Bioscience Reports 5, 453-461 (1985)
Printed in Great Britain
#53
Functional prokaryotic gene control signals
within a eukaryotic rainbow trout protamine promoter
3acek M. 3ANKOWSKI, Eva WALCZYK and Gordon H. DIXON
Department of Medical Biochemistry, Faculty of Medicine,
Health Sciences Centre, The University of Calgary,
3330 Hospital Drive N.W., Calgary, Alberta T2N 4NI, Canada
(Received 13 May 1985)
F o l l o w i n g the c o n s t r u c t i o n of a series of pSV2=cat
d e r i v e d p l a s m i d s containing the chloramphenicol
acetyltransferase (CAT) gene under the control of a
eukaryotic trout protamine promoter, it was noted that
Escherichia coli, transformed with these plasmids,
developed resistance to chloramphenicol (CM).
This
result suggested that the eukaryotic trout protamine
promoter possessed significant prokaryotic promoter
activity.
Modification of the trout protamine promoter
region by removing the region containing the eukaryotic
Goldberg-Hogness box in the plasmid p525-cat increased
the expression of the CAT gene almost to the wild-type
level and conferred strong CM resistance.
Sequence
c o m p a r i s o n s of the plasmid series indicate that
prokaryotic promoter elements are present in the trout
protamine promoter and that their similarity to the
p r o k a r y o t i c promoter consensus sequences and the
distance between the two elements is more favourable
in p525-cat~ the plasmid which conlers the greatest CM
resistance.
The g e n e t i c code a p p e a r s to be universal for Prokaryota and
Eukaryota, although there is an alternative use of certain codons in
mitochondrial genes (5).
It should be possible, therefor% to express
p r o k a r y o t i c p r o t e i n s in eukaryotic cells~ if appropriate eukaryotic
p r o m o t e r signals are juxtaposed to such genes.
A good, recent
e x a m p l e which c o n f i r m s this prediction is the expression of the
bacterial enzyme chloramphenicol acetyltransferase (CAT) in monkey
CV-I cells (6).
However, the DNA sequence signals that control the
synthesis of RNA in Eukaryota~ and which involve three distinct RNA
polymerases~ appear to be quite different and easily distinguishable
from those in Prokaryota~ where only a single RNA polymerase is
involved.
N e v e r t h e l e s s ~ if p r e s e n t - d a y E u k a r y o t a evolved from
ancestral organisms that also gave rise to present-day Prokaryota~ it
should not be surprising that traces of these ancestral control signals
m i g h t be found in E u k a r y o t a .
For exampl% the presence of a
bacterial promoter within the eukaryotic genome of the SV#0 virus has
been reported by Zain et al. (20)7 Dhar et al. (#) and Rosenberg et
al. (15).
/45"4
3ANKOWSKI ET AL.
In this paper~ we report the presence of functional Escherichia
gene c o n t r o l signals within the eukaryotic promoter of the
rainbow trout protamine gene previously cloned and characterized by
States et al. (17) and 3ankowski and Dixon (9) which allow the
transcription of the CAT gene in a bacterial host.
coli
Materials and Methods
Preparation of plasmid DNAs
E. c o l i strains HBI01 or DH1 were used as host bacteria.
Plasmid
DNAs were prepared by lysozyme-Triton X-100 lysis ( l l ) and cesium
chloride - ethidium bromide equilibrium gradient centrKugation (lt~).
Enzymes
R e s t r i c t i o n e n z y m e s were obtained from New England BioLabs,
Bethesda Research Laboratories or Pharmacia - PL Biochemicals; T4
DNA ligase, kinase and Bal 31 exonuclease from New England BioLabs.
Preparation of DNA fragments
Restriction
endonuclease
d i g e s t i o n s w e r e c a r r i e d out as
recommended by the suppliers.
Cohesive ends were made flush-ended
by incubation with Bal 31 exonuclease (7), ligated with a Hind III
synthetic linker (New England BioLabs) and then digested with Hind III
endonuclease. DNA fragments were separated by agarose gel electrop h o r e s i s and v i s u a l i z e d by e t h i d i u m b r o m i d e s t a i n i n g ; s e l e c t e d
fragments were purified by the low melting agarose method (13).
Bacterial transformations
F r a g m e n t s were l i g a t e d by T4 DNA ligase overnight at 0~
Transformations of E. c o l i were performed using the calcium chloride
method (2).
Colonies were selected on plates containing ampicillin
(100 pg/ml).
Assay of CAT activity
One ml of an overnight bacterial culture was diluted to 100 ml of
LB medium (Gibco) and grown at 37~
(on a rotary shaker).
