CP
CP
AP3
C TCC CAA GA
AP3
CP
CP
AP3
AP3
CP
CP
AP3
AP3
CP
CP
AP3
NSTSRPSQSSSNSGRSRHSTRIIAOTSVDA
S A s S R N Q S S Q A v L T L
GGCCAT AA A TGC C C AG CTG T GM C CAG C G G CCL CCT TG G A
?C YG r EC, y ;TT FG ?A ;CT y ;AT ;CG ;TT YC ;AC ;'A ;GT ;'A GTG CGT GTC ACT AGT GAT GTT AGC GGA GAT CIA CAG
VRVTSDVSGDQQ
E L N E Y D K L E A Q R G P P "
ffiG C C C G
CCT AGG TC~" GA: aai GT: it; AZA GCT TAG :T: CA: CAT ATT CAG MA 66: AM cd AT: caa &A TTT GGT TGC ~TG TTG GCC :T: GAT
RSDKVTTAYLHHIQKGKL~QPFGCLLALD
E . I Q T
G G GC T C C TC G C G A CT AC C G A C TGT AT
r ;AA ;CA FTC 7" ;TT :,T FG YT :GT r" ?T ;CC CpCT r" ;TG :TG ;CC ;TG GTG AGC CAT :CT GTC CCA AGC ATG G&S GAT ::C
v s H VPSNGDY
E S N F T v D P
AP3
CP
CP
AP3
AAGG G G GT AC MG CIA CAT
CCT GTT CTT ffic ATT GGC AC: &T GTA ki :CT :TT TTC Act GCA CCT AGT GCT TCT GCA CTG TTG MG GC: :T: GG: TTT Gz: GAE GT:
PVLGIGTDVRTIFTAPSASALLKALGFGEV
R N s L 5 D Q G T H A D
AP3
CP
CP
AP3
AP3
::
AP3
AP3
CP
CP
AP3
TTTG G T G A A C G T C A CA TTT GGGGA
ACA CTT CTT AAT CCT ATC CTG GTG CAT TGC MG ACT TCT G&A MA CCC TTC TAT GCA ATT GTT CAT CGT GTT ACT GGA AGC TTA ATC ATT
TLLNPILVHCKTSGKPFIAIVHRVTGSLII
5 a A C V"
A ACA n T CC T G TG G C C G T G A ATC AG
GAC TTT GAG CCT GTG AAG CCT TAT GAA GGT CCA GTG ACT GCA GCT GGA GCT CTA CAA TCA TAT AA1 CT, GCC GCC AA1 GCG ATT ACT AGA
DFEP"KPYEGP"TAAGALQS"KLAAKA,TR T F A s K
AC A AG AGGT A CAT TG GAG C C T C G CAG
TTG CAG TCT TTG CCT AGT GGA AGC ATG GCT AGG CTT TGT WC ACA ATG GTT CAA GM GTT TTT GAA CTA ACA GGT TAT GAT CG1, GTT ATG
LPSLPSGSMARLCDTMVQEVFELTGYDRVN
1 G E V N V K D
AP3 C G T A C T G AT c AA T T G T A cc c
CP GCT TAT MA TTC CAT GAT GAT GAT CAT GGG GM GTG ATC.TCT GM GTC GCA MG CCC GGC CTT CAG CCL TAT CT1 GGT TTG CAT TAT CCA
CP AYKFHDDDHGEVISEVAKPGLQP"LGL""P
AP3 E F 1 T E
AP3
CP
CP
AP3
C C A CAGC CT G C A A TG C G G TCC A G C
GCA ACT GAC ATT CCT CAA GCT GCA CGT TTT TTG TTC ATG MA MT MG GTC CGG ATG ATT GTT GAT TGT CGT GCA AAA CAT TTA AAA GTA
ATDIPQAARFLF"KNKVRMIVDCRAKULKV L C R S I
AP3 ATGG C GC CC CC AT GCC GACC G A T CCT G
CP CTC CIA GAT GAG AAA TTA CAG TTT GAT CTA ACT TTA TGT G4i~ TCA ACT na AG4 GCT cca cat aGT TGC CAT TTA CAG TAT ATG w MC
CP LQDEKLQFDLTLCGSTLRAPHSC"LQYNEN
AP3 I E A A P I s A
AP3
:;
AP3
AP3
::
AP3
AP3
CP
CP
AP3
G T A CCT C T G AAT A G T G CT GAG TCT GM AA C A GCA
ATG AAC TCT ATA GCC TCT TTG GTT ATG GCA GTT GTG GTT MT GAA GGG GAT GAA GM MT GAA GGC . . . . . . . . . CCT GCT TTG CAG CAG
MNSIASLVNAVVVNEGDEENEG P A L Q Q
N E D D A;iiQPA
G A AG A C CCCT T C CC WG C A CT G GCT
CAA MG AGA AAG AGA TTA TGG GGC nG GTA GTA TGT CAT MT TCA AGT CCC AGA TTT GTT CCG TTT CCT CTT AGG TAT GCT TGT GAG TTC
PKRKRLYGLVVCHNSSPRFVPFPLRYACEF
K K L H E Y
T A G G GC C G GTAAGA GTGCGT G c A MGT A AG
CTA GCT CIA GTA TTT GCT ATT CAT GTG UC AAG GM TTA GAG nG GAA MT CAA ATT ATA GM MG MT ATT CTG CGT ACG CAG ACA CTC
LAQVFAIHVNKELELENQ,IEKNILRTQTL
V R F K L R K N N
AP3
CP
a'p;
CC CT T TGTC A A C TCT CCGAC C A AG CC C T C c A G T
nG TGC GAC ATG CTA ATG CGT GAT GCT . . . CCT na GGT ATT GTG TCG AGG AGT CCT MC ATA ATG GAT CTT GTC AAA TCT GAT G&G GCT
LCDNLNRDA PLGIVSRSPNINDLVKSDGA
S F E ; T G T C
AP3
:;
AP3
AP3
:;
AP3
AP3
:;
AP3
AP3
:p'
AP3
TCTCG GG GG GA TCGC TAATGT A CG G CT AAAT T C C TC A A T GT
CCC TTG TTA TAT MG MA MA ATT TGG CGA TTA GGA TTG ACA CCT MT GAC TTC CAG TTG CTG GAC ATA GCT TCG TGG CTT TCC GAG TAT
