Volume 14 Numb** 2i 1986
Nucleic Acids Research
Conserved sequence motifs upstream from the co-ordinately expressed vfteUogenin and
apoVLDLII genes of chicken
Fred van het Schip, Rein Strijlcer, John Samallo, Max Grubcr and Geert AB
Department of Biochemistry, Groningen University, Nijenborgh 16, 9747 AG Groningen, The
Netherlands
Received 15 July 1986; Revised and Accepted 30 September 1986
ABSTRACT
The v i t e l l o g e n i n and apoVLDLII y o l k p r o t e i n genes of chicken are
transcribed 1n the l i v e r upon estrogenization. To get Information on putat i v e r e g u l a t o r y elements, we compared more than 2 kb of t h e i r 5' f l a n k i n g
DMA sequences. Connon sequence m o t i f s were found 1n regions, e x h i b i t i n g
estrogen-Induced changes 1n chromatin structure. Stretches of alternating
pyrinidines and purines of about 30-nucleot1des long are present at roughly
s i m i l a r positions. A d i s t i n c t box of sequence homology i n the chicken genes
also appears to be present at a s i m i l a r p o s i t i o n 1n f r o n t of the v i t e l l o genin genes of Xenopus laevis, but 1s absent from the estrogen-responsive
egg-wh1te protein genes expressed i n the oviduct. In f r o n t of the v i t e l l o genin (position -595) and the VLDLII gene (position -548), a DNA element of
about 300 base-pairs was found, which possesses structural characteristics
of a mobile genetic element and bears homology to the t r a n s p o s o n - l i k e V1
element of Xenopus laevis.
INTRODUCTION
The induction by estrogen of yolk p r o t e i n synthesis 1n the l i v e r of
oviparous vertebrates provides an attractive model for the study of hormoneregulated and tissue-specific gene expression (1-4). The vitellogenin (Vtg)
and apo Very Low Density Lipoproteinll (VLDLII) genes encode the major yolk
protein precursors synthesized In chicken l i v e r .
Expression of these genes
appears to be similarly regulated 1n many respects. Both genes are normally
t r a n s c r i b e d only 1n the l i v e r of l a y i n g hens, but can be a c t i v a t e d in the
l i v e r s of roosters, Immature chicks or even 11-days old embryos by injection
of estradiol (5-8). Induction of transcription Is very rapid and completely
r e v e r s i b l e . Transcripts from the genes appear w i t h i n a few hours a f t e r
e s t r a d i o l a d m i n i s t r a t i o n and r a p i d l y decline to undetectably low levels
after hormone withdrawal (6,9). In addition, preferential stabilization of
Vtg and VLDLII mRNAs by estrogen has been observed (9).
Estrogen-induced
expression
© IR L Press Limited, Oxford, England.
of the Vtg and VLDLII genes In l i v e r is as8669
Nucleic Acids Research
sod a ted with structural changes In the chromatin encompassing these genes.
Nuclease-hypersensitive sites, which are correlated with expression, appear
In equivalent regions In front of both genes shortly after estrogen Induct i o n (10-12).
These common characteristics suggest that the genes might share similar
regulatory sequence elements. The molecular organization and sequence of the
5'-end regions of the VLDLII (13,14) and Vtg (11,15,16) genes have been
described e a r l i e r . In the present paper we compare more than 2 kb of 5'
flanking DNA of both genes. These sequences and their homologies are discussed and compared to regulatory elements I d e n t i f i e d 1n other systems.
MATERIALS AND METHODS
DNA cloning and sequencing
B g l l l - f r a g m e n t s of 2.0- and 3.0-kb, covering the VLDLII 5' gene r e g i o n ,
were I s o l a t e d from X Charon4A clone 12 (see Ref.13 and Refs. t h e r e i n ) and
subcloned In pBR322. Sau3AI fragments of these subclones were cloned 1n
phage M13mp9 (17) and sequenced according to the chain-termination method
(18). Sequences 1n the 2.0-kb B g l l l fragment were p a r t i a l l y determined by
the sequencing s t r a t e g y of Hong (19), using randomly generated d e l e t i o n
mutants.
