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Volume 14 Number 20 1986 Nucleic Acids Research
Comparison of the late HI histone genes of the sea urchins Lytechinus pictus andStrvngelocentrotus purpuratus
James A.Knowles and Geoffrey J.Chiids*
Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY10461, USA
Received 24 July 1986; Revised and Accepted 11 September 1986
ABSTRACTWe have i s o l a t e d and sequenced a gene encoding a l a t e HI h i s tone subtype
from the sea urchin spec ies L_. p i c t u s . The primary s tructure of the l a t e HIsubtype encoded by t h i s gene I s 209 amino ac ids in l eng th , and has a netp o s i t i v e charge of 67. This gene i s present in a s i n g l e copy per haploldgenome and encodes an mRNA of 752 n u c l e o t l d e s . Late HI t ranscr ip t s aredetected in the u n f e r t i l l i e d egg and are most prevalent in gas tru la tingembryos. Comparison of 375 bp of 5' f lanking sequences of the L_. plctua l a t eHI gene and the Hl-gaauna gene of a d i s t a n t l y re la ted sea urchin s p e c i e s , £ .purpuratus, revea l s large blocks of sequences that are i d e n t i c a l between thetwo genes . To determine i f these conserved 5' sequences are present in othermembers of the sea urchin HI gene family, the analogous region of £ .purpuratus Hl-alpha, an ear ly HI gene, was sequenced. The homology betweenthe flanking sequences of the ear ly and l a t e f a m i l i e s was H a l t e d to consensussequences which are found upstream of a l l HI genes . The poss ib le regulatoryimpl icat ions of these f indings are d i scussed .
INTRODUCTION
Sea urchin h i s tones are encoded by several multlgene f a m i l i e s which are
regulated in both a temporal and t i s sue s p e c i f i c fash ion . The two best
characterized f a m i l i e s are those encoding the ear ly and l a t e h i s tone subtypes.
The ear ly h i s tone gene family contains c lus tered sequences encoding the alpha
subtypes of a l l f i v e h i s tone prote ins which are repeated in several hundred
nearly i d e n t i c a l tandem arrays ( 1 - 4 ) . Lytechinus p i c t u s contains three
separate ly maintained tanden arrays of ear ly genes , the A-D, B-C, and E
subfami l i e s , which d i f f e r primarily in t h e i r spacer sequences ( 5 - 7 ) .
Transcripts of these ear ly h i s tone genes are found as stored mRNAs in the
u n f e r t i l i z e d egg, are synthesized during c l eavage , and become most abundant In
the ear ly b ias tula stage of development ( 3 , 4 ) .
The genes encoding the l a t e core h i s tones have been cloned and are
repeated only 8-10 times in the sea urchin genome. Unlike the i r ear ly gene
counterparts , the l a t e core hi stone genes are not arranged In tanden arrays
( 8 - 1 0 ) . The coding regions of the ear ly and l a t e H3 genes of L. p i c tus d i f f e r
©IRL Press Limited, Oxford, England. 8 1 2 1
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by 19Z In nucleotlde sequence (11). Late gene transcripts for the core
histories are also stored In eggs, but are 100-fold less abundant than early
gene transcripts (12). The genes encoding the late core histories are
activated in cleavage stage embryos at the same time as the early hi stone
genes, but late gene transcripts continue to accuoulate up to the onset of
gastrulatlon, long past the the peak of early hi stone oRNA accumulation. At
least part of this accumulation can be accounted for by an Increase in the
relative rate of transcription of the late gene family at about the bias tula
stage (7-14 hrs. post fert i l izat ion) (12).
Recently, a gene encoding the S. purpuratua late HI subtype, Hl-gamma,
has been isolated (13). Unlike Its core hi stone gene counterparts, Hl-gamma
i s not clustered with other late hi stone genes, and i s present In only a
single copy per haplold genome. The temporal pattern of expression of
Hl-garma Is similar to that of the late core histories. Hl-gamma aRNA Is
stored in the unfertilized egg, and Is aost abundant during gastrulatlon.
This report detai ls the cloning and sequencing of a late HI gene from the
distantly related species, L_. plctus. A comparison of the upstream region of
the two late HI genes revealed striking homologies. To ascertain whether
these conserved sequences are common to a l l members of the sea urchin HI class
of genes, the upstream region of the S. purpuratua early HI genes was
sequenced. The upstream region of the early gene shares l i t t l e homo logy with
upstream regions of either of the late genes. The significance of this
observation to a model for the regulation of these genes i s discussed.