ALLYKKKIURLGLTPNDFQLLDIASULSEI
G G V R N A T E S I H F D v
C G C 7 CC cccc T C T A GC CT ATG A T A
MT 5" :CG GGG TTG AGT ACT GAC AGT TTG TAT GAT GCA GGA TAC CCT GGA GCT ATT GCT TTA GGT GAT GAA GTG TGT GGG ATG
D GLSTDSLYDAGYPGAIALGD;;cGN H A
TG CT A C AC TCC G T T C CAG A T A T
GCA GCT GTG AGG ATA ACT MT MT GAC ATG ATT TTT TGG TTT CGA TCT CAC ACT GCT k: Gd AT: :GA TGG GG: GG4 GCA MG :AT d
AAVRITNNDNIFYFRSHTASEIR!dGGAKHE
V A K N S K I L A N D
CA KG G C T C AG GG C T G TG A T TG AG C
CAT GGT CM MG GAt GAT CCC AGA MA ATG CAT CCA AGA TCA TCT TTC MG GCT TTC CTT GAA GTA GTC MG ACA AGA AGT TTG CC: TGG
HGQKDDARKNHPRSSFKAFLEVVK;RSLPU
P s D II S R L K
-111
-1
180
M
270
ul
360
120
CM
1.50
540
180
630
210
720
240
810
270
wo
3cm
990
330
107l
357
1161
387
1251
417
1336
446
1428
476
1518
546
1608
53.5
1656
566
Fig. 1. Nucleotide and derived aa sequences of Cucurbitu phytochrome clone pFMD1 (CP) and comparison with Avena type 3
phytochrome (AP3) sequences. Dideoxy sequencing was performed as described (Biggin et al., 1983) utilizing the phage vectors
M 13mp 10 and M 13mp 11 (Norrander et al., 1983). DNA and protein sequence analysis and alignment were performed using computer
programs provided by the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). Numbering of the nt and
aa sequences refers to the Cucurbitu sequences and begins at the initiator methionine. The complete Cucurbita phytochrome sequences
289
AP3
CP
CP
AP3
AP3
CP
CP
ALP3
GT A TT T A ACA CC AGC AG CC AGCGGGA GCT
MG GAC TAT GAG ATG GAT GCA ATC CAC TCT TT:G CA-A UT AT: CT," :GA M": ACT nT ME GAT it; GLT GCA ACT GAA ATA MT AGA MA
: DYEMDAIHSLQLILRNTFKDTDATEINR~ G LN .ASKPKRE
AGT T A G T AC CAG A T C T CTT GCT C CGGC G C A T TC G A
TCA ATT CIA ACA ACA CTT GGT GLC CTA AAA ATT GM GGG AGG CIA GAA TTG GAA TCA GTA ACA AGT GAG ATG GTC C&4 TTA ATC GAG ACA
SIPTTLGDLKIEGRPELESV~SEMVRLIET
L D N Q I L D L A Q A N
AP3
:p'
AP3
A TACGAA GGC A ACGGC C CAG GCA G G G AA&A T
GCT ACT GTG CCG ATT TTA GCT GTT WT TTA GAT GGG nA ATT MT GGG TGG MT ACA AM ATT GET &AA TTG ACT GU CTG CCT GTG G&T
ATVPILAVDLDGLINGUNTKIAELTGLPVD G N V a A R
AP3
CP
CP
AP3
GT A TAG CA CCT G C C cc CA G CA ATCA TC G TA
?A ,"T ;X ?C c;'" ;A1 :,, :,, :CG y YG p &AT ;CA :CT GTA p GTT GlC AGG PG ATG TTG ;TC nG GCA TTG CM GGA CIA
D V v v R N L LALQGP
D R 1 P Q R Y K
AP3
CP
CP
AP3
AP3
CP
CP
AP3
AGGA G T GA T T CCGAG GG TA TCAGT T TG G C
GAA GAG CIA AAC GTT CAA nC WG ATC AAG ACA CAC GGT TCT CAC ATT GAG GTT GGC TCC ATC AGC CTA GTT GTA MT GCT TGT GCA AGT
EEQNVQFEIKTHGSHIEVGSISLVVNACAS
K E R V P K R D D P v I
C CTA TC TTA Gc T C G TC T T G T GGT
AGG GAC TTG CGT MA AAT GTC GTG GGT GTG nT TTT GTA GCA CAA GAT ATC ACT GGT CAG MG ATG GTA ATG GAC AAG nC ACT CGA TTA
RDLRENVVGVFFVAQDITGQKNVNDKFTRL
H D H C II v H L V
AP3
CP
CP
AP3
G T C CAT C C C cc T TGT C TG
GAA GGC GAT TIC AA," GC:. ATT GTA CIA MT CC: AAT CCA TTG ATC CCT CC: ATA TTT GW TCA GAT GM TTT GGA TGG TGC TCA GAG TGG
EGDIKAIVPNPNPLIPPIFGSDEFGVCSEY I H A
AP3
CP
CP
AP3
AP3
CP
CP
AP3
AP3
CP
CP
AP3
AP3
CP
CP
AP3
G AC GTG AAT A A T GC CT TA G AC AG AG TGT
MT CCT GCA ATG GCG MA CTA ACT GGG TGG TCA CGT GM GM GTA ATC GAT AAG ATG CTT nG 661, GLG GTT TTT GGT GTT CAC MA TCA
NPAMAKLTGYSREEVIDKNLLGEVFGVHKS
A T N D L D S S N A
CC CC G CAG T A A G TTG CTAC G ATAGCC GG AAA CA
