volume 7 Number 81979
Nucleic A c i d s Research
Purification of the mRNA for chicken very low density lipoproteirtjj and molecular cloning of its
full-length double-stranded cDNA
B6 Wieringa, Willem Roskam*, Annika Arnberg, Janneke van der Zwaag-Gerritsen, Geert AB and
Max Gniber
Biochemisch Laboratorium, The University, Nyenborgh 16, 9747 AG Groningen, The Netherlands,
and *Unite de Ge"nie Genetique, Groupe de Biologie Moleculaire du Gene, Institut Pasteur, 28 Rue
du Docteur Roux, 75724 Paris, Cedex 15, France
Received 18 October 1979
ABSTRACT
The mRNA coding for the small apo-Very Low Density Lipoprotein
(apo-VLDL ..) from chicken serum was highly enriched by oligo(dT)
chromatography and preparative gel electrophoresis of estrogenised liver RNA.
Double-stranded cDNA was synthesised by the subsequent actions of reverse
transcriptase and DNA polymerase, and used for a preliminary characterisation
of the structural gene. Molecular cloning of dC-tailed ds-cDNA into the Pst I
site of plasmid pBR 322 yielded several recombinant clones. Five chimeric
DNAs were selected and characterised by restriction enzyme mapping and
electron microscopy of R-loops. At least two of them (pVLDL 3.33 and
pVLDLi;[ 4.82) contain an almost full-length da-transcript of VLDLj-j mRNA in
which no more than 10-20 bases at the 5'- end are missing.
INTRODUCTION
In birds, the Very Low Density Lipoprotein (VLDL)complex is synthesized
exclusively by the liver and secreted into the blood (1-7). This circulating
VLDL is the main source of lipoprotein used for egg-yolk formation in the
ovary (I, 4-6, 8 ) . The major, and probably the only, constituents of the
protein moiety of VLDL are a large 350,000 dalton protein (apo-lipoprotein B)
and a small 9,500 dalton protein, called apo-VLDL.^ (4, 5, 7). Apo-VLDLjj. has
been isolated from serum and egg-yolk of chicken. Its amino acid sequence has
been determined independently by Jackson et al. (9) and Inglis and Burley
(10); some minor discrepancies still have to be resolved.
Male and immature chickens have a small but distinct level of VLDL
synthesis. The administration of estrogenic steroids results in a 10-400 fold
increase over the basal hepatic output (2-5, 11, 12). Concomitantly, the
synthesis of the major yolk protein precursor vitellogenin (see for a review
ref. 13) is induced from zero level. The simultaneous study of estradiol
effects on apo-VLDL-j. and vitellogenin synthesis may throw more light on the
action of this hormone on gene expression in the terminally differentiated
hepatocyte. Previously we reported the purification of the mRNA for
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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vitellogenin (14); recently incomplete DNA copies of the mRNA sequence were
cloned in bacterial plasmids (V.d. Boogaart, unpublished results). In this
paper we report the cloning of the entire structural gene sequence of VLDL^j.
by insertion of full-length ds-cDNA into the Pst I site of plasmid pBR 322.
The isolated clones are useful probes for the study of hormonal effects on
transcription and/or translation and the organisation of the yolk protein
genes in chromosomal DNA.
MATERIALS AND METHODS
Materials. AMV DNA polymerase was a generous gift of Dr. J.W. Beard and the National
Cancer Institute, USA. T,-polynucleotide kinase and DNA polymerase were
obtained from Boehringer. Information about isolation and sources of terminal
deoxynucleotidyltransferase, plasmid pBR 322 and bacterial strain C^QQ (rk »
UL. ) can be found in ref. 16,17. Restriction endonuclease Hae II, Hap II,
Hha I, Xho I, Xba I, Ava I, Bel I and Bgl II were purchased from New England
Biolabs. Other restriction enzymes were isolated by the procedure of Greene
et al (18). (32P)ATP, (3P)dCTP and (32P)dCTP were from the Radiochemical
Centre, Amersham. All other chemicals were as described previously (14-17,19).
Polysomes and RNA. Large scale preparations of polysomes were obtained as
described earlier (14, 15, 20). To obtain higher yields of the small polysomes,
modified discontinuous gradients of 1.5 ml 161 (w/w); 4.5 ml 35Z (w/w) and
3 ml 65Z (w/w) sucrose were used in rotor SW-27 (Beckman) (3.5 h at 27,000
rpm). Alternatively, RNA was extracted directly from liver by a procedure
developed by C. Auffray and F. Rougeon using direct homogenisation of tissue
in 6 M urea and 3 M LiCl (unpublished). Fractionation of RNA on oligo(dT)cellulose and by preparative polyacrylamide gel electrophoresis was as
described (14). The VLDL - mRNA used for colony hybridisation received an
additional purification step on a 3Z agarose gel in urea-citrate pH 3.5 (21).