Cells
were collected when the AGo 0 reached 0.6 and were washed with 10
mM Tris/HCl, pH 8.0, and 1 mM EDTA. The pellet was resuspended
in 4 times its weight of 0.1 M phosphate buffer pH 7.2. The cells
were disrupted by sonication and CAT activity was determined by the
colorimetric method according to Shaw (16).
Results
and Discussion
Construction of plasmids pP5-cat and p525-cat with the chloramphenicol acetyltransferase (CAT) gene under the control of the
trout protamine promoter
As outlined in Fig. I the entire trout protamine promoter fragment
whose sequence is given in Fig. 2 was obtained by digestion of the
p B R 3 2 2 - d e r i v e d plasmid pP5, containing the entire protamine gene
promoter fragment Bgl II-Ava II (constructed previously and described
TROUT
PROTAMINE
A,
EcoRr
PROMOTER
ACTIVITY
IN E. COLI
455
B,
Hindlll
Aval~
TATA
Amp~
Amp R
o~"~r~
Ndel - protamlne
promote~
Ndel
Aa{ll
i
"Hlnd[ll
2, Bal3l exo
linker
NdL,
'l'
/
I
HI!dill
\
~
Hindlll
Ndel
v
lndIII
CAT
TATA
N
~
/
Amp
A
TATA
Ndlel
Hraclll{
~ Hlndlll. Ndel
"rATA
......... /
4. Hindlll
Ndel
Ndel - -
8gill
Amp R
on
or
Na~l
--
"H~.dm
Nd,~ ~
"f......
Aslll HIndlll
Fig. I. Construction and restriction maps of series
of plasmids used in this paper,
pP5 contains 396 bp
of a Bgl II-Ava II fragment containing the entire
protamine gene promoter inserted between Hind III
and Xho II 1666 sites of pBR322 ( 9 ) . pP5-cat is a
derivative of pSV2-cat (6) in which the SV40 early
genes promoter has been replaced by a protamine g e n e
promoter (Bgl II-Ava II fragment from pP5 plasmid).
The plasmid p525-cat is derivative of pP5-cat in
which
Aat I I - H i n d III r e s t r i c t i o n
fragment
(containing the protamine gene TATA box) has been
deleted.
Bgl II
I0
20
30
40
50
AGATCTGGT~GATTATTTC~AACATGGTT~ATTGGGTTG~CCAAAGAAA~AGATTGTTA~
60
70
80
90
I00
110
GGGTGTAAA~TGGCACAGT~ATGCCATCT~'TTGGTAAAT~TTCATTACT~CAACTCATG~
120
Hpa I - ~ 1 3 0
140Hpa II 150
160Hsa I
170
GTTTTACCGGTGTGCTTGA~ATACCCGGA~GTATTGTGA?GTACTGAAC~AGACTGGTT~
180
190
200
210
220
230
CTCGCATCA%TGCCTCTCT~GTCATTTAA~ATTCACACA~AGATCACTA?TTAAAATGAE
240
250
260
270
280Aat II
290
AAAATAAAAATATCATTAT~ACATCATCC~GCCACTGCT~CTATGACGT~ACATAATTC~
300
310
320
330
340 ~
350
GATGTTTTC~CAATTTAAA~TGTCTTTAA~ACTTATTGC~TCATTTATC~CATAATGACA
360
AZU I
370
380
390Ava
II
400
410
420
TCACTCCAG~TCCCCTCCA~CCC~GGACCACC~CCCGTCTAA~CATTTTATC~
Fig. 2. Nucleotide sequence of the trout protamine gene
promoter (after States et al. [17]).
The TATA box of
protamine gene is boxed.
The vertical arrow indicates
the end of the trout DNA sequence in p525-cat.
.......
456
3ANKOWSKI ET AL.
in reference 9), with Hind III and Nde I. The 1024 bp Hind III-Nde I
restriction fragment containing the protamine promoter regions from
Bgl II to Ava II was ligated into the large Nde I-Hind III fragment of
plasmid pSV2-cat previously described by Gorman et al. (6) and which
contains the CAT gene. The pSV2-cat plasmid was kindly supplied by
Dr. Bruce Howard, Laboratory of Molecular Biology, Division of Cancer
Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20205.
In the new plasmid, pP5-cat, the CAT gene is now under the control
of the trout protamine gene promoter.
A second plasmid, p525-cat,
was also constructed by digestion of pP5 with Aat II, shortening by
digestion with Bal 31 exonuclease, blunt end ligation with synthetic
Hind III linkers and finally digestion with Hind III and Nde I.