MATERIALS AND METHODS
P r e p a r a t i o n of RNA
SNA was prepared from L_. p l c t u s embryos a s p r e v i o u s l y d e s c r i b e d ( 1 2 ) .
Isolation of Genoalc Clones
S_. purpuratus late HI cDKA was subcloned Into rapl8 (13), radiolabeled by
primer extension (12), and used to screen several L. plctus genomic l ibraries
(BamHl and EcoRl) as described (14). Purified phage were grown In liquid
culture and DNA was purified as described (15).
DMA Sequencing
A 3.5 kb Xbal-EcoRI fragment of ALplHl-1 was subcloned into M13mpl8 and a
series of nested deletions was constructed as described (16). To provide
clones to sequence the opposite strand, restriction fragments of the 3.5 kb
Xbal-EcoRI fragment were subloned Into appropriately cut H13 vectors. For ^.
purpuratus early HI, an 800 bp Smal-EcoRI fragment of pSplO2 (17) was cloned
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into H13mpl8. Ml3 clones were sequenced by the dideoxy sequencing technique
(18). DMA and protein sequences were analyzed using the BIONET National
Computer Resource.
Filter Hybridisations
All hybridizations were done in 50Z formamide, 5X SSC, 5X Denhardt's
solution, 50mm sodium phosphate pH 6.8, and 0.1 ng/nl sonicated salmon sperm
DMA (50% formamide hybridization buffer) (15). S_. purpuratus la te HI cDKA
(pJK-1) was hybridized to h. plctus genomic libraries at 37°C in this buffer,
and the f i l t ers were washed at 65°C in 0.5X SSC, 0.1Z SDS, 1Z sodium
pyrophosphate (0.5X wash buffer). Genomic DHA hybridizations were carried out
at 42°C with the addition of 102 dextran sulfate. A nick translated (19)
SspI-EcoRI fragment was used as a probe (figure 1). For northern blots , an
SspI-EcoRI fragsent was subcloned Into pGem2 and the resulting p la said was
linearized with Hind III yielding a 1.2 kb template. The Sp6 probe was made
as described (20) and hybridized to the blot at 58°C In 50Z formamide
hybridization buffer. The blot was then washed at 65°C In 0.5X wash buffer.
Autoradlographlc Intensity was quantltated using a Cambridge Instruments
Quantlnat 920 Image Analysis Systea.
A EH XbXo BS**• ilnlHI-1 Hi JJ iL_XLplHH
BXbXoHSXbEHE XbEH XbXoH B 1 U
XLplHl-2 N^ II I Iff 1 * M I I
°- E HKHI Si SU XbHn SmXo
pLplH-1 M l I I LJLJT _500 bp
Figure 1. Restriction endonuclease maps of DNA segments containing an L .plctus late HI gene. Bars denote hi stone HI coding sequences and arrows thedirection of transcription. Each nap Is aligned such that the l e f t and rightarms of phage DNA are on the l e f t and right of the figure, respectively. Hapswere generated from examination of single and double enzyme digestions ofphage and plasmid DNA. A.) Two overlapping lambda clones containing a late HIgene are shown. ALP1H1-1 was Isolated from an EcoRI generaic library(generously provided by Dr. Eric Davidson) while ALP1H1-2 was Isolated from aBanHl library (8 ) . The map for ALplHl-1 contains only those s i t e s detected byhybridization of pJK-1 to appropriately digested ALplHl-1 DNA. B.) pLplHl-1Is a subclone containing the 3.5 Xho-EcoRI fragment of ALplHl-1 cloned Intothe vector pUC9. The Sspl s i t e was determined from the DNA sequence.Abbreviations used for restriction endonucleases are, B-BamHI, E-EcoRI,H-Hindlll, HII-Hindll, K-Kpnl, S-SacI, Sm-Smal, Ss-Sspl, Su-StuI, Xb-Xbal,Xm-XmnI, Xo-Xhol.
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Hours
Figure 2. Accumulation of late HI mRNA during development. A.) RNA wasprepared from embryos at various times after f er t i l i za t ion . This RNA wasfractionated on a 2X agarose gel containing formaldehyde, blotted tonitrocellulose, and probed with an Sp6 transcript complementary to the late HImRNA. B.) Relative autoradiographic ueuoiLiio vsrc determined from this andlonger and shorter exposures of this blot and then plotted.