TGT TGT CGT TTA AAG AAT CIA GAA GCT nT GTT MT CTT GGG ATT GTC nG MC MT GCC ATG TGT GGT CM GAT CCT GAl\ AAG GCT TCC
CCRLKNPEAFVNLGIVLNNANCGQDPEKAS
S P R D S c v L I S L A E E T P
C C ACAA GT A A AT G CA CA CGAAAGAAA GG G TCTCAC T AGA
;T ;GT ;TC y ;CT ;GG FC y p :,C FG r" :,r :TT :" :,r y MT MG ATC nG GAT AM &AT GGC GCG GTT ACA GGG TTC
NKILDKDGAVTGF
F D S K 1 S A RKENEG L I V
CT ATTGG C T CGGG G AA GCC CG GTGCA A GCC AG
nT TGC TTT TTG CAG CTT CCT AGT CAT GAG TTG CM CAA GCA CTA AAT ATC CAA CGC nA TGT GAG CAA ACT GCA TTG AAG AGA nG A&,
FCFLPLPSHELPQALNIQRLCEQ~ALKRLR
I H V A H a v Q A S S K
AP3
CP
CP
AP3
T C TCC G G CAT GC CAC C C A C GC AC TA A GC A AA
GCA TTG GGA TAC ATA MA A&A CAG ATA CAA MT CCT CTT TCT GGG ATA A.TC nT TCA CW Aw. nA TTG GAG CGC AC: GA; TTG 2: G+A
ALGVIKRQIPNPLSGIlFSRRLLERTELGY
F S N R H A N II L Y K A K N D N E
AP3
:::
AP3
AP3
CP
CP
AP3
AP3
CP
CP
AP3
AP3
c";
AP3
G TG A G AG A T A G, G A AT AAT ccc A AA T CA C TG
GAA CA1 AAA GAA CTT CTG CGT ACT AGC GGA CTC TGT CAA AAG CAG ATC TCC AAG ",: CTC GAT GAG TCA GA: ::: GAT A: AT: A:: GA:
EPKELLRTSGLCQKOISKVLDESDlDKlID
M K Q I H V G D N H H N I A D L Q S T E
AAA TCT A C GC T G CT A CTG T CTGTGA
. . . . . . GGG Tn ATT GAT TT," d ATG GAC GAG TTT AC1 TTG CA: GAA GT: :TG :GTG GTA TCA ATT AGT CM GT: :TG :TA Ai: :,": hi
i ; : : L
IDLENDEFTLUEVLNVSISQVNLKIK
A L Q D V V A A V L ITC a
A G AGA C TC TGC A C CTG GAW TT AAG C TAGC G GTCA C G A CC
GGA MG GGT ATC CAG ATA GTT MT GAG ACT CC1 GAA GAG GCT ATG TCC GAG ACC TTA TAT GGA GA7 AGT TTA AGG CTT CAA CAG GTC nG
GKGIQIVNETPEEAMSETLYGDSLRLQQVL
R S C N L R F K Q S V G V I
T TCT G T GAG
ADFLLISVSYAPSGGQLTISTDVTKNQLGK
S F K F S V S V E S K L s I E
AP3 AAC C T TATGCCT ACT C AGCCCM A CAGA CCC. GCA C A
E;
WIG AGGA
TCG GTC CAT CTG GTG CAT TTG GAG TTC AGG ATA ACA TAT GCT GGA :A GG: :Ti CCG G4G TCG TTG CTG UC GAG ATG TTT . . . GGA AGC
SVHLVHLEFRITYAGGGIPESLLNEMF
AP3 G S
N L I D L K H Q L V A E M A Q i E D
:,’
AP3
AP3
:p'
AP3
ALP3
CP
iP3
AP3
AC1 G CA
GAG GAG GAC GCG TC: GA: GAG GG! n: ~6: CT: CT: !T: ~ZT AGL, AACG CTG ZTG A~G CT: ATG MT 6: ~4: GT: cw" FAT ir2 AGG GAA
EEDASEEGFSLLISRKLVKLNNGDVRVMRE
N K E 9 L V N L R H L
TGT AC
GCE GGG AAG TCG AGC TTC ATC AT: AC: ST GA: CTT GCT :C: GCT C:: A:: &A AGG ACA ACG TAG GCCAAGACA ATTTGMGCA TCAACCTTGT
T GG CM GA TW GCCAG TGGMGT T CA CTTA G
AGKSSFIITVELAAAHKSRTT+
V T A S P T A II G P l
TCATCAAATG TTC GT TGA A TCC AGTC ACUTAGCCG TWGCATTGG TUGTTGGTG GCATGTTCCT GGGGACGAGG AlGG4ATGTG CTGCAGCCTG TAGTGTAGTC
ATGAATTGCT GCATTCTATG TTGAAAG
TTGCAGCTTG GTACTTCCGC TGTTGTTATG TTTCTGTCAT CCTACTGTGT AAGGTTCAAG TTTGAATTTA CCATGAATM AGTGTGCAGA TGTACCTGCA CTTTGGTCTC
AAAAAAAMA AAAAAMAAA AAAMMAAA AM
1788
5%
1878
626
1968
656
M58
686
2148
716
2238
746
2328
776
2416
en6
2508
836
2.5%
866
2688
8%
2778
926
2868
9%
2952
984
3042
lM4
3132
1044
3219
1073
3309
1103
?z
are presented, including the correction in codon 223 derived from clone pFMD5 (see section b). Only nt and aa in the Avena sequences
that differ from those in Cucurbita are shown. Gaps (indicated by dots) have been introduced in both the Cucurbitu and Avena sequences
in order to maintain colinearity within the coding regions (see section c). No gaps have been introduced in the 5’ and 3’ non-coding
regions. The 5’ non-coding region of AP3 is not full-length (unpublished data; see Hershey et al., 1985).