EcoRI-fragments of 0.6, 1.36, and 0.54 kb, covering the 5'-end region of
the Vtg gene, were I s o l a t e d from X Charon4A clone 22 (20). A 3.6-kb BamHI
fragment, p a r t i a l l y overlapping these fragments , was subcloned 1n pBR322
and digested w i t h Sau3AI and P s t I ; the same holds f o r the 1.36-kb EcoRI
fragment. Subsequently, a l l fragments were cloned Into the M13 derivatives
mp8 and mp9 and sequenced. To determine sequences from both strands, short
stretches of DNA were occasionally sequenced by the chemical modification
procedure of Haxam and G i l b e r t (21) a f t e r 3 ' - or 5 ' - e n d - l a b e l l 1 n g of the
appropriate fragments (see F1gs. 1 and 2).
DNA sequence analysis
The nucleotide sequences were analyzed by using a two-dimensional dotmatrix program prepared by Dr. P. Terpstra 1n our laboratory as well as the
program NAQ of the Protein I d e n t i f i c a t i o n Resource system (22).
RESULTS AND DISCUSSION
We have sequenced and compared 5 ' - f l a n k 1 n g DNA of the chicken v i t e l l o genin gene and the VLDLII gene, spanning the regions from p o s i t i o n s -2030
8670
Nucleic Acids Research
Yl
-2500
gaattctact
tggagacatg
tagtggatcc
atctcttgtt
cactgacaca
tttactacga
gaggggctgc
tgactgcaaa
ccgagtggat
ctagcaacta
tatgggtacc
ttttgtgtga
tgtgctcttg
gttcattctc
ctagaacagg
catcagttca
cctcttgcct
ttacagaatg
ccgtgatttc
atacatgaat
gagtatagtc
aacataacct
agtagcacat
cagaaaaagg
aaagaggagt
taaaagtctc
-2000
acttgtgtgc
cttacttcct
aaaaactgat
tgctgttcct
gtactcatat
tacataacat
ccgtgtattg
tccctgcaac
ttgaagccca
ctgaaatagc
ctgggaacag
tttgaaggcc
ttcttgttga
ttagttcact
cgattttctg
gctacttggc
tactgctgag
cttatgaaac
aataaataca
agcaaaggtt
aatgtagatt
gggcaaaacc
caattttaaa
ttttttatca
gtttacataa
accatgcctg
ctttcttttt
taactgcccc
ccttgaaaga
atcaaagctg
tcctttattc
ataagtttgc
ttgtgaatag
aacgacatag
gatgaaatcc
aaacgtactg
aaataaagtg
acaatctatt
tgcaactgcc
ctttgtttat
aatgtgttct
tgcattttgt
aatctctgca
ctttgttcat
tatgttccat
tatctggatt
atactgattg
agtctcatct
tttattgtaa
gagatgccaa
actgataaaa
gcagagccct
-1500
tcttttttct
cccacgtcta
agcattcaaa
caatttagtt
aagtttttgt
tatgaagaaa
tttatcaact
tcttctgatg
agttgaaaaa
cattgtggtc
actgaagagc
gcactgctgg
acccagcttg
ctcctatttc
tgtgttatca
atttggttct
ggttttaatt
ttaaaaatat
atattgtttc
agcctctggc
ctgattgggt
gtggcaggac
atgccgtagt
ggtattattt
aacttgagaa
—•—r
-1000
tttttttttt
atctggaata
ttgtttgata
aaagcatcag
tagtagcctt
tggtcaacta
gaggcacaga
tgaattctgt
gtcaatgcct
attaaaacac
cagtatgtca
aagttgcacc
ctttggagga
tgagggtacc
atataaatca
gtaggaaatg
gttcacattt
tcctggtcag
tcagtagcct
ctgcaggaat
tattatcagc
catgtaccag
agaagtgttt
gattttcttt
ttcagcagaa
attttggagt
aaatagatga
gtttatttat
tccatggtgt
ggatttcttt
caaaaaaata
ttacacttgt
agcagttccc
gtgggactga
tttgccacaa
tagattttga
tccaaaagca