RESULTS and DISCUSSION
I go la t Ion of a Late HI gene
A pJK-1 cDNA clone encoding a £ . purpuratus late HI protein (13),
wts used to screen several L_. pIctus genomlc l ibraries . Several highly
homologous clones were Isolated. Restriction endonuclease maps were generated
for two of these clones (figure 1), and each appear to be overlapping DNA
segments encoding the same HI gene. Neither clone Is homologous to any of the
core hie tone genes (data not shown). Therefore, the _L. plctus late HI gene,
like the £ . purpuratus Hl-gamma gene (13), Is not tightly clustered with other
his tone genes. In order to further localize the region homologous to pJK-1
a 3.5 Xho-EcoRI fragment of ALplHl-1 was sub cloned and mapped In more detail
(figure 1).
Temporal Expression of Late HI aRNA
When the HI gene encoded by XLplHl-1 i s used to probe a northern blot
containing L_. plctus RNA from several developmental time points, a single RNA
of about 750 nucleotldes that accumulates In a temporal pattern typical of the
late his tone gene family (13,21-23) i s observed (figure 2) . Like the core
late hlstone mRNA a (12) and S . purpuratus Hl-gamma mRNA (13), the L_. plctus
late HI mRNA Is detected in the egg as well as a l l subsequent developmental
stages. Longer exposures clearly show that the HI mRNA detected by this probe
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Individual*!
am HI
CD
coRI
UJ
s•DCX
individual #3
XEom
coRI
U l
H•DC
X
Individual #4II . ,
XEom
i—i
oUJ
1
C
X
kb
23.1-
9.4-
6 .6-
4.4-
2.3-2.0-
-.1-
Figure 3. Genoalc organization of I,, plctua late HI genes. Sperm DNA fromthree Individuals was digested with the restriction endonucleases Indicated;the digestion products were separated on a 0.6Z agarose gel , blotted tonitrocellulose, and hybridized to late HI as described In Materials andMethods.
Is the same size as late HI (data not shown). Therefore, this gene encodes a
sonatlc HI protein (either Hl-gamma or Hl-beta) that Is also expressed
whenever the embryonic early HI gene Is active.
Genoalc Organization of the Late HI gene
The late HI gene was used to probe genomlc DNA by Southern blotting
(figure 3 ) . One or two bands were detected with restriction endonucleases
that do not cleave within the HI gene, while three or four fragments were
detected when genomlc DHA Is cut with Hind III , (an enzyne that cleaves within
the HI gene). In the case where three bands are detected (Individuals tl and
#4), one of the bands Is twice as Intense as the other two. This result Is
most consistent with there being only a single Hl-gasuna gene. HindiII cleaves
within Hl-gasma (Figure 1) normally yielding two bands of equal Intensity.
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10 20 10 40 SO 60 70TSTTCTCTCA TETTATCTAT CTETTTTTCT SBAAAATATT ATTTTACACA ASATTTCAOC eTTTSTTTTT
SO 90 100 110 120 130 140SSATTTTAAA CT6CCATTGT CAAAOTSTAA aTTTCaAGaT AAATTACTCT CCAATAOAAT AACCATSGTT
1 M 160 170 1S0 1W> 200 210ATTTTTTAAO ATOTCAAOTO AAASATTQAA ASCTCCTCTT TC8TTT0AT8 06TATTTTTTC TCCTTACC6T
USE_U_120 230 240 240 280 270 280
ASCACSAACA ABAAACAAAA ACOATATATT CCAA£A£CTT ACTAA£A£AT CCAAAAA£A£ OATTSCCAAA