290
chrome molecule that are involved in photocon-
version and in mediating the resultant changes in
gene expression are just begirming to be elucidated.
Purification of native, unde~aded ph~oc~ome
from etiolated tissue of the monoeot plant Arena
sativa (oat) has led to detailed characterization of
this molecule and correlation of several structural
domains of the pol~eptide with its photo~he~c~
and biochemical properties (Vierstra and Quail,
1982a; 1983). For instance, a light-induced confor-
mational change in Avena phytochrome has been
demonstrated by protease sensitivity studies which
indicate that discrete cleavage sites are differentially
exposed in the Pr and Pfr forms of the molecule
(Vierstra and Quail, 1982b; Jones etal., 1985;
Lagarias and Mercurio, 1985). In addition, it has
been shown that the N-terminal 6-lO-kDa region of
the Avena phytochrome polypeptide is critical to the
spectral properties and the ~~t-~d~ced confor-
mational changes of this molecule (Vierstra and
Quail, 1982b; Jones et al., 1985) and that dimeriza-
tion of Plvena ph~oc~ome monomers involves a
site(s) within a 42-kDa domain in the C terminus of
the protein (Jones and Quail, 1986). The primary
sequence of Arena ph~~~orne has been deter-
mined (Hershey et al., 1985) so that structural and
functional data can now be correlated with a precise
aa sequence.
Dicot ph~~hrome, including that from C~curb~ta
(zucchini), is similar to oat phytochrome with respect
to monomer M,, subunit structure, spectral proper-
ties, and sensitivity to proteolysis (Vierstra et al.,
1984; Cordonnier and Pratt, 1982a; Vierstra and
Quail, 1985). However, it has repeatedly been
observed that, although antibodies raised against
phytoehrome from monoeot species cross-react we11
with other monocot ph~o~~omes, they react poorly
with dicot phytochromes and, likewise, anti-dicot
phytochrome antibodies recognize monocot phyto-
chromes poorly (Cordonnier and Pratt, 1982b;
Cordonnier et al., 1984). Since there are many struc-
tural and functional similarities between monocot
and divot phytochromes but little immunolo~c~
cross-reactivity, comparison of phytochrome pri-
mary sequences from these two groups of plants
should be useful in defining conserved regions of the
protein. We report here the first nt sequence and
derived aa sequence for a dicot ph~o~hrome cDNA
clone and the comparison of the oat and zucchini
phytochrome molecules.
EX~ERI~E~AL AND DISCUSSION
(a) Isolation of ph~~hro~ cDNA cJones
The construction of the Cucurbita pep I.,. cv.
Black Beauty cDNA library and selection and
characterization of phytochrome cDNA clones will
be described in detail elsewhere (J.L.L., J. Colbert
and P.H.Q., in preparation). Briefly, poly(A)+ RNA
was isolated from etiolated Cucurbita hypocotyl
hooks and size-fractionated; cDNA copies of this
RNA were prepared as described (Hershey et al.,
1984). These cDNA copies, annealed into pBR322
and cloned in ~~~~~~~a cdi, were colony-screened
using a nick-translated 3.0-kb Kpnnf-Sad insert from
the Avena ph~~hrome cDNA clone pAP3.2
(Hershey et al., 1985) as a probe.
(b) Sequence aadysis of a C~lcurbir~ pbyt~b~~rne
cDNA clone
The cDNA insert from the largest Cuc~r~jta
phytochrome cDNA clone isolated, pFMD1, was
sequenced and found to contain the entire aa-coding
region (Fig. 1). A computation arising from this
analysis was that the QRF of pFMD1, shown to
contain a cysteine encoded by a TGT codon at aa
position 223 in Fig. 1, was in fact interrupted at this
position by a TGA translation t~na~on codon.