ccaacttttt
tgatgagctg
cagttagtga
caaaagttct
tgctcccatt
cgtgaccgga
cttaaatcat
ggccataaac
tagataacaa
cagccagccg
tactgtgata
attcgccgtg
aacagccacg
taccatgaag
taaagaatga
tatctgttgc
atgctatgtt
tatttgtgca
aaattagtta
ctgcttgagc
aaaacagagg
aaatcc^gaa
aacactcttg
attagaatat
tcctccaatg
tgtagggttt
tttccacact
tgaagttggc
agctgacctg
tactttggaa
gctgaaagaa
gtgcgttggt
caaagctgag
cttgggtcag
tgacccaatc
cattgaaact
aagagaattt
tgttcctgaa
cttttgtggg
aagggagatg
gatatgtctg
ttacttttca
aggcttaaaa
aaaatgtagg
ataaattggt
taggagaact
agagcagttg
tggtgagtcc
aactgaatgt
cagaagaccg
acaggaaccc
cctgacattt
tgcaagcctg
cacttctatc
gataaaatat
cacattgatc
gcacatatga
ggaagaggga
gtgccaggtc
taggaaagca
tctggtcaat
atgattgcaa
cattcttcca
-1951
-1871
-1791
-1711
-1631
-1551
-1471
-1391
-1311
-1231
-1151
-1071
- 991
- 911
- 831
- 751
- 671
- 591
- 511
- 431
- 351
- 271
- 191
- Ill
- 31
1
Figure 1 Nucleotide sequence of 2030 bp upstream of the Vtg gene.
The restriction map and sequence strategy are outlined above the sequence.
Sequences determined by the chemical modification procedure are Indicated by
• (5'-end-labeled fragments) and o (3'-end-labeled fragments). The numbering
Indicates positions relative to the cap site.
and -2594 to the transcription start sites (+1) of the respective genes
(Figs. 1 and 2).
Our data extend and correct sequences published by us and others (11,1316). By sequencing both strands, we can decide unambiguously between minor
conflicts 1n the earlier data. One major discrepancy between the VLDLII
sequence we report here, and that published by Hache £ t ^ 1 ^ (14), deserves
further comment. Upstream from the Haelll site at position -254 their
sequence completely deviates from ours. This Is caused by their positioning
at -254 of a Bglll-Haelll fragment which In our sequence lies much further
upstream, viz. -2594 to -2363. Since we have read the sequence through the
8671
Nucleic Acids Research
=
Hi
-2500
-2000
<
gatctggtcc
tatgccttgc
gctataaatg
ctttttaaaa
ttctaggttg
aagaaataaa
cacctaagaa
atgccatgtg
attaaagaac
tgccattcca
gaaagtgctg
gacaaaagct
ctcaggcagc
gacacttgag
gtgccttgtg
gaatttataa
tgtttgcacg
atctgtcttt
caagacagaa
tgtgacctca
acaatacaca
agagagtcca
tgttttgttg
ccatattgca
ggctctcgga
gttccctagg
agcctgcgca
ttggttaata
ggtcggcttt
tttggtcagt
cacgtctcct
tccatcctgc
ttctgcataa
acaaaccact
aactgcaagc
aaagcctttg
gagagagtaa
tactttactc
tatgatctca
tgaattttgt
gaacagataa
tatgtatttt
ggaatgaagg
aaaggccctg
accatagcac
acagcagttt
gttatattaa
caggtcttct
ttaaaaatga
agaagtggaa
ttttgcttcc
gataccttta
gttgtatcac
aagctccatg
tctgttagtt
gatctcacat
aattttctgc
gattgagcta
cagtctgggt
aaaaggcttg
catttgttgg
caacatgagc
ttatgaaagg
gtgctcaggt
tgagcagaat
atgccagtgt
-1500
1
gtggttcata
agacagaaaa
caatggcact
attttgcagc
atttttaact
tttgtgagtt