200 100 310 320 330 340 350ATACACSOTA CATSTACAAA ACAACCCflCC TCTACACACe CTATASCC8A CTT8ACTeCA TT8TTTTCCC
ISO 370 ISO 380 400 410 420CCACSTCCGC ' n " ' O T T ^ IAIAICTATQ CSATAOCGCS BAAATTSAAC_ATIA£BTTTC TCOCTCACTS
430 440 450 4SSACAACACASC CSSAATCTAT CTCACTCACC ATB TCT SCC SCC AA8 CCA AAA ACC SCA
UET tar All Ala Lyi Pro Lyi Thr All
4S0 4S3 510 925AAS AAB SCC CCT 8CT OCC CCA CCA CAC CCA CCT ACC TCT CAB ATS ETA QTT OCTL y i Lya A H Arg A l l A l l Pro A l l H I . Pro Pro Thr Ssr B i n Uot V i l V . I A H
540 55.5 570 ?»«acr ATC ACC acc CTC AAS BAB COT BET BET TCC TCA AAC CAB sec ATC AAS AAEA l l I I * T h r A l l L « i L y a B l u A r g S l y B l y t a r t a r A a n S i n A l a I l a L y a L y a
800 816 ( 3 0TAC ATC BCT 8CC AAC TAC AAB BTT SAC ATC AAC AAB CAB OCT ACT TTC ATC AABTyr I la A l l A l l Aan Trr Lya Val A m I la Aan Lya Bin Ala Thr Pha I la Lya
S4S 8*0 875 BOOCST BCC CTS AAB 6CT 6flT ETT SCC AAT GET ACC CTC STC CAA ETC AAA SEA AAEArg A l l Lau Lya A H Bly V . I A H A m Bly Thr Lau Val Bin V i l Lya Sly Lya
705 720 718S B A e C C A B T e a A T C T T T C A A e C T C B E C A A a a T C A A a a C T S S C A A S A C C S A S E C Ca i y A l l Sar Ely ta r Pha Lya Lau Sly Lya V . I Lya Ala 8 l y Lya Thr filu A H
750 70S 780 7»5CAB AAS sec car SCT sec sec AAB AAS sec AAB err BCT acc AAS AAS AAS SAASin Lya A l l Arg A l l A H A H Lya Lya Ala Lya Lau Ala A l l Lya Lya Lya Olu
010 825 840 855C A S A A f i e A e A A e A A a S C T C C T A A e A C C A A S S C C A f i e A A B e A e A A A C T A S C C S C Ca m Lya S lu Lya Lya A l l A H Lya Thr Lya A l l Arg Lya e i u Lya Lau A l l A H
870 SSS gooAAeAAcecTecAAAaAASeccaccAAaAAeaTTAAaAAscccacceccAASEccLya Lya Ala Ala Lyi Lya A l l A l t Ly» Lya Vi l Lya Lya Pro AH AH Lya AH
»16 810 945 get)
AABAAaCCAeCTAAEAASSCABCCAAaAAeCCCSCCSCCAASAASaCASCCAABLya Lya Pro A l l L Y . I in AH A l . Lv. I v . p™ A H A I . l y . i , i > i . M . i Y 1
•76 990 1005
AABCCCBCCECTAAeAAaCCABCTAASAASSCCBCCAAaAABCCTSCTBCTAASL»« Prn A l l A l l Lyi Lya Pro A l l Lv« I V . A l l A l l Irn L Y I f ro Ala A l l Lya
1020 1015 1060 \oe>
AAaeCCeCCAABCCASCAAAEAASeCAECCAAEAAECCCeCCaCCAA6AABaCALya Ala A l l Lya Pro A I » m l . i A H A I . L Y . I V . trr, Ala Ala Lya Lya AH
1080 1091 1103 1113 1123 1133SCC AAE AAB TAA ATTCTTASC8 CCACTTEETS TATTSAOCTT TTTCAECTCC ACCCCAACSBAH Lya Lyi .
1141 1U3 1183CTCTTATCAB ACrT-Arry.il ACTTCAAKAA tCAATTr
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However, due to the large amount of genomlc polymorphism (41 sequence
difference) seen between Individual sea urchins (24,8) , the two bands of lower
Intensity l ikely result from polymorphism In the flanking HindiII s i t e s .
Consistent with this Is the observation that each of the three Individuals
contain many bands that differ In s i ze . If the Hl-gamma gene were present In
more than one or two copies per haplold genome, polymorphism would have surely
resulted In the generation of a greater number of hybridizing bands as Is the
case with the late core hlstone genes (8 ,9 ) . The faint bands on the
autoradlogran are oust l ikely due to cross-hybrldlzatlon with an HI gene
encoding a different late subtype.