The predominant
band was excised and VLDL
mRNA was recovered from the gel
slices (22). Nucleic acids were analysed under denaturing according to
references 21 and 23.
5'-end labeling of RNA. RNA (3 ug) was partially hydrolysed with 0.1 M NaOH
for 45 min at 0°C, neutralised with 1 M Tris, pH 7.5 and alcohol precipitated.
P-labeling of the free 5'-OH termini with T,-kinase (4 units) was performed
according to Doel et al. (24).
cDNA-mRNA hybridisation.
H-labeled ss-cDNA was synthesized as described for
the first-strand reaction of ds-cDNA synthesis, in presence of 50 Ug/ml
actinomycin D. Hybridisation of cDNA with excess RNA in 0.3 M NaCl, 10 mMTris,
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pH 7.1, 2 mM EDTA, 0.2Z SDS at 68°C and determination of the percentage S ( nuclease resistance was as described (14).
DNA electrophoresis. Flat bed agarose gels (25) (O.7X-2.5Z) in 40 inM Trisacetate, 1 mM EDTA, pH 7.7 were used. DNA samples were pretreated and loaded
in agarose beads according to Schaffner (26) and run in presence of ethidium
bromide (0.5
yg/ml).
Synthesis of ds-cDNA. cDNA containing a high proportion of full-length transcript was made according to Uickens et al. (27) with modifications. The
reverse transcriptase reaction was carried out in 50 mM Tris-HCl, pH 8.5
(25°C), 8 mM DTT, 10 mM MgCl_, 70 mM KOAc, 500 uM each of dATP, dGTP, dTTP
and 100 yM of dCTP, including 175 yCi (32P)dCTP, 10 yg/ml oligo(dT)
and
7.5 yg mRNA in a final volume of 300 yl. After 5 min at 0 C reverse transcriptase (15 units) was added and incubation was for 1 h at 42 C. The
complete reaction mix was heated for 3 min at 100 C, quickly cooled in liquid
N_. Denaturated protein was removed by centrifugation. Second-strand
synthesis was initiated by immediate addition of 200 mM Hepes, pH 6.9, 500 yM
each of dXTPs including 100 yCi (3H)dCTP, 10 mM MgCl , 8 mM DTT and 70 mM KCL
and 250 units (60 yl) DNA polymerase in a volume of 300 yl. The final reaction
mix (600 yl) was incubated for 2 h at 15 C. Double-stranded cDNA was purified
by phenol-chloroform extraction and Sephadex-GlOO chromatography.
S -nuclease digestion, was performed in 30 mM NaOAc pH 4.6, 300 mM NaCl, and
3 mM ZnCl- for 40 min at 35°C, and optimized for each batch of ds-cDNA to
determine the minimal amount of S
required to open the hairpin structures.
dC/dG tailing of ds-cDNA and pBR 322. 0.3 yg ds-cDNA was incubated for 3 min
at 35°C in 0.2 M potassium cacodylate, pH 7.2, 1 mM CoCl-, 0.1 mM 2-mercaptoethanol, 100 yM dCTP and terminal transferase in a total volume of 100 yl.
Pst I-linearised pBR 322 (20 yg/ml) was tailed with dGTP (20 min at 35°C);
8 mM MgCl. was used instead of CoCl- (16,17). Reactions were stopped by the
addition of EDTA and SDS (17) and DNAs were used directly for annealing or
gelfractionation.
Isolation of full-length ds-cDNA. After electrophoretical separation in one
dimension DNA of desired length was recovered by binding to hydroxyapatite
(Biorad) and chromatography on Sephadex G-75 as described by Tabak and Flavell
(28). Ethidium bromide stain was removed from the DNA-containing fractions
(adjusted to 0.1 M NaOAc, pH 6) by 5 successive extractions with butanol-1,
and DNA was alcohol precipitated in presence of tRNA carrier. Yields were
generally in the 80-100Z range.
Annealing of ds-cDNA to pBR 322. Four ng of size-fractionated dC-tailed
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ds-cDNA and 40 ng of dG-tailed pBR 322 were annealed following Rougeor.
and
Mach (16) and Roskam and Rougeon (17).
Transformation and colony selection. All manipulations were carried out under
L3-B1 conditions according to the rules set by the French National Control
Committee. Transformation of C a d . treated E. Coli C,-_ (r. ,m, ) cells was
Z
oUU
K
K
performed according to Glover et al. (17, 2 9 ) . Individual growing colonies
were selected for tetracycline resistance on Tc containing L-agar plates,
replated and maintained on fresh Tc L-plates. Colonies grown on nitrocellulose
filters were processed for hybridisation essential as described
(17, 3 0 ) .