gtatgaaagt
ggagatccac
ttaatttgag
acaaggactg
ttgctatctg
aaacctcttc
aaaacacacc
attcaccccc
gaacgttatc
gagaacttaa
ctgaaaattc
attgggattt
ataaatactt
tgctgcactc
aagaaaagtc
gaaattcatg
tgtaggtttc
ctcctnyttca
tgattgagat
agaagttatg
gctacgggga
aaacagaggg
cttcccacag
ggcctctatg
cagactgacc
ttgcttgcaa
ctcagatgag
-1000
»
aagcactgat
gtaggaagga
ggtcaatacc
tgaaagactg
ttttagccat
attttagact
gcctagatag
gctgctcatg
taaaaaaggg
ctttttgtgc
tggggctgga
agatatacat
cttaatatgg
acaggtaatg
tcagctgtaa
aactcttgtt
tctgttctga
ttttggatca
catcgagaca
actgatttgg
ttctagaaaa
tcagtgactt
atgtatgcct
atggttttat
tgagctacat
cttcccctgt
aaaatgaact
tgaactgtga
gaacagtggg
acatggttgc
ttccattacc
aagagcaaat
catc
cagtggtctg
aatagactac
acgtagactt
cacagtgaaa
tcciaaaagc
tttgtttctt
ccactggagt
tcagctgctt
cttgcaatat
aatcattgat
ttcatgatag
attactacgt
ccagagactg
actgcttatt
aagcaggcag
gttcccgtgt
cttctagaag
aacgttcttc
gtggaaaaac
ctgccatatg
acgtgttggg
agtaggtggc
catgcctgca
gtgtgagata
tccacagcat
gagatcttgg
gtgagagtac
atgagcataa
agcaaaaatc
ctgaaaatgt
aaatccgaac
ggggaaacaa
<
-500
'
tgaaataatt
actttgaacg
tatttaacag
gaacagtagg
tgatgtttgt
cctgtcgtgc
tatctgtgtt
tccacctcag
ctggctttct
ttgggcttgg
cactgggtaa
agggcagcta
ccatctgttg
gctatttctg
taaatctgta
gttcttccta
taaccttgtt
atgtttcagg
tgggaggcca
tgtaaaaggg
ctataaataa
taatactact
ccttttcctc
ctgatggttg
ggcactacag
caagtggctg
Cgtatgtata
tagacacaaa
agtccagctg
agggggctca
aacaggtcca
agcaggacct
aaaaaggaaa
cttatttctg
tgagattaca
aaatagttga
gtgtcaccca
aggtgaagga
aaaataaaga
accaaagatt
tgcttatttg
caattgcctg
agttagacaa
aattcccttt
acctgattta
ttaatttgaa
gagtctcctt
gaccataaat
agacattagt
gggctttttc
tgggcagccc
taatgcttac
agatactggt
ttatgtcact
agctaatgcc
accttactga
atgctttctg
agatttacac
catatacctg
taatttgcag
tagctacagt
gtgacccagg
gagtcctaca
ttgacccctc
tctatatata
tttttaatta
aggcctttga
gcttcattct
tttgtgatga
atatacctta
ataggtgact
cataagaaag
cagagcactc
attggcagta
acttagcagg
gttttactac
gaagagagat
tgcctgtaat
agtgattgta
tgctttttag
aataattttc
tttgtttttc
tctttaatga
tgtttggttt
tgagcttttc
gacataagat
atttgttgct
agacccaatg
actgggcaca
ccattttgtc
tgtgcaccag
ctgtcaaagt
taatgtggaa
agctgccttc
cagcttcttg
actatattag
^2515
-2435
-2355
-2275
-2195
-2115
-2035
-1955
-1875
-17 95
-1715
-1635
-1555
-1475
-1395
-1315
-1235
-1155
-1075
- 995
- 915
- 835
- 755
- 675
- 595
- 515
- 435
- 355
- 275
- 195
- 115
- 35
1
Figure 2 Nucleotide sequence of 2594 bp upstream of the VLDLII gene. The
r e s t r i c t i o n map and sequence strategy are o u t l i n e d above the sequence.
Sequences determined by the chemical modification procedure are Indicated by
• (5'-end-labeled fragments) and o (3'-end-1abeled fragments). The numbering
Indicates positions relative to the cap site.
H a e l l l s i t e at -254, we I n f e r that our arrangement Is the c o r r e c t one.