Nucleotlde Sequence of the L. plctus Late HI Gene
The entire late HI coding region and 400 bp of 5' flanking sequence of
ALplHl-1 was then sequenced (figure 4) . The nucleotlde sequence contains an
open reading frame of 209 ami no acids. Based on the overall similarity of Its
prlaary structure to that of other HI proteins, this HI protein probably has the
tripartite structure (nose, head, and ta l l ) typical of this class of hi stones
(25-26). The N-tennlnus of the protein Is a variable region that Is very basic
and rich in alanine and prollne. The central domain Is the most highly
conserved region of HI proteins and has an anlno acid distribution typical of
most globulin proteins while the C-terminus i s extreaely variable In size and
sequence but Is highly basic. Comparison of the I., plctus late HI protein to
Hl-gamna of S_. purpuratus Indicates that the two proteins are only 75Z
homologous. Although they differ significantly In primary sequence, a plot of
the hydropboblclty of the two proteins Is nearly Identical (figure 5) . The two
late HI proteins therefore contain a large number of conservative amlno acid
changes. Like Hl-gamna (13), the L_. plctus late HI C-terminal ta l l does not
contain serlne and Is composed of repeating amlno acid sequences. The
octapeptlde, AKKAAKKP, i s repeated five t ines, linked together by pentapeptldes
similar to those of £• purpuratus Hl-gamma. The large number of lyslne residues
gives this protein a net positive charge of 67.
Examination of the DKA sequences bordering the open reading frame reveals
several conserved sequences that define the 5' and 3' ends of most hlstone
mRNAs. The 5' end of the mRNA Is probably coincident with bp 401, which l i e s in
the middle of a degenerate form of a conserved cap box sequence (CATTAC) seen in
Figure 4. DMA sequence of the L . plctus late HI gene. The derived amlno acidsequence Is shown below the corresponding nucleotlde sequence. Conserved DNAsequences are underlined and discussed In the text. The mRNA cap s i t e Isdenoted by an arrow.
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x -i .
IBB
A mi no Acid
Figure 5. Comparison of the hydrophoblcltles of the protein encoded by the L .pIctus late HI gene to £ . purpuratus Hl-gamma. Hydrophoblclties werecalculated using the parameters of Kyte and Doollttle (38) and plotted using aTextronlcs graphics plotter.
many sea urchin hi stone genes (27 ,4) . The 3' end of the non-polyadenylated oRNA
Is assumed to be at the adenlne residue at bp 1152, following a highly conserved
region, capable of forming a hairpin loop, observed at the 3' end of many sea
urchin hlstone mRNA a (4) . Therefore, this late HI gene probably encodes a
transcript of 752 nucleotides. Downstream of the mRNA Is another conserved
elenent, CAAGAAAGA, which appears to be necessary for the binding of U7 snRNPs
Involved In the processing of the 3' end of the mRNA (28).
Comparison of the nucleotlde sequence of the L. plctus late HI gene to
the £ . purpuratus Ul-ganma gene (13), reveals that the two genes are 801
homologous In their coding regions. The nucleotlde sequence homology
comparable to the affllno acid sequence conservation reflects a bias In codon
usage In the genes of these two species. When the 5' upstream regions
(to -375) of these two genes are compared they are 77Z homologous over the
entire region, with long sequences of near 1001 homology (figure 6 ) . This Is
quite suprlslng as these two sea urchin species diverged froo a common
ancestor more than 65 million years ago (29). The Hl-speclflc upstream
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* •» • .« • • • • •» « «- 3 7 3 TTTTCGGGAAAACGTTITTAATA--TAAGATATT-GGCGTTrATTTTCGGATTTrAAABT- 3 7 6 TTTTCTGGAAAATATTATT-TTACAaUl6AT-TTCAGCBTTTGTTTTTG6ATTTTAAAGT
MMM MM
S. p. GC-ATTGTCAAAGTGTAA8TTTCGTGeTAAATTTCTCTATGATGAAATAATTATGTATGAL. p. GCCATTGTCAAAGTGTAAGTTTC6AGgrAAATTACTCTCCAATAGAATAACCAT6-GTTA
CTTTTAAGG-TGCCAAGTGGAACATTGAAAGCTCCTCTTGATTT-GAGfiflTATTTT--CATTTTTTAAGATGTCAAGTGAAAGATTGAAAeCrCCTCTTTCGTTTGATGGTATTTTTTCT
CCAGTTTTGATTTAGC-CAA-CAAAAGAGAAAAACAATATTTTCCAACACCTTACTAACAC--G—TTACCGTAGCACGAACAAGAAAGAAAAACGATATATTCCAACACCTTACTAACA
USE II USE I
CATTTAAAAACACGATTGCCAAAATACACACTACGTGCACAAAACAABCGGGCTGTACACCATCCAAAAACACGATTGCCAAAATACACGfiTACATGTACAAAACAAGCGfiGCTGTACAC
GTCCTACGGGCGACCTCACCGTACCGTTTTCCCCCACGTCCGCAAeAA—CGTTATAT—ACCCTATAGGCGACTTGACTGCATTGTTTTCCCCCACGTCCGCAAAAAGGCGATATATCT
ATGCCCGAGAAGCCGCGGAAATCAAACATTACQATTTTTGTTGA +17ATGGG—A-TAG-CGCG6AAATTGAACATTACGTTTCTCGCTCA
RNA START
Figure 6. 5' upstream homology of the L. plctua late HI gene and the £ .purpuratus Hl-ganoa gene. The underlined sequences are discussed In thetext. The top sequence Is that of the £ . purpuratua Hl-gamma gene and thebottom the L . plctus late HI. (*) denote mismatched bases between the twosequences and an arrow denotes the mRNA start s i te for the £ . purpuratus genedetermined by SI nuclease analysis (13).