Filters were pretreated with 0.21 ficoll, 0.2Z polyvinylpyrrolidone, 0.02X
bovine serum albumin, 0.2Z SDS in 3xSSC (Modified Denhard solution; 3 1 ) .
32
5
Hybridisation wiuh 5'-end (
P)-labeled mRNA (0.5-1x10
dpm per filter) in
this solution (BSA omitted), and washing was as described by Jeffreys and
Flavell (32).
Isolation of plasmid DNA. Plasmid DNA was amplified by chloroamphenicol
treatment (M--medium), and cells were lysed with Triton-X 100 (33). High
plasmid yields were obtained from phenol-chloroform extracted, cleared
lysates by direct chromatography on DEAE-cellulose (DE 52, Whatmann). Columns
were washed with 50 mM Tris, pH 7.5, 100 mM NaCl and 5 mM EDTA and plasmid
DNA and RNA was eluted by increasing the NaCl concentration to 1.5 M.
Additional incubation with heat-treated
( 5 1 , 90 C) pancreatic RNAse (20 yg/ml)
for 30 min at 37 C and phenol/chloroform extraction yielded almost pure DNA.
Alternatively, supercoiled DNA was isolated by CsCl/ethidium bromide plasmid
centrifugation (34).
R-looping of chimeric plasmids for electron microscopy. R-loops were formed
by hybridizing Eco Rl-linearised plasmid DNA (1.5 yg) with VLDL
mRNA at
10 yg/ml in a volume of 100 yl as described by Woolford and Rosbash (35).
Spreading of R-loops was done in 2.5 M urea, 62Z formamide (v/v), 25 mM Tris,
pH 8.5, 2 mM EDTA, 40 mM NaCl. Further conditions and measurements were as
reported (14, 15).
Restriction enzyme digestion, was performed in 90 mM Tris, pH 7.5, 6 m M M g C l . ,
6 mM 2-mercaptoethanol
for Eco RI; 6 mM Tris pH 7.5, 6 mM NaCl, 6 mM MgCl.,
6 mM 2-mercaptoethanol
for Pst I, Kpn I and in 10 mM Tris, pH 7.5, 50mM:<aCl,
6 mM MgCl., 6 mM 2-mercaptoethanol
for all other enzymes used.
RESULTS
Isolation of the V L D L J T mRNA.
Polysome profiles of estrogenized hen liver (20, 36) show a distinct
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size class representing structures of 4-6 ribosomes per mRNA chain (Fig. 1A,
peak I ) . This polysomal sub-class synthesizes a protein, which is regulated
by estradiol and is identified as Very Low Density Lipoprotein II (data not
shown). The VLDL
-mRNA can be visualised as a distinct peak in a gel profile
of poly(A)-containing RNA (Fig. IB, peak I) isolated from polysomal preparations enriched in these oligosomes. The RNA isolated by two sequential
oligo(dT)-cellulose binding steps shows two additional mRNA peaks which
contain the chicken serum albumin- and vitellogenin raRNAs (Fig. IB, peaks II
and III respectively) as reported earlier (14, 15, 3 6 ) .
The VLDL - mRNA can be substantially purified by fractionation of the
poly(A)-containing RNA on a preparative polyacrylamide gel (14). The RNA
fractions were analysed by translation in the mRNA-dependent reticulocyte
lysate. Immunoprecipitation of the translation products with a specific antiserum against serum apo-VLDL
allowed us to identify the VLDL
mRNA
containing fractions (data not shown). Judicious collection of fractions,
taking only the RNA eluted in the middle of the 9-10S peak, routinely yielded
a RNA preparation in which V L D L ^ ™RNA represented 5O-70Z of the RNA. This
POLTSQKE SIZE InuniiK of
TOP
BOTTW
- std&Mnfatlon
3
7 ca
algralkjn
Figure 1. Sucrose gradient centrifugation of polysemies (A) and polyacrylamide
gel electrophoresis of poly(A)-containing RNA (B) from estrogenized chicken
liver. (A) Polysomes were isolated as described in Materials and Methods and
centrifuged on a 15-40Z (w/v) sucrose gradient for 30 min at 40,000 rpm
(rotor SW 41, Beckman).
(B) Poly(A)-containing RNA was obtained by two binding cycles on oligo(dT)cellulose, heat denatured and analysed on a 2.6J polyacrylamide gel as
described previously (14).
Polysome and RNA peaks containing VLDL j. mRNA ( I ) , serum albumin mRNA (II)
and vitellogenin mRNA (III) are indicated.
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value should not be taken as an absolute measure of the purity of the
^j
mRNA preparation since the percentage found strongly depends on the salt
conditions for translation and i s moreover influenced by the nature
of the
contaminating mRNA species (manuscript in preparation).