Moreover, our sequence and arrangement agrees with the r e s t r i c t i o n map of
the genoraic ONA determined by the other group (23) and ourselves (24). A
cloning a r t i f a c t Is therefore excluded. As a consequence, some of the homology elements noted by Burch (11) are affected: Element 2, constituting an
Imperfect Inverted repeat of element 1 (position -200), was I n i t i a l l y pos1-
8672
Nucleic Acids Research
.1000 .900
.800
.700
.600
.500
_<00
.380
.200
J00
1 (cop)
via.
repeots
1-»
enhancer ewes
i-,
v
- — 2 9 4 bp
"tronsposon"
«-i
v
Py/Pu
"20bp"
VLOLD
hss
»
repeats
enhancer cores
»
2-» 2-»
w
i«
276 bp b H
"Ironsposon"
p w,H
/
Py/Pu
(
»
?
"20 bp"
F i g u r e 3 Common s t r u c t u r a l f e a t u r e s 1n the 51 f l a n k i n g regions of the Vtg
and VLDLII gene. Nuclease-hypersens1t1ve s i t e s (hss) are given by black
t r i a n g l e s ( • ) . Open t r i a n g l e s ( V ) I n d i c a t e p o s i t i o n s of sequences r e sembling enhancer core elements: 5'-CTTTCCTAGA-3' (at Vtg -282),
5'-ACTGGTTTTG-3' (at Vtg -337), 5'-GTGGAAGG-3' ( a t VLDL -199),5'-GTGGGAAG-3'
(at VLDL -334), 5'-GTTGAAAG-3' (at VLDL -348) a l l 1n the lower strand and
5'-GTGGAATT-3' (at VLDL -280) 1n the upper strand. Arrows Indicate repeated
sequences: 5'-GTTTTTTATCAG-3' ( a t Vtg - 8 9 , -181 and -393); 5'-TGAACTGTGA-3'
(at VLDL -404 and -480). Py/Pu, °20 bp° and "transposon" refer to sequence
elements discussed 1n the text.
tioned around -290 but Is actually located about 2 kb further upstream.
Common structural elements In the regions exhibiting estradiol-induced
changes In chromatin structure
From studies on the chromatin structure of genes I t has emerged that the
upstream regions proximal to active genes, or genes which can be activated
1n the tissue studied, often contain sites which are hypersensitive to
DNAsel (25-27). These sites are believed to mark nucleosorae-free regions
Involved In the control of gene expression (25-27). The regions 5' to the
Vtg gene (10,11) and 5' to the VLDLII gene (12) contain several nucleasehypersensitive sites which are Induced by estradiol and only occur In liver.
The Vtg gene 1s marked by six sites, five of which have already been reported (11) and the sixth one recently been observed In our laboratory (K.Kok
£ t iU, to be published) , a l l mapping within the f i r s t 900 base-pairs
proximal to the cap s i t e . The four sites found for the VLDLII gene map
within the 500 nucleotide sequence preceding the transcription I n i t i a t i o n
site (12). In a search for common sequences that might have a bearing on the
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Nucleic Acids Research
GRE--
VLDLII
-2332
TTTG(ftGCTGAAAGA]CTGCACA6 fGAAAGAACA 3TAGGAAATAGTTG
VITELL06ENIN
TTCCTGGTCAGCGTGACCG<£GC fGAAAGAt\CA :ATTGATCCCGTGA
-631
1
ER-blndlng oito- -
J
Figure 4 Putative estrogen-receptor binding sites within the Vtg and VLDLII
51 flanking regions. Homologous decanucleotides are boxed similarly. Direct
and Inverted repeats are Indicated by arrows. The putative estrogen-receptor
(ER) binding site 1s Indicated. A sequence showing 82% (VLDLII) and 94%
(Vtg) horaology to the consensus sequence of the glucocort1co1d-respons1ve
element (GRE) 1s Indicated.
special chromatin organization of these regions, we have analyzed and compared their nucleotide sequences.
In front of the Vtg as well as the VLDLII gene we find about 30-nucleotides long stretches of alternating pyrimidines and purines at roughly
similar positions viz. -533 and -468, respectively (F1g. 3). Alternating
pyrimidine/purine sequences may promote the Z-conformat1on of DNA (28) which
prevents binding of nucleosomes (29). Such sequences have a potentiating
a c t i v i t y on transcription 1n which capacity they are found 1n the SV40
enhancer region (30), which 1s essentially nucleosome-free (31). Horeover, a
9-bp alternating pyrimidine/purine element has been found to be essential
for transcription of the hunan metallothionein gene (32).