Sequencing of a second, overlapping cDNA clane in
this region (pFMD5; J.L.L., J. Colbert and P.H.Q.,
in preparation) showed 100 % nt sequence homology
with pFMD1 for 300 nt except for the third nt of
codon 223 which was T instead of A, indicating that
the TGA codon was very likely a reverse tran-
scriptase error (Battula and Loeb, 1974). With this
correction, the 3.4-kb cDNA insert from pFMD1
defines an mRNA consisting of an QRF encoding
1124 aa bordered by 143 nt and 56 nt, respectively,
of 5’- and 3’-untranslated sequence. The absence of
a polyadenylation signal and a poly(A) sequence in
the 3 ‘-untranslated region make it unlikely that
pFMD1 constitutes a full-length cDNA copy of a
mature Cueurbita phytochrome mRNA. It is notable
that a nearly concensus translation initiation
sequence (CCACCAUGC) is located within the
5 ‘-untranslated region of the mRNA represented by
pFMDl, 88 nt upstream from the start codon for the
phytochrome ORF (Fig. 1). An in-frame UGA
translation termination codon is located 27 nt down-
stream from this first start codon. This situation, a
highly favorable translation initiation sequence
between the 5’ end of an mRNA and the actual
translation start codon, has been observed previ-
ously in eukaryotic mRNAs and several studies have
demonstrated that initiation at the correct, down-
stream site is dependent on having a stop codon in
frame with the first AUG and upstream from the
second (Dixon and Hohn, 1984; Kozak, 1984).
The calculated M, of the Cucurbita phytochrome
monomer, from translation of the large ORF of
pFMD1, is 124950 (after subtraction of the initiator
methionine residue). This is approx. 5 kDa greater
than estimates based upon the electrophoretic
mobility of the Cucurbita monomer in SDS-PAGE
(Vierstra et al., 1984). The calculated isoelectric
point of the derived monomer is 6.2 which is lower
than the observed range of 6.5 to 7.0 for the immuno-
purified Cucurbita protein (Cordonnier and Pratt,
1982a) and closer to the calculated and experi-
TABLE I
Phytochrome amino acid composition
291
mentally determined value of 5.9 for Avena phyto-
chrome (Vierstra and Quail, 1982a; Hershey et al.,
1985). The aa composition of the derived polypeptide
is consistent with that determined for immuno-
purified Cucurbita phytochrome (Table I).
(c) Comparison of Cucurbitu and Avena phyto-
chrome nt and aa sequences
Hershey et al. (1985) determined the nt sequence
of two complete ORFs, designated type 3 and type 4,
from Avena phytochrome cDNA and genomic
clones. Within the coding region, these two
sequences are approx. 98% homologous at both the
nt and aa levels. The Cucurbita phytochrome coding
region from pFMD1 is approx. 65% homologous to
the Avena type 3 and type 4 phytochrome sequences
at both the nt and aa levels (Fig. 1; unpublished
data). The calculated M, of the Cucurbita phyto-
chrome polypeptide (124 950) is very close to the M,s
of the two completely sequenced Avena polypeptides
Amino acid Cucurbita
residue (from sequence in Fig. 1) Cucurbita
(by hydrolysis)” Avena
(We 3) b
A%? 54 48 53
Lys 65 64 64
Asn
Asp
Gln
Glu
40
63 103
51
14 125 1
110
128
45
68 113
49
17 126
His 29 24 34
Pro 41 - 46
Tyr 25 25 21
Trp 10 8 10
Ser 87 II 89
Thr 58 54 43
GUY 15 82 69
Ala 76 79 96
Met 34 28 34
Cys 20 22 23
Phe 45 41 44
Leu 126 120 124
Val 81 12 83
Ile 69 59 56
Total 1123
a Cordonnier and Pratt (1982a); proline content was not reported.
b Hershey et al. (1985).
1128
292
(124 870 and 124 949) though the Cucurbitu sequence
is 5 aa shorter. The aa composition of phytochrome
from these two plant species is also strongly con-
served (Table I). ~~~rb~t~ phytocbrome is signifl-
catty less negatively charged than the Avena mole-
cules, having a calculated net charge at neutral pH
of -18 as compared to -28 for Avena.
The sequence alignment in Fig. 1 has been done in
such a way as to optimize conservation of sequence
at both the nt and aa levels within the coding regions.
The gaps in the nt sequences in Fig. 1 are based upon
comparison and alignment of the aa sequences and
represent the simplest arrangements possible.
Several more complex patterns of gapping in both
sequences, resulting in higher local nt and aa
sequence homology, are possible at some of these
sites but no evidence currently exists that such
arrangements should be preferred. No gaps have
been introduced in the non-translated regions of the
sequences in Fig. 1. The 56 nt of the Cucurbita 3 end
that are present on pFMD1 are not homologous to
the 3 ‘-untranslated regions of Avena type 3 or type 4
phytochrome transcripts (Fig. 1). This result might
be expected since the 3’ ends of the type 3 and type 4
Avena sequences are not themselves highly con-
served (Hershey et al., 1985). In contrast to the
3’ ends, the 5’-untranslated regions of the Avena
type 3, 4, and 5 phytocmome mRNAs are greater
than 90% homologous with one another (Hershey
et al., 1985). The Cucurbita 5’ end is not homologous
to these sequences (Fig. l), indicating that, if there is
a regulatory function associated with the conser-
vation of Avena phytochrome mRNA 5’ non-coding
regions, that function is not conserved between these
two species at the sequence level.
In order to better visualize the relatedness of the
oat type 3 and zucchini phytochromes, the local level
of conservation of aa sequence between these two
polypeptides is presented in Fig. 2A as a linear plot
of percent homology within a 9-aa moving window.