Further purification of the VLDL
mRNA was achieved by electrophoresis
on a 3% agarose gel in 6 M urea. Careful excision of the predominant RNA band
and elution of the mRNA from gel slices resulted in a VLDL-.. mRNA preparation
which is more than 70Z pure as judged by in vitro translation. Since this
second electrophoretic purification step is rather laborious and results in
only A0-60J recovery, most of the experiments mentioned in the following
paragraphs were performed with the VLDL
mRNA preparation obtained from the
preparative PAA-gel.
Characterisation of the mRNA.
The translation products of the 9-10S mRNA fraction in a wheat germ and
a reticulocyte cell-free system contained one major component of 11,000
dalton as shown by electrophoresis on different
types of polyacrylamide slab
gels in SDS (Fig. 2). This protein is inmunoreactive with anti-VLDL^j. serum.
In addition other protein products of M
are observed which
15,000 (double band) to M 23,000
however are not immunologically related to VLDL-. (Fig. 2,
lane 3). The main component synthesized in both in vitro translation systems
is significantly
larger than the apo—VLDL
isolated from chicken serum which
migrates as a polypeptide of M 9,500. I t has the size of the precursor of
RETICULOCYTE
LYSATE
WHEAT
50 —
40 —
50403020.
21SM
GERM
Figure 2. Acrylamide/SDS gel
electrophoresis of translation
products on 9-10S mRNA in vitro.
VLDL mRNA-containing fractions obtained from preparative
gel electrophoresis were translated in the mRNA-dependent
reticulocyte lysate and the
wheat germ cell-free system
(14,19). -TJ-leucine labeled
total products (lanes 1 and 2)
or H-labeled products, immunoprecipitated with anti-VLDL
serum (lanes 3), were separated
on a 5-2OZ (w/v) gradient gel
(left) or a 15Z (w/v) linear
gel (right) and fluorographed
for prolonged time. Migration
of the main translation product
(M 11,000, large arrow) and
apo-VLDLl:[ marker (M 9,500,
small arrows) are indicated.
Nucleic Acids Research
apo-VLDL
which contains a s i g n a l peptide of 23 amino acids preceding the
N-terminus of the 82 amino acid polypeptide chain of VLDL_-. (4, 37).
To estimate the p u r i t y in a way which does not depend on t r a n s l a t i o n , we
determined the RNA complexity of our p r e p a r a t i o n . Aliquots of ( H)-labeled
cDNA synthesized on the 9-10S fraction were hybridized to t h e i r template RNA
under conditions of RNA excess. Analysis of the data showed t h a t the cDNA
consisted of one predominant species representing about 65X of the m a t e r i a l ;
—4
the Rot, value i s 7 to 8x10 M.s. (Fig. 3 ) . Correction for the 65Z p u r i t y
-4
leads to a Rot, for the pure VLDL.j. mRNA of about 5x10 M.S. Under i d e n t i c a l
r e a c t i o n conditions pure v i t e l l o g e n i n mRNA (7000 bases) has a Rot, of
-3
4.6x10 M.S.; t h e r e f o r e , VLDL - mRNA has a length of approximately 750 + 100
bases.
An independent, and more precise, size determination was done by
measuring the electrophoretic mobility on 3Z agarose in urea at low pH, using
rabbit globin mRNA and 18S rRNA as markers (Fig. 4 ) . VLDL . m RNA was found to
be somewhat larger than globin mRNA and was calculated to consist of 700 + 50
bases. The rather disperse distribution of the VLDL mRNA is probably a
reflection of length heterogeneity of its poly(A) tail (see table I ) .
Electrophoresis on agarose gels under completely denaturing conditions in
presence of glyoxal and DMSO, with DNA restriction fragments of phage PM_ DNA
as internal markers, yielded a length of 680-700 nucleotides for the VLDL
mRNA chain (Fig. 4) and 610-620 nucleotides for rabbit globin mRNA (not
shown). We conclude that the mRNA contains, in excess of 315 nucleotides of
its coding sequence, 300—400 nucleotides, including the poly(A) tail, in
non-coding regions.
HYBRIIJISAIIUN
(V.) 1
Figure 3. Kinetics of association of VLDL TI mRNA-containing
RNA and its cDNA. VLDL
mRNA
.3ng/I0yl) and 175 pg
-cDNA (15.106cpm/yg) were
mixed and incubated at 68°C for
various times. Percentage of
hybridisation was determined
by S|-nuclease digestion
followed by trichloroaceticacid
precipitation and counting.