Ch
Ch
XI
XI
XI
XI
Vtg
VLDLII
Al
A2
Bl
B2
Consensus:
T TTAC
275 «- T T T . C
98* T T T G C
107 - T T T A C
202 -• T T T . G
203 - T T T . G
99 -
TTT
C
• G
A A A C T G AT A
A AA
A A A C T G AC C
A AT
A A C CT G A T A
C AA
A A C CT G AT A
C AG
A A A GT G AC C
A G G
A A A G T G AC G
A G G
AA
T A
AC
T G A
CG
C C
A AG
C G A
Figure 5 A sequence element conserved I n yolk protein genes. Numbers r e f e r
t o the 51 n u c l e o t i d e of the sequences shown. The d i r e c t i o n of the a r r o w s
I n d i c a t e whether the sequence I s I n t h e upper (-») or i n the l o w e r ( « - )
s t r a n d . N u c l e o t i d e s I n v a r i a n t i n a l l s i x genes are boxed. Ch: c h i c k e n . X I :
Xenopus l a e v i s ; sequences were taken from r e f . 41.
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Nucleic Acids Research
In addition to the alternating pyrimidine/purine sequences, the Vtg and
VLDLII 5'-flank1ng regions contain other motifs which are reminiscent of
enhancers. F i r s t l y , four elements occur In the -190 to -350 region of the
VLDLII gene which, 7 out of 8 nucleotides , match the SV40 enhancer core
sequence (33). Similarly, the Vtg gene 1s flanked by two segments matching
the IgCjj (34) or IgCk (35,36) enhancer core elements by 9 out of 10
positions. Secondly , multiple repeated sequences are present; two copies of
a 10-bp repeat in front of the VLDLII gene and three (Im)perfect copies of a
12-bp repeat 1n front of the Vtg gene are most conspicuous. All elements
discussed above are depicted in F1g. 3. The presence of several structural
features which are attributed to potential enhancers 1n the regions of
nuclease-hypersensitive sites may f i t Into the modular structure proposed
for enhancers (for a review see Ref. 37). In this context, the elements
found In front of the Vtg and VLDLII genes might have a role, although
perhaps not an essential one , as modulators of a larger enhancer region
regulating transcription.
Distribution of putative estrogen receptor binding sites within the
viteliogenin and VLDLII 5' flanking regions
The region around -600 of the Vtg gene, containing the estrogen-dependent
hypomethylated Hpall site (38,39), has been Implicated 1n the estrogen
induction of the gene (40). In vitro DNAsel protection experiments have
Indicated the sequence running from -621 to -597 as a possible target site
for the estrogen-receptor (see also Fig. 4). This sequence segment overlaps
the symmetrical element -GGTCANNNTGACC- which has also been found In front
of the chicken VLDLII gene as well as In front of the four Xenopus laevis
Vtg genes (41), although positions and copy number differ even within the
Xenopus Vtg gene family. Moreover, at position -587 to -604 1n the lower
strand of the chicken Vtg gene, a sequence resembling the glucocorticoid
response element Is located (42).
In view of these data, we screened the DMA flanking the VLDLII gene for
s i m i l a r i t i e s to the -0.6 kb Vtg gene region. When the sequence of the
proposed estrogen-receptor binding site was considered, only sequences of
limited homology (624) were found, viz. at positions -1629 and -1452.
However when we searched for subsets of the sequence more striking similarities were noticed. Two different decanucleotides, which 1n Vtg overlap and
form part of the receptor binding s i t e , are also found in front of the
VLDLII gene.viz. at -2.3 kb where they are 8 bp apart (see Fig. 4). In both
genes these sequences are adjoined by Inverted repeats. Interestingly, a
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Nucleic Acids Research
sequence resembling the glucocorticoid-responsive element Is also present In
the -2.3 kb VLDLII gene region In the same configuration as 1n the Vtg gene
(see F i g . 4).
A s i m i l a r regulatory role of these sequences Is not excluded by t h e i r
vastly different distances relative to their genes. Steroid-responsive elements generally have not been found at fixed positions In front of genes and
have been located sometimes as far as 2.6 kb as observed f o r the r a b b i t
uteroglobin gene (43).