Al AMINO ACID SEQUENCE HOMOLOGY ---_-_____-_--____,
L .‘I, ““4 ‘1 .“I’< “““,
0 ; loo 200 300 400 500 600 700 800 900 1000 1100 ;
AMINO ACID RESIDUES :
I :‘: I I
6 4 64 55
Fig. 2. Sequence homology and hydropathy difference for the Cucurbita and Avena phytochrome polypeptides. (A) Local level of aa
sequence homology between Cucurbita and Avena type 3 phytochromes. For the aligned polypeptides in Fig. 1, the number of identical
residues within a moving window of nine aa has been expressed as percent homology (O/9 = O%, 9/9 = 100%) and plotted at the middle
position of the window. For this reason, the plot begins with the fifth aa of both mature polypeptides. All gaps in the alignment in Pig. 1
have been counted as mismatches. Shaded regions correspond to regions of greater than 50% homology. (B) Hydropathy difference
between Cucurbita and Avena type 3 phytochromes. The hydropathy profiles in Fig. 3 were superimposed and the difference between
them at each data point was calculated in arbitrary units. This difference is plotted here without regard to whether it represents increased
or decreased hy~ophobicity of one pol~ptide relative to the other. Panels A and B have been divided into three domains (I* II, III)
based upon consideration of the levels of sequence homology and the hydropathy differences (see section e). A peptide map of Avena
phytochrome is shown at the bottom, indicating the position of the chromophore (solid rectangle) and approximate sizes of peptide
fragments in kDa generated by proteolysis (see Daniels and Quail, 1984).
In this plot, 0 of 9 aa matches is plotted as 0%
homology for the residue at the middle position of the
window and 9 of 9 aa matches as 100% homology.
It is clear from this plot that, based upon identity of
aa residues, conservation of phytochrome poly-
peptide sequence is not evenly distributed over the
length of the molecule. Instead, localized regions of
high conservation (80-100% homology over the
window) are observed throughout two-thirds of the
molecule beginning at aa approx. 60 and ending at aa
approx. 800, while the extreme N terminus and the
C-terminal one-third of the molecule have relatively
low levels of ~onse~ation.
To further compare their predicted structural
properties, hydropathy profiles for the Avena type 3
and Cucurbifa phytochrome polypeptides, again cal-
culated for a 9-aa moving window, are shown in
Fig. 3. It is clear that many features of these profiles
are conserved between these two species. Fig. 2B
shows a plot of the relative difference in hydropathy
between oat and zucchini phytochromes in arbitrary
units at each aa position, disregarding whether the
difference is due to increased or decreased hydro-
phobicity of one protein compared to the other.
Consideration of both the aa homology and hydrop-
athy comparison plots (Fig. 2,A and B) provides a
preliminary picture of the interspecific conservation
of phytochrome structure wherein three domains can
be recognized. First, an N-terminal domain
(region I) which is poorly conserved for aa identity
293
but is only moderately divergent in hydropa~c
properties. These data reflect the conservative nature
of most of the aa substitutions that have occurred in
this domain. Second, a large domain (region II)
which consists of localized regions of high sequence
conservation interspersed with non-conserved
regions. Within this large domain, the hydropathy
comparison further delineates a region of very high
structural homology between aa 150 and aa 400
which contains the chromophore attachment site.
Finally, the C-terminal portion of the molecule
(region III) is clearly more divergent in both
sequence identity and hy~opat~c properties than
are the first 800 aa. This domain-like picture is also
discernable in comparison of the secondary struc-
tures of the oat and zucchini phytochrome poly-
peptides as predicted by the method of Chou and
Fasman (197X). As expected, such an analysis shows
extensive regions of conserved structure within
region II, most notably from aa 150 to aa 400. Far
less conservation is observed within regions I and III
(unpublished data).
(da) Correfation of conserved regions with phyto-
chrome functional domains
Over the entire phytochrome protein sequence, the
most highly conserved stretch of consecutive aa is at
the chromophore attachment site, cysteine 321 of the
mature Avena phytochrome sequence (Hershey
AMINO ACID REStDUES
0 100 200 300 400 500 600 700 800 900 IO00 1100
;I I 1 t t I t / / I 1 I , I
AVENA
6 ‘4’ i
64 I
55
Fig. 3. Hydropathy profiles of Cucurbitu and Avena type 3 phytochromes. Hydropathy analysis was performed according to Kyte and
Doolittle (1982) using programs supplied by J. Pustell, Harvard University. Solid peaks indicate hydrophobic regions; unshaded troughs
indicate hydrophilic regions. The profiles have been aligned in the same way as the sequences in Fig. 1, with gaps introduced at the same
positions. A peptide map of Avena phytochrome is shown at the bottom (see legend to Fig. 2).
294
et al., 1985). There is complete conservation for 29
consecutive aa residues around cysteine 322 (exclud-
ing the initiator methionine) of Cucurbitu phyto-
chrome. This strongly indicates that cysteine 322 is
the chromophore attachment site and that, like the
Avena molecule, Cucurbita phytochrome has only
one such site per monomer. In addition, a region
N-terminal to the chromophore attachment site (aa
approx. 150-300) is very highly conserved in both
hydropathy and aa identity (Fig. 2). The sequence
and structural conservation in these two hydro-
phobic regions adds support to the notion (Hershey
et al., 1985) that they are directly involved in forming
the hydrophobic cavity in which the tetrapyrrole
chromophore is postulated to reside in the native
phytochrome molecule (Song, 1983; Hahn et al.,
1984). A high degree of conservation of those regions
of the polypeptide which interact directly with the
chromophore is expected given the almost identical
spectral properties of phytochrome from these two
divergent species (Vierstra et al., 1984; Vierstra and
Quail, 1985).