70503010—I
-5
1
'
-3
-2
-1
Log Crt
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UREA
Figure A. Agarose gel
electrophoresis of
purified VLDLJ-J. mRNA
under denaturing conditions. Left: RNA was
dissolved in 6M urea, 25
mM citrate, pH 3.5 and
electrophorized on a 3Z
agarose gel in ureacitrate. Two ug each of
three different V L D L ^
mRNA preparations(lanes
1-3), a mixture of 2 yg
VLDL
mRNA and 3 yg
crude globin mRNA (lane
4) and 3 yg crude globin
mRNA isolated from rabbit
reticulocytes (lane 5)
were analysed. Right:
RNA and DNA-markers were
treated with glyoxalDMSO (rcf.23) and applied
on a 3Z agarose gel in
10 mM sodium phosphate,
pH 7.4.
mRNA preparations (lanes 1-3) and
Electrophoresis of 3 yg of different VLDL
II
of 1.5 yg PM.DNA fragmented with restriction enzymes Hindll and Hindlll are
shown. PM, fragments of 2000-,1800-,1300-,570-,560-,470- and 400 bp are
indicated by arrows (lane 4 ) .
Synthesis of ds-cDNA and preliminary characterisation of the structural gene.
Double-stranded cDNA was prepared by a procedure in which the purification
of the single-stranded intermediate can be omitted (27). In two different
experiments ds-cDNA yields of 12Z and 19Z of the input V L D L ^ mRNA preparation
were obtained as calculated from the incorporation of H?-dCTP
into the first
strand or ^1-dCTP activity into the second strand. 81Z and 88Z of the firstand second strand, respectively, were protected from S.-nuclease digestion.
The cDNA synthesized contains a high proportion of products migrating as
duplex DNA molecules of 600-620 basepairs visible as a predominant band after
autoradiography (Fig. 5A lane 1-3 and 7-9) or ethidiumbromide staining (Fig.
5B). In addition shortly exposed autoradiograms reveal several distinct bands
of intermediate size (Fig. 5, lane 3,9). Probably they reflect premature
termination of first-strand transcription at preferential sites near the
5'-tenninu8 of the mRNA, a phenomenon also observed with ovalbumin- and
globin mRNA (38,39). A substantial amount of full-length ds-cDNA molecules
are in the open configuration, i.e. have lost their single stranded hairpin
loop at the 3'-end of the first strand. This feature is illustrated in Fig. 5A
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1 i 2 i 3 i 4 i 5 i 6 I 7 1 8 1 9 I 10 I 11 112 i
I
600
600 6 0 0
225
Ll.OO
,50,
32
Figure 5. Autoradiogram of
P-labeled ds-cDNA electrophoreaed on 2,5Zagarose.
A) (32p)ds-cDNA was applied native (lanes 1-6) or after prior treatment with
150 mM NaOH (lanes 7-12). Long term exposures of total ds-cDNA (lanes 1,7)
treated with Sj-nuclease (lanes 2,8) or short term exposures of ds-cDNA
digested with EcoRI (lanes 3,9), Haelll (lanes 4,10), PvuII (lanes 5,11) and
Alul (lanes 6,12) are shown. The positions of the full-length 600 bp (ds and
ss), and intermediate size 550- and 480 bp (ds), molecules are indicated.
B) VLDL-. ds-cDNA (0.1 yg) was separated on a agarose gel (left) and the predominant band (ethidium bromide stained) recovered by hydroxyapatite binding.
Purified full length ds-cDNA was digested with restriction enzymes EcoRI
(lane 1), Alul (lane 2 ) , PvuII (lane 3 ) , Haelll (lane 4 ) , Bell (lane 5 ) , BamHI
(lane 6) and Kpnl (lane 7). Lengths of the fragments are listed at the bottom
of each slot, partial- or undigested fragments are indicated with an asterix.
Weak band visible in slots 1, 2, 3 and 7 may be generated from contaminating
ds-cDNA present in minor amount. C) Preliminary restriction map of ds-cDNA.
where native ds-cDNA is compared with cDNA subjected to strand separation at
high pH prior to electrophoresis. A large part of the molecules in the predominant band does not snap back in the neutral gel, and migrates as the
S -nuclease treated molecules shown in Fig. 5A lane 8. The molecules of
discrete, intermediate size seem to bear open structures to a much lesser
extent since they do not show a shift in mobility after NaOH treatment.
Fig. 5A shows an autoradiogram of the labeled cDNA fragments generated by
the action of the restriction enzymes Haelll (lanes 4,10),PvuII (lanes 5,11)
and Alu I (lanes 6,12). The length heterogeneity of the DNA molecules resides
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at the end corresponding to the 5'-terminus of the mRNA and is due to incomplete transcription by reverse transcriptase as was explained above. This
feature conveniently allowed us to determine the orientation of some restricttion sites within the DNA sequence. The Haelll site is situated close to the
end, namely 50—60 nucleotides from the 3'-terminus of the mRNA, since this
enzyme reduces all bands in size to about the same extent (lanes 4 and 9 ) .