sequence elements (USE elements) 1 and II (30,31) that are found In the S_.
purpuratus Hl-ganma gene (13) are 100X conserved In the L. plctus late HI
gene. A core sequence of element II (AACAC) Is repeated upstream twice oore,
within a 62 bp conserved region, with the same phasing In both late genes.
Further upstream In a region of high homology between the two genes are three
copies per gene of a 10 nucleotlde consensus sequence, GTGGAAGATT. This
sequence Is s lul lar to the core consensus sequence (GGTGTGGAAAG) derived from
several enhancer elements (32). This Is quite different from what Is known
about 2 non-allel lc pairs of late H3 and H4 genes from _L. plctus. When the
sequences of these genes are compared the coding regions are 96Z homologous,
but the 5* flanking sequences are l ess than 40Z homologous, with the exception
of short DSE homologles (11). The large degree of sequence conservation
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10 20 30 40 SOGGATATTAGG GTSACCATeC AGGGCAAAAT ATTATCTTTT TAGTCSGCAT
80 70 80 90 100ATTAATSCSA AACTATGAAT ATSCAATTTC CCATGAATTT CAA6A6GGGT
110 120 130 140 1S06ACGATCAGG GGTTGATGGT GTTATCGCCA TTTGATTTCA TTTTTTTCTT
100 170 180 190 200CAAATTCATA TAATGATTSA GTACATTATA CGTGGTAGGA GAGGATGAAG
210 220 230 240 2S0AAOAAQAGGQ ATGAGAAGAA GACGGAGAAA GAGAAGAAGG A8AAGGCGGT
260 270 280 2S0 300GGTGGAATAA GASCATGTAT GGATGGAAAA GAAfiGGAGAA AAGTTTCTTT
USE II USE I310 320 330 340 350
AAACAAACAC AAACTQCCAA TCCACTCATC acfiCCaflACC ACCCGGGACT
380 370 380 3«0 400QTCTCCTCCC ACCTACOCAA CAATCCCTTA T^TTGACCQT TGCCSAGCCG
410AT6CTTA.TTC
Figure 7. DNA sequence of the 5' flanking region of the £ . purpuratuaHl-alpha gene. The underlined sequences are discussed In the text. The DNAsequence from bp 345 to bp 410 was determined by Sure» et al (27).
upstream of the late HI genes indicates that some of these sequences oust be
crucial to the functioning of these genes.
To determine whether these striking homologles were a common feature of
sea urchin hi stone HI genes or specific to the late ganes, the 5' upstream
region of an Hl-alpha (early) gene was sequenced (figure 7) . The upstream
sequence element IX of the early gene contains only one copy of the AACAC
sequence, in the middle of a complete USE II . This DSE II Is 521 homologous
to tha consensus sequence of Perry et al (TTTTGAGACTCTAGAAACACAGACTG) (31).