Interestingly, the two regions of the phytochrome
polypeptide to which at least a partial function has
been assigned in both Avena and Cucurbita are the
least conserved regions. The N-terminal 6 to 8 kDa
of both oat and zucchini phytochrome are removed
by endogenous plant proteases in crude extracts and
this cleavage results in similar alteration of the spec-
tral properties of these molecules (Vierstra and
Quail, 1982a; 1983; Vierstraet al., 1984; Jones et al.,
1985). The C-terminal 40-55 kDa portion of both
oat and zucchini phytochrome, again generated by
proteolytic cleavage, has been shown to contain the
contact sites for dimerization of the monomeric
subunits (Vierstra and Quail, 1985; Jones and Quail,
1986). Overall, both of these regions are conserved
to a lower degree than is the central portion of the
molecule (Fig. 2) but, within both of these general
regions, there are local segments of high sequence
conservation which may represent the polypeptide
domains that are critical to these functions. In this
regard, it is notable that one of the type 1 monoclonal
antibodies isolated by Daniels and Quail (1984)
recognizes an antigenic domain within the N-termi-
nal 6 kDa of Avena phytochrome and also cross-
reacts with undegraded Cucurbita phytochrome,
indicating that there is a conserved antigenic deter-
minant within this region. Within the N-terminal
6-kDa peptides of Avena and Cucurbita phyto-
chromes, the only region of significant contiguous aa
sequence homology is found at aa 37-46 (Figs. 1
and 2). This site corresponds to a peak of hydro-
philicity in the hydropathy plots for both molecules
(Fig. 3). Though not predicted to be an antigenic
determinant by computer analysis (Hopp and
Woods, 1983 ; unpublished data), this region is
clearly a candidate for containing the epitope for that
type 1 monoclonal antibody. In addition, since
dimerization is a conserved feature and there is
evidence that the dimerization site(s) lies within
40 kDa of the C terminus (i.e., between aa 765 and
aa 1128) of Avena phytochrome (Jones and Quail,
1986), the region between aa 765 and aa 800
becomes a candidate for this site, given its high level
of conservation relative to remainder of the C-termi-
nal region (Fig. 2).
ACKNOWLEDGEMENTS
We thank Sue Ford and Willow Ealy for their help
in preparing this manuscript and M. Smith,
J. Devereux and F. Blattner for assistance in per-
forming computer analysis of sequence data. R.A.S.
was supported by postdoctoral fellowship DMB-
8508836 from the National Science Foundation.
J.L.L. is the recipient of a National Science
Foundation Graduate Fellowship. This work was
supported by grants to P.H.Q. from the National
Science Foundation (DMB-8302206) and the U.S.
Department of Agriculture, Science, and Education
Adminstration (8 l-CRCR- 1-744-O and 85-CRCR-
1-1578).
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Communicated by J.L. Slightom.
... W e have describ ed a n o th er P E S T sequence for phytochrom e from etio lated o at seedlings at residues 537-547 [18]. This region, co n trary to th e region n ear to the ch ro m o p h o re is n o t conserved, as a PEST sequence, how ever, in phyto ch ro m e from etio lated cucum ber seedlings [19]. It shall be in terestin g to know w h eth er e ith e r one of the indicated P E S T sequences will be fou n d in the so-called "g re e n " or type II p h y to ch ro m e which is found in green plants and does not show any Pfr d estru ctio n [20]. ...
... m ains to be show n w h eth er th ere is lack of p h y to chrom e m icroheterogeneity on the p ro tein level e.g. in cu cu rb ita w here no genetic polym orphism has b een found [19]. ...
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... W e have describ ed a n o th er P E S T sequence for phytochrom e from etio lated o at seedlings at residues 537-547 [18]. This region, co n trary to th e region n ear to the ch ro m o p h o re is n o t conserved, as a PEST sequence, how ever, in phyto ch ro m e from etio lated cucum ber seedlings [19]. It shall be in terestin g to know w h eth er e ith e r one of the indicated P E S T sequences will be fou n d in the so-called "g re e n " or type II p h y to ch ro m e which is found in green plants and does not show any Pfr d estru ctio n [20]. ...
... m ains to be show n w h eth er th ere is lack of p h y to chrom e m icroheterogeneity on the p ro tein level e.g. in cu cu rb ita w here no genetic polym orphism has b een found [19]. ...
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Chapter
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Native phytochrome from Avena sativa L. is homogeneous with a monomeric molecular weight of 124 kdalton; 6-10 kdalton larger than the heterogeneous "120" kdalton preparations previously considered to be undegraded (Vierstra and Quail, 1982, Proc. Natl. Acad. Sci. USA, 79: 5272-5276). The phototransformation difference spectrum (Pr-Pfr) of 124 kdalton phytochrome measured in crude extracts has a minimum in the farred region at 730 nm, the same as that observed in vivo. These spectral properties contrast with those of "120" kdalton phytochrome purified by column immunoaffinity chromatography where the difference minimum is at 724 nm. When 124 kdalton phytochrome is incubated as Pr in crude extracts, the difference minimum shifts progressively to shorter wavelengths (from 730 to 722 nm) concomitant with the proteolytic degradation of the chromoprotein to the mixture of 118 and 114 kdalton species that comprise "120" kdalton phytochrome preparations. These two effects are inhibited in concert by the serine protease inhibitor, phenylmethylsulfonylfluoride, and or maintenance of the phytochrome in the Pfr form. These results provide further evidence that 124 kdalton phytochrome is the native molecule in Avena and indicate that the peptide segments removed by proteolysis of the Pr form are important to the pigment's spectral integrity. The present data thus resolve the previously unsettled question of why the Pfr form of "120" kdalton phytochrome isolated by various procedures from Avena has been found to absorb at shorter wavelengths than that observed in vivo. Previous spectral studies with "120" kdalton phytochrome preparations are open to reexamination.