Full-length ds-cDNA did not contain recognition sites for the restriction
enzymes EcoRI (lanes 3,9), Hhal, HapII, Hind III, Aval, Xhol, Xbal and Haell
(not shown). Since none of these enzymes caused a detectable reduction of the
intensity of the predominant bands, we may conclude that no species of ds-cDNA
other than VLDL
-cDNA hiding under the main band was present in considerable
amount. PvuII digestion (lanes 5,11) yielded main fragments of about 325 and
150 bp. Alul digestion yielded fragments of 200 and 100 bp (lanes 6 and 12).
More accurate mapping was achieved when only full-length ds-cDNA molecules
were used. For this purpose the predominant DNA band of a 2.5Z agarose run was
recovered by hydroxyapatite binding, and subjected to incubation with EcoRI,
Alul, PvuII, Haelll, Bell, BamHI and Kpnl (Fig. 5B), and AosI (not shown). The
Kpnl site is 50 bases from the 5'-end and about 500 basepairs from the Haelll
site which is situated at 70 bases from the 3'-end. A provisional map of the
structural VLDL - gene (Fig. 5c) was used to identify the chimeric DNAs as
VLDLII containing plasmids.
Construction of recombinant VLDL
DNA-plasmid pBR 322 molecules.
The double-stranded DNA copy of the mRNA sequence was inserted into the
Pst I site of the Ap
locus in pBR 322 using the "G-C" tailing technique (40).
The dG-dC tails generated by terminal deoxynucleotidyltransferase contained
approximately 40-50 dG or dC residues as was found by analysis of various
chimeric VLDL
DNA-plasmids (not shown). In order to clone only full-length
molecules the largest "tailed" ds-cDNA fraction was purified by electro-phoresis on a preparative agarose gel and binding to hydroxyapatite.
A clear decrease in the mobility of the predominant
DNA band was observed after extension with dC residues. The DNA band was
shifted from 600-620 to 700-750 nucleotides and was more disperse. Chimeric
plasmids were formed by annealing this purified full-length dC-tailed DNA to
dG-tailed pBR 322. Transformation of E. coli C600 cells to Tc resistance was
detected by plating on L-agar plates containing tetracycline (17). The transformation efficiency was 1.5-2x10
transformants per pg of DNA. Out of 660
colonies 124 had retained their Ampicillin resistance. All 660 colonies
obtained were transferred to fresh plates with nitrocellulose filters on top,
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and screened by hybridisation to (
P)-labeled VLDL.-.-rich mRNA. Twelve
strongly positive colonies were chosen for further characterisation and
32
double-checked for VLDL DNA-inserts by a second hybridisation to ( P)labeled VLDL.
mRNA which had been purified by agarose-urea gel electro-
phoresis and was more than 75Z pure.
From the cleared lysates of small scale cultures the plasmid DNA was
isolated using direct phenol treatment and subsequent DEAE-cellulose chromatography. This procedure is time-saving, results in high yields and the plasmids
obtained are suitable for digestion with restriction enzymes (Fig. 6) and
electron microscopy of R-loops (Fig. 8).
All twelve recombinants showed a
lower electrophoretic mobility than pBR 322 when analysed on 0.71 agarose
flat gels after linearisation with the restriction enzyme EcoRI or BamHl.
Ultimately, five clones in which the length of inserted DNA ranged from 500
to 750 base pairs, were chosen for further characterisation by restriction
enzyme mapping. Identification of the fragments containing the inserted DNA
was done by visual comparison of the fragment patterns of the chimeric DNAs
1 84 la 73b.33Ua3J6ia\pBf>327
273
HHA I II KPN I
- PVUII HIN Fl
PVU I
KPN I
Figure 6. DNA fragments produced by digestion of V L D L T T C D N A plasmids with
various restriction endonucleases. DNA was digested and electrophoresed on
0.7Z (BamHI digests) or 2.51 agarose (other digests) as given in Materials
and Methods. Single digests with BamHI, and Haelll of the different plasmids
indicated, and double and triple digests of plasmid pVLDLji 4.82 are shown.
Fragments containing the inserted DNA are indicated by arrows. The lengths of
the different V L D L T J DNA-containing fragments can be derived from Fig. 7 and
the data for pBR 322 presented by Sutcliffe (41).
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with restriction fragments of the vector pBR 322. The complete restriction
map of pBR 322 as presented by Sutcliffe (41), was used to verify our results
and to determine the exact sire of the fragments. Restriction enzyme Hhal was
known not to cleave the structural gene sequence in VLDL ds-cDNA. Therefore
the sire of the DNA inserts could be derived from the size of the Hhal bands
containing the insert. The length of the cloned cDNA including G-C linkers is
530-, 710-, 76O-, 760- and 620 bp for the plasmids 1.84, 2.73, 3.33, A.82 and
6.78, respectively. Only the largest plasmid, called pVLDLjj.4.82 will be
discussed in detail. PstI generated four fragments of 250, 205, 150 and 125
bp; they add up to 730 bp. This value corresponds well with the insert length
of 760 bp derived from Hhal digestion. The Haelll site adjacent to the 3'-end
of the mRNA sequence is about 105 bp away from one of the reconstitued PstI
sites. The unique Kpnl site which, as cDNA mapping showed, is close to the
5'-end was found to be located 75-80 bp from the other recovered PstI site.