A sequence element conserved within the 5' flanking region of genes
controlled by estradiol 1n l i v e r
Comparative analysis of the Vtg and VLDLII gene reveals a 20- nucleotide
block of sequence horaology, lying In opposite orientation, within the -100
to -300 region (F1g. 5). We found a s i m i l a r sequence 1n the Xenopus laevis
vitellogenin genes (41) at corresponding p o s i t i o n s . The derived consensus
sequence contains a I0-nucleot1de motif that 1s perfectly conserved within
the six yolk protein genes (see F1g. 5). However, this motif 1s absent from
the egg-wh1te protein genes, which are also estrogen-responsive but only expressed In the o v i d u c t , v i z . the ovalbumin, ovalbumin-Hnked X and Y,
conalbumin (44), ovomucoid and the lysozyme gene, (Genbank, release 26.0).
LENGTH
294 kp
Ch«tg
ATTTTC [TJA) ATGTSTTCTT6TGT
_^
-591
CTGAAAGAACACAT ITGAI TCCCCTGATTTCAATAAA
276
ChtLOLII
-827
^_
GTTTTC i t t l S GATCTCACATTGTA
_^
-544
TCCCCTGTGA6ATC ITTGI GCAAGT66CTGAGATTTA
448
X1A1/B1
CACAAC [TTAj G6C6CACATTTACT
ACrAAAIfiI£CCCC fcAA) ATGTGTGTGtATTATATT
189
IlBJLf
-727
CTACAC ICtAl GGGGCACATTCACQ
«
-S31
AcfAAATGTGCCCC fcTA| ATGTAGCA6ATTTTCTTC
CTACAC (£TA) tCCGCACATATACT
AGTAAATGTGCCCC ETAI ATGTAGCAGATTTTCTTC
X1A2
CAACAC Rf*l CCGGCACATTTACT
TCTAAATCTGCCCC WTAIATGATATAGATT6tATTT
11A1
CCGCAT |TGT] TCSGCAAAUTACC
GGCAAATTTGCCCAG^ST] TGATAAATGAGCCCCACT
112
-692
^__
Figure 6 Transposon-Uke elements 1n chicken and Xenopus laevis yolk protein
genes. The bounding Inverted repeats are Indicated by arrows above the
sequence. The flanking direct repeats are boxed. The length given for each
element Includes the Inverted repeat. Positions, where known exactly, are
given relative to the cap sites. Short sequence motifs that are frequently
present 1n regulatory sequences are underlined. The Xenopus sequences were
taken frora Schubiger et a]^ (45).
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Nucleic Acids Research
Nor 1s the element found 1n the liver-specific, but hormone-Independent
albumin gene. This sequence element common to yolk protein genes forms the
core of a block of homology between the chicken and Xenopus Al and A2 Vtg
genes noticed earlier (41). In that study, the homology between the chicken
gene element and those of the Xenopus B genes ( at position -90) was rather
poor. More consistent results are obtained with a sequence track around -200
of the B genes (see Fig. 5),y1eld1ng better homology to the chicken genes
and to all four Xenopus genes as well.
In the chicken Vtg and VLDLII gene, this conserved element coincides
with an expression-1Inked nuclease-hypersensitive site at position -120 and
-270, respectively, suggesting a regulatory function. Since the conserved
element flanks estrogen-controlled genes expressed 1n liver only.1t might
Influence their expression 1n a tissue-specific manner.