Article
The relative molecular mass (Mr) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the Mr of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (ΔAr/ΔAfr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different Mr degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-Mr polypeptide segments cleaved by Pr-specific proteolysis are located at the NH2-terminus of the chromoprotein and are adjacent to a 64000-Mr polypeptide that contains the chromophore.
Article
The spectral properties of peptides generated from etiolated-Avana, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 nm, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structureal integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722-724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6-10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 63-kDa species leads to aberrant spectral properties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/114-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule.
Article
In this review areas of currently active research are considered which have demonstrated that a plant's response to light involves changes in the expression of specific genes at the level of RNA. The regulation of gene expression by phytochrome and the UV-sensitive photoreceptor have been studied most extensively at the molecular level, and this review particularly focuses on such studies in higher plants. Some of the observations made on the differences in gene expression between light-grown and dark-grown plants are also included, although the photoreceptor(s) responsible for the differences may not have been ascertained. In some of these cases, phytochrome involvement has been or may be demonstrated in later studies, while in others the observed differences may be a result of the action of other photoreceptors or of multiple light-affected processes. One such process is the development of chloroplasts, a major developmental step triggered by light in angiosperms. In addition, many of the genes whose expression is changed by light and which have been studied at a molecular level encode chloroplast proteins. 156 references.
Article
We present three lines of evidence that etiolated Avena phytochrome with a monomeric molecular mass of 124 kilodaltons (kDa) is a dimer in solution. First, data obtained by utilizing low-speed sedimentation equilibrium centrifugation indicate that, over the full concentration range examined (0.1-0.35 mg/mL), phytochrome behaves as a single, uniform population with a calculated molecular mass of 253 kDa. When 1 M NaCl is present, the population becomes heterogeneous with an average molecular mass of 155 kDa, indicating partial subunit dissociation. Second, when phytochrome over a concentration range of 6-66 μg/mL is covalently cross-linked with glutaraldehyde or one of a family of imido esters, the major product has an apparent molecular mass of approximately 225 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Third, when a sample of phytochrome containing a proportion of cross-linked products is chromatographed on a size-exclusion column under nondenaturing conditions, cross-linked and un-cross-linked molecules comigrate. Data from size-exclusion chromatography (SEC) provide evidence that both the intact dimer and the dissociated subunits have an elongated shape. These data indicate further that dimerization probably involves some ionic bonds and that if disulfide bridging is involved it does not account exclusively for the dimer configuration. We also present evidence that the contact site(s) between the monomers lie(s) within 42 kDa from the carboxyl terminus. First, when phytochrome is digested with trypsin and subjected to SEC under nondenaturing conditions, all peptides containing the carboxyl-terminal 42 kDa migrate as species with molecular masses 2-3 times their monomeric masses calculated from SDS-PAGE. In contrast, tryptic peptides derived from the remaining 82-kDa, ammo-terminal portion behave as globular monomers. Second, glutaraldehyde cross-links the 42-kDa fragment-containing peptides at ratios of glutaraldehyde to phytochrome 2-3 times lower than for those peptides lacking the 42-kDa fragment. The higher efficiency with which these carboxyl-terminal peptides are cross-linked is interpreted to indicate that these domains are adjacent to each other in the native dimer.
Article
Two procedures for the purification of 124-kdalton (kDa) phytochrome from etiolated Avena seedlings are described. These procedures involve combinations of existing protocols but with two key modifications aimed at precluding previously encountered proteolysis: phytochrome is maintained in its far-red absorbing form (Pfr), and the serine-protease inhibitor phenylmethanesulfonyl fluoride is included in all media until proteolysis is no longer a problem. The initial steps in both procedures are identical but are followed in one case by Affi-Gel Blue chromatography and in the other by immunoaffinity chromatography. The phytochrome preparations obtained by either procedure are >95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lack detectable levels of the proteolytically degraded 118- and 114-kDa species that constitute preparations obtained by previous protocols. The spectral properties of 124-kDa phytochrome purified by the Affi-Gel Blue procedure contrast with those of the extensively characterized 118/114-kDa products in several major respects. The purified 124-kDa molecule has a Pfr absorbance maximum at 730 nm, negligible dark reversion with or without sodium dithionite, and enhanced absorbance at 730 nm of the spectrum (predominantly Pfr) obtained after saturating red irradiation relative both to the 673 nm shoulder of this spectrum and to the absorbance maximum of the Pr spectrum at 666 nm. The difference spectrum, with a spectral change ratio (ΔAr/ΔAfr) of 1.07, is indistinguishable from that determined in vivo, indicating retention of the spectral properties of the native molecule through the purification procedure. Immunoaffinity-purified 124-kDa phytochrome on the other hand is spectrally denatured and has been used here only for compositional analyses. The amino acid composition of 124- and 118/114-kDa preparations does not differ significantly on a mole percent basis, but in contrast to published data for the 118/114-kDa species, 124-kDa phytochrome has a blocked NH2 terminus. These data provide further evidence that the proteolytic conversion of 124- to 118/114-kDa phytochrome involves removal of polypeptide segments critical to the structural and spectral integrity of the native molecule and indicate that at least part of this proteolysis involves removal of the NH2-terminal residue(s).
Article
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Article
The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.