Restriction enzyme PvuII recognised two hexanucleotide sequences in the
inserted DNA, confirming the data found from the ds-cDNA digestion. One PvuII
site must be indistinguishably close to a PstI site, since a double digestion
with both enzymes yielded five fragments from the inserted DNA. The 250 bp
PstI fragment was split into two fragments of 105- and 145 bp while the other
fragments are not significantly different from the fragments obtained with
PstI only. Detailed information about the mapping results will be available
upon request. Some of the digestion patterns are depicted in Fig. 6 and the
data obtained are compiled in a partial restriction map (Fig. 7 ) .
Restriction analysis thus far revealed no sequence variation in the five
chimeric DNAs. Fig. 7 shows that in pVLDL
T
1.84 a sequence at its 3'-end was
missing. Probably this is caused by dC tailing at an internal nick in the
ds-cDNA followed by in vivo excision of the protruding DNA tails. Plasmid
VLDL
6.78 was found to be incomplete at its 5'-end and contained an
unrecovered PstI site at its 3'-end. Besides variations at the termini, the
maps of all recombinant VLDL
plasmids are colinear.
Electron microscopy was used to analyse R-loops formed between
linearised plasmid p V L D L u 2.73, p V L D L ^ 3.33 or pVLDLjj 4.82 and our
VLDL ..mRNA preparation. Almost all DNA molecules observed possessed a R-loop
structure when hybridisation was performed according to Woolford and Rosbash
(35) in mRNA excess. Upon close examination of R-loops with VLDL
2.73 DNA,
only one protruding RNA tail of heterogeneous length could be detected, which
was always oriented towards the short arm of the assymetrically split pBR 322.
R-loops from clone 3.33 and 4.82 showed a similar phenomenon with the RNA
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0 - -KJ.11Q
40/TL4143
S3JM-SO*!
, I I V
_T
(
pVLOL
__,
mmt—
-CTQCAQ-
1 84
8.78
1.73
" 3 J ,
4.S2
Rni
-CAOCTQ-
PvuII
PvuII
-cfturrc-OOTACC-
Kpol
100 tap
Figure 7. Partial restriction endonuclease cleavage map of the VLDL^. DNA
insert in plasmid pBR 322. The fragments sizes are the best estimates from
a number of experiments (as shown in Fig. 6 ) . The restriction sites of the
five different chimeric DNAs are oriented in parallel. The positions
providing the best fit with the putative sequence for the mRNA, derived from
amino acid sequence data (ref. 9, 10, 37), are shown. Amino acids are
numbered starting at the methionine in the signal peptide (37). Restriction
endonucleases Eco RI, Hindlll, BamHI, Hhal(Haell), AvaI(HapII and Xhol), Hindll,
Bell, Hpal and Bglll were found not to cleave the VLDL
DNA. Alul cleaves at
the internal four bases of the PvuII sites. Other Alul sites are probably
present near the 5'-terminal Hinfl site, between both PvuII sites and close
to the 3'-proximal PstI site, but could not be located exactly.
tail oriented in the opposite direction towards the long arm. We conclude
that this RNA tail represents the 3'-poly(A) sequence of the mRNA. This
allows us to orient the insert within the plasmid as shown in Table I. Data
are in agreement with the restriction mapping of the 3'-proximal Hae III site.
Furthermore we conclude that all three clones examined contain a fairly
complete ds-cDNA sequence because 5'- non hybridisable mRNA tails more
than 50 bases in length should have been detected in our analysis. Table I
compiles the data for the three plasmids analysed by electron
microscopy.
DISCUSSION.
Estrogen administration to roosters causes an increase in apo-VLDL
II
synthesis, from a rather low level to 8-12Z of the total hepatic protein
production. For several days after hormone injection the cytoplasmic VLDL
II
mRNA concentration is sufficiently high to allow its isolation from either
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Nucleic Acids Research
Figure 8. Electron micrographs
of R-loops formed between VLDL
mRNA and chimeric plasmid DNAs.
Hybrid molecules were prepared
and spread as described in
Methods. Lengths were determined
in relation to pBR 322 DNA,
linearised with EcoRI. Plates
and schematic representations
are: From the top, pVLDL
2.73,
pVLDL
3.33 and pVLDL
£.82.
The bars shown in the photographs
represent 0.2 ym equal to 527
nucleotide pairs.