The upstream region of this gene also has an upstream sequence element I that
Is similar to the consensus sequence (61Z) (ATGGGCGGGGT) (31). Both the DSEs
of this gene are 1001 homologous to the shorter consensus sequence of Coles
and Wells (USE I-GGGCGG; USE II-AAACACA) (30). Within the USE I there i s a
potential Spl transcription factor binding s i t e (GGGCGG) (33), which Is not
present in the USE I of the late HI genes. One additional sequence that Is
shared between the early and late HI genes Is a 16-mer (consensus;
CCCACGTNCGCAANAA) centered at -45 (between the TATA box and USE I) that Is 88Z
conserved between the early and la te HI genes. This appears to be a sea
urchin specific sequence. The upstreao sequence of the early HI contains no
other regions of homo logy to the two late HI genes, but does contain two long
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purlne rich regions. The f i r s t of these Is just upstream of USE II and
consists of 19 consecutive purlnes (bp 275-293). Further upstream, there Is a
region that Is 63 nucleotldes In length that contains 95Z purlnes (bp
187-249).
In one model for the differential temporal expression of multlgene
families during developoent, different members of the gene family compete for
Halt ing amounts of shared positive transcription factors (34). As
development proceeds, ce l l division exponentially Increases the number of
genes In the embryo, but the quantity of transcription factor remains constant
or declines. The genes best able to compete for the limiting quantities of
transcription factors are able to form stable transcription complexes and
therefore continue to be expressed later In development. The Xenopus 5S gene
ays ten Is the best documental example of this type of regulation (34).
If one aspect of the early/ late hlstone gene switch entails competition
for common transcription factors (34), then either a greater number of binding
domains (35), or binding domains of higher aff inity for regulatory oolecules
would be predicted to occur within the late gene consensus sequences. The
OSE I and II regions or the 16-oer centered at position -45 are logical
candidates for binding domains of coamon regulatory proteins. Consistent with
this hypothesis, the late HI genes contain three copies of a sequence, AACAC,
In DSE II , whereas the early HI gene contains only one copy of this sequence.
Conpetltlon for a single shared transcription factor goes a long way to
explaining the mechanism of the late gene switch, however, several aspects of
the early to late hlstone gene transition during eabryogenesls are
Inconsistent with a simple nodal where one positive transcription factor
becomes Halt ing during embryogenesls. The nost sal ient observation that Is
Inconsistent with this model Is the a 5-6 fold Increase In the relative rate
of transcription of the late genes between 7 and 14 hours post-fertl l lxatlon
(12). Two gene families competing for a gene specif ic transcription factor,
as In the case of the 5S genes (34), would be either transcribed or s i lent but
the rate of transcription would not be modulated. It Is reasonable to assume
that the late genes contain at least one sequence not present In the early
genes that serves either as a s i t e for a negative regulator that H a l t s the
transcription rate until early bias tula or for an additional late gene
specific positive transcription factor appearing at early bias tula (7-14 hrs.
pos t - fer t i l i sa t ion) . The three repeated copies of the late gene specific
consensus sequence GTGGAAGATT, or any of the other hooologles shared between
the I-. plctus and £ . purpuratuB late HI genes represent candidates for such
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cis-icting sequences. Site directed mutageneais of these conserved DNA
sequences followed by assays In sea urchin In vitro transcription systems (36)
or transient expression In embryos following Injection Into sea urchin eggs
(37) should Identify these els acting regulatory sites.
ACKNOWLEDGEMENTSWe would l i k e to thank E r i c Davidson f o r p r o v i d i n g the EcoRI genomlc
l i b r a r y of L_. p l c t u s DNA and to Ken Krauter f o r h e l p f u l comments. T h i s workwas s u p p o r t e d by g r a n t #GM30333 from the NIH. Jim Knowles I s suppor ted by ap r e d o c t o r a l M.D. /Ph .D . t r a i n i n g g r a n t NIH g r a n t # T32GH7288. Computerr e s o u r c e s p r o v i d e d by t h e BIONET N a t i o n a l Computer Resource f o r M o l e c u l a rB i o l o g y whose funding I s p r o v i d e d by NIH g r a n t f 1U41 RR-01685.
T o whom correspondence should be addressed
REFERENCES1 . Conn, R . H . , Lowry, J . C . , and K e d e s , L.H. ( 1 9 7 6 ) . C e l l 9 , 1 4 7 - 1 6 1 .2 . S c h a f f n e r , W., G r o s s , K . , T e l f o r d , J . , and B l r n s t l e l , M. ( 1 9 7 6 ) . C e l l 8 ,
471-478.3. Kedes, L.H. (1979). Ann. Rev. Blocheo. 45, 537-070.4. Hentschel, C.C. and Blrnstlel , M.L. (1981). Cell 25, 301-313.5. Cohn, R.H. and Kedes, L.H. (1979). Cell 18, 843-853.6. Cohn, R.H. and Kedes, L.H. (1979). Cell 18, 855-864.7. Holt, C.A. and Chllds, G. (1984). Nucleic Acids Res. 12, 6455-6471.8. Chllds, G., Nocente-McGrath, C., Lelber, T., Holt C , and Knowles, J.