A transposon-11ke DNA element within the upstream regions of the
vitellogenin and VLDLII gene
When we surveyed the 5' flanking regions of the Vtg and VLDLII gene our
attention was drawn to a common structural feature at similar positions. I t
1s a sequence track of about 300 basepairs bounded by perfect Inverted
Vtg
-900
CGATTTTcEfiS-ATGTGTTCTTGTGTTATCAATATAAATCACAGTTAGTGATGAAGTTGGCTGCAAGCCTGCATCAGT*****
** *
*** *
**
* * **
**
**********
*
TT6TTTT6ffT6TGATCTCACATTGT
A6GTTTCAT6TAT6CCTCATGCCT6CACCTTTT
VLDL -835
l
2
--TCAGCTACTTGGCTGCATTTTGTATTTGGTTCTGTAGGAAATGCA'MAGTTCTAGCTGACCTGCAC
******* *
CCTCAGCTAATGC
***** **
*
* *
CATTT-GTTGCTCCATAT
******
+***
**
****
TTCTATCCCTCTTG--*
** ***
*
TGCAAATTTTCTGCCT--CCTGTTCAATGGTTTTATGTGTGAGATAC
CCTTACTGCTGAGAATCTCTGCAGGTTTTAATTGTTCACATTTTGCTCCCATTTACTTTGGAAGATAAAATATTTACAGA
******** **
* **
**** *
• **
*
* * *
*****
TGATGGTTGACCTTACTGAAGACCCAATGGGCTCTCGGAGATTGAGCTATGATTGAGATTGAGCTAEATTCTABAGCATGGCACTACAG-
ATGCTTATGAAACCTTT---GTTCATTTAAAAATATTCCTGGTCAGCGTGACCGGAGCTGAAAGAACACATtTGA(TCCCGTGATTT
******
*
****
*
* *
****
**
*
*
* *
*
*
***
ATGCTTTCTGACTGGGCACAGTTCCCTAGGCAGT
-582
*
-CTGGGTA6AAGTTAT6CTTCCCCT6T6A6ATC-trnJ6CAA6T6GCT6 -535
Figure 7 Nucleotide sequences of the transposon-like elements within the
upstream region of the Vtg and VLDLII gene. Dashes were Inserted to achieve
optimal alignment of homologous sequences. The Inverted repeats bounding the
elements are Indicated by thick arrows. The flanking direct repeats are
boxed. Corrispondingly numbered arrows Indicate direct or Inverted repeats.
An 8-bp sequence Identical to the enhancer core consensus (33) 1s boxed.
Consecutive stretches of homology with the B2 Lf V1 element of Xenopus
laevis (45) are Indicated by bold lines.
8677
Nucleic Acids Research
repeats, which In turn are flanked by s1te-spec1f1c three base-pair direct
repeats(F1g. 6), a structure reminiscent of mobile genetic elenents.
Interestingly, a transposon-Uke DNA element (V1 element) Interspersed 1n
the Vtg locus of Xenopus laevis has recently been described (46). Both
chicken elements bear homology 1n structure, length of the direct repeats
and sequence of the Inverted repeats to these V1 elements (F1g. 6).In
Xenopus, seven copies of the V1 element are present near or 1n the Vtg genes
whereas a total of about 7500 copies 1s dispersed throughout the genome
(45). We found no evidence for a multiple occurrence of these elements 1n
the chicken genome, when Southern blots of cloned DNA containing sequences
of the Vtg or VLDLII element were probed with nick-translated genomic DNA
at low stringency (data not shown).
The transposon-11ke elements 1n chicken and Xenopus differ 1n length;
however they share homologous nucleotide stretches up to 12 basepairs as
11 lustrated 1n F1g. 7 for the chicken Vtg and VLDLII genes and the Xenopus
B2 gene. As yet we do not know the functional significance of these elements. However, the presence of a demethylation site (see above), the
occurrence of direct and Indirect repeats and a sequence Identical to the
enhancer core consensus (see Fig. 7) as well as the recurrence of the socalled recognition sequences TGTG/CACA and GAGA/TCTC (46) In the Inverted
repeats bounding the elements (see F1g. 6), argue 1n favour of a possible
regulatory role.
Our sequence analysis has uncovered a number of elements conserved In the
estrogen-responsive genes expressed exclusively In liver. Their precise role
In the hormone- and tissue-specific expression of these genes, which are
transcribed at high frequency, has s t i l l to be established. The availability
of the sequence makes direct studies of the significance and Importance of
these elements, of their Interaction with factors, and of their possibly cooperative function possible.
ACKNOWLEDGEMENTS
We are g r a t e f u l t o Dr. P. Terpstra f o r assistance i n the computer analys i s . We thank B. Dikkeschel for assistance I n the i n i t i a l p a r t of t h i s work.
This I n v e s t i g a t i o n was c a r r i e d out under auspices of the Netherlands Foundat i o n f o r Chemical Research (S.O.N.) w i t h f i n a n c i a l a i d from the Netherlands
Organization f o r the Advancement of Pure Research (Z.W.O.).
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