Table I:
Clones:
pVLDL
2.73
pVLDL T 1 3.33
pVLDL TT 4.82
Hybrid length
632+79 (30)
632+79 (28)
658+79 (36)
737+105(36)
R-loop length
711+105(30)
71 1 + 105(28)
RNA length
71 1 + 79 (34)
763+79 (47)
763+105(34)
82-263 (13)
87-316 (21)
5O-3IG (23)
Distance to EcoRI site
737+79 (30)
684+79 (27)
737+53 (36)
Orientation
5'- 3'
3'- 5 1
3'- 5'
RNA "tail"
poly(A) length
Molecules were linearised with EcoRI, purified and hybridised with VLDL rich mRNA as described in Methods. R-loops were frozen and stored in liquia
N o to minimise branch migration until analysis. The orientation of the mRNA
sequence is read clockwise in the pBR 322 map presented by Sutcliffe (41).
Mean lengths are given as number of nucleotides or basepairs and calculated
from the number of determinations given in parentheses.
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Nucleic Acids Research
polysomal or total RNA. Our purification procedure involving oligo(dT)cellulose partition and preparative gel sizing results at best in a
preparation with 70-80Z V1DL
mRNA. The presence of various other, less
abundant mRNAs is evident from gel analysis of the translation products in
vitro and from physico-chemical characterisation as shown in Figs. 2-4. Our
results are at variance with findings of Chan et aL, (4) who claim to have
purified the VLDL
mRNA, which in their case is only 400 bases long, by a
series of sizing methods which each have low individual resolving power.
For a detailed study of the many aspects of steroid hormone control of
VLDL.., synthesis pure probe in sufficient amount can be obtained by recently
developed cloning procedures. Our cloning "strategy" was based on three
successive steps: First, the generation of complete ds-cDNA in high yield on
a VLDLj-mRNA preparation of 6O-70Z purity. Second, the selection of the
predominant full-length dC-tailed ds-cDNA molecules after S -nuclease
treatment and terminal transferase tailing, just prior to annealing to the
pBR 322 vector. Third, the ultimate selection of VLDL T recombinant clones
32
by colony hybridisation with highly purified (5'P) labeled mRNA.
The plasmids pVLDL
3.33 and pVLDL ^.4.82 probably contain the entire
structural gene sequence. From the amino acid sequence of apo-VLDL .(9, 10)
and signal peptide (37) putative nucleotide sequences of the V L D L T T structural
gene could be derived. If the hypothetical restriction sites of these
sequences are compared with the experimentally determined restriction map
(Fig. 7), excellent agreement appears. Both PvuII sites can be correlated
with the nucleotide sequence inferred from the amino acid sequence at amino
acids
40-43
and 90-91. The putative sequences would allow for three PvuII
sites maximally. The PstI site and PvuII site which locate within an
experimental error of 5 bases at the same position, can only be determined
by the sequence -GCXGXCGXCGXC- which codes for the unique amino acid
sequence ala-ala-ala-ala- at position 40 to 43 in the polypeptide chain
(9, 10). Moreover, the positions of both Hinfl sites - two out of nine
theoretical sites - can be exactly aligned with the nucleotide stretches
coding for the amino acids val-ile-leu and arg-leu at positions 10-12 and
33-84, respectively. This provides evidence for faithfull cloning of the
VLDL
DNA sequence.
The 5'- proximal Hinfl site is 110-120 bases away from the
reconstitued PstI site. This stretch includes the dC-dG linker which is
30-50 basepair in length. Therefore the inserted ds-cDNA extends to 35-55
bases in the 5' non-coding region. Furthermore we conclude that the coding
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Nucleic Acids Research
sequence begins at 80 to 90 - and terminates at A00 bases distal from the
PstI site.
Amongst the five chimeric plasmids finally selected for further
characterisation no sequence heterogeneity could be detected, whether with
restriction enzyme mapping or with electron microscopy of R-loops.
Apparently, VLDL
mRNA is either transcribed from one gene, or from several
genes with only minor heterogeneity which so far escaped detection. Study
of the gene(s), including sequence determination will provide a
straightforward answer.
ACKNOWLEDGEMENTS
We are very grateful to Dr. F. Rougeon for giving us the possibility to
use laboratory facilities at the Pasteur Institute and for his generous
support during the course of the cloning experiments. B.W. was supported by
an EMBO short-term fellowship. S -nuclease and various restriction enzymes
were generously supplied by Drs. P. v.d. Boogaart, F.C.P.W. Meijlink,
T. Havinga and R. Dijkema, respectively. Special thanks are given to
Dr. Willems for performing the wheat germ translation and to Mr. J. v.d.
Heuvel, N. Panman, G. Turksema, J. Bouwer for help in different technical
problems and to Ms. T. Uneken and Mrs. Y. Tempelaar for excellent secretarial
assistance. This investigation was carried out with financial aid of the
Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and
the Netherlands Foundation for Chemical Research (S.O.N.).
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