(1982). Cell 31, 383-393.9. Maxson, R., Mohun, T. , Gromeiano, G., Chllds, G., Kedes, L. (1983).
Nature 301, 120-125.10. Kaumeyer, J.F. and Welnberg, E.S. (1986). Nucleic Acids Res. 14, 4557-
4576.11. Roberts, S.B., Weisser, K.E., and Chllds, G. (1984). J. Mol. Blol. 174,
647-662.12. Knowles, J.A. and Childs, G. (1984). Proc. Nat. Acad. Scl. 81,
2411-241513. Knowles, J.A., Lai, Z.C. and Chllds, G. (1986). Mol. & Cell B lo l . ,
submitted.14. Benton, W.D. and Davis, R.W. (1977). Science 196, 180-183.15. Maniatls, T., Fritsch, E.F. and Sambrook, J. (1982). "Molecular Cloning:
A Laboratory Manual." Cold Spring Harbor Laboratory, Cold SpringHarbor, New York.
16. Dale, R.M.K., McClure, B.A., and Houchins, J.P. (1985). Plasmld 5,31-40.
17. Sures, I . , Lowery, J . , and Kedes, L.H. (1978). Cell 15, 1033-1044.18. Sanger, F.S. , Nlcklen, S . , and Coulson, A.R. (1977). Proc. Nat. Acad.
Scl. 74, 5463-5467.19. Rigby, C.W., Dleckaann, M., Rhodes, C , and Berg, P. (1977). J. Mol.
Biol. 113, 237-251.20. Melton, D.A., Krleg, P.A., Rebagllatl, M.R., Maniatls, T., Zlnn, K., and
Green, M.R. (1984). Nucleic Acids Res. 12, 7035-7056.21. Rudernan, J.V. and Gross, P.R. (1974). Dev. Blol. 36, 286-298.22. Cohen, L.H., Newrock, K.M. and Zweldler, A. (1975). Science 190, 994-997.23. Chllds, G., Maxson, R. and Kedes, L.H. (1979). Dev. Biol. 73, 153-173.24. Britten, R., Etton, A., and Davidson, E. (1978). Cell 15, 1175-1186.
8132
Downloaded from https://academic.oup.com/nar/article-abstract/14/20/8121/1081298by gueston 06 April 2018
Nucleic Acids Research
25. Bustln, M. and Cole, R.D. (1968). J. Blol. Chem. 243, 4500-4505.26. Hartnan, P.G., Chapman, T.M., and Bradbury, E.M. (1977). Eur. J. Blochem.
77, 45-51.27. Sures, I . , Levy, S. , and (Cedes, L.H. (1980). Proc. Nat. Acad. Set. 77,
1215-1269.28. Strub, K., Gall l , G., Bussllnger, M. , and Birnetiel , H.L. (1984). EMBO
Journal 3, 2801-2807.29. Snlth, A.B. (1981). Paleontology 24, 779-801.30. Coles, L.S. and Wells, J.R.E. (1985). Nucleic Acids Res. 13, 585-594.31. Perry, M., Thomson, G.H. and Roeder, R.G. (1985). J. Mol. Biol. 185,
479-499.32. Khoury, G., and Gruss, P. (1983). Cell 33, 313-314.33. Dynan, W.S. and Tjlan, R. (1985). Nature 316, 774-778.34. Brown, D.D. (1984). Cell 37, 359-368.35. Reeder, R.H. and Roan, J.A. (1984). 38, 39-44.36. Morris, G.F., Price, D.H. and Marxluff, W.F. (1986). Proc. Nat. Acad.
S c l . 83, 3674-3678.37. McMahon, A.P., Novak, R.T., Britten, R.J. and Davidson, G.S. (1984).
Proc. Nat. Acad. Scl. 81, 7490-7494.38. Kyte, J. and Dool l t t le , R.F. (1982). J. Mol. Blol. 157, 105-132.
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