The
r e s u l t i n g Nde I-Hind III fragment now contains only the 5' distal
regions of the protamine promoter but does not contain the TATA box
region.
This s h o r t e n e d f r a g m e n t was also ligated to the large
Nde I-Hind III f r a g m e n t derived from pSV2-cat, and the resultant
p l a s m i d , p 5 2 5 - c a t , was checked for correct ligation by restriction
e n z y m e mapping, and the e x a c t j u n c t i o n d e t e r m i n e d by DNA
sequencing (12).
Chloramphenicol resistance of E. coli transformed with plasmids
pP5-cat and p525-cat
A functional assay for the expression of the CAT gene in the
plasmid constructs is provided by measuring the chloramphenicol (CM)
resistance of E. c o l i cultures transformed by this series of plasmids.
The ability to grow in CM-containing cultures is proportional to the
inactivation of CM in the medium by an acetylation reaction catalyzed
by CAT. Therefore, E. c o l i cultures were tested for growth on CM
plates with increasing concentrations on the antibiotic (5, 12.5, 50 and
100 lJg/ml) a f t e r t r a n s f o r m a t i o n with both plasmids pP5-cat and
p525-cat as well as controls of the plasmid pBR325, which contains
the intact CAT gene with its own promoter and pSV2-cat with the
CAT gene under the control of an SV40 promoter (6).
As seen in Fig. 3, the control plasmid, pSV2-cat, did not allow
growth even at the lowest concentration of CM. This suggests that
the eukaryotic SV40 early transcription region, the promoter region in
pSV2-cat, cannot function in Prokaryota while E. c o l i transformed
with pBR325 with the wild-type CAT gene can grow at all CM
concentrations. E. c o l i transformed with plasmid p525-cat was also
able to grow at all concentrations of CM, although at the highest
antibiotic level (100 IJg/ml) colonies were not seen until 48 h (data
not shown). At 12.5 ~g/ml CM, colonies of bacteria transformed with
p525-cat were easily detected overnight but those transformed with
pP5-cat were not able to grow.
However at 5 IJg/ml CM, pP5-cat
could grow.
These results imply that there is stronger prokaryotic
promoter activity in the shorter protamine promoter region and hence
a higher level of CAT expression and consequent CM resistance in
p525 than in the complete region in pP5-cat.
In fact, by this assay
p 5 2 5 - c a t a p p e a r s to have almost as effective a promoter as the
natural CAT promoter in the control plasmid pBR325, which can grow
at all CM concentrations (sector 4 in Fig. 3A-E).
A more sensitive measure of promoter efficiency is provided by the
g r o w t h c u r v e s of b a c t e r i a t r a n s f o r m e d with the same series of
TROUT PROTAMINE PROMOTER ACTIVITY
IN E. COLI
457
Fig 3.
A comparison of the chloramphenicol
resistance of E. coli transformed with a series of
plasmids:
(I) pSV2-eat, (2) pP5-cat, (3) p525-cat,
and (4) pBR325, on various concentrations
of
chloramphenicol in plates:
(A) I00 ~g/ml, (B) 50
~g/ml, (C) 12.5 ~g/ml, (D) 5 ~g/ml, and (E) contains
I00 ~g/ml of ampicillin instead of chloramphenicol.
plasmids.
B a c t e r i a which e x p r e s s CAT can only begin t h e i r
exponential phase of growth after the level of unmodified CM has
fallen to a point that is no longer inhibitory. The length of the lag
phase is, therefore, roughly proportional to the concentration of CM at
the time of inoculation (16).
The length of the lag phase is also
inversely proportional to the level of expression of the CAT gene and
h e n c e to t h e e f f i c i e n c y of t h e p r o m o t e r r e g u l a t i n g CAT gene
expression.
Thus, the relative efficiencies of different promoters
c o n t r o l l i n g t h e e x p r e s s i o n of the CAT gene can be compared if
bacteria carrying the CAT plasmids are inoculated under the same
conditions and at the same concentrations of CM and the length of
the lag phase measured.
As shown in Fig. 4A, bacteria transformed
with a plasmid (pPS-cat) containing the C A T gene regulated by the
complete protamine promoter show a substantially longer lag phase
than those with a shorter protamine promoter lacking the eukaryotic
T A T A box region (p525-cat).
As seen in Fig. 4B, the efficiency of
expression of the C A T gene in this latter plasmid (p~25-cat) is almost
as high as with the intact C A T gene under the control of its own
g58
3ANKOWSKI ET AL.
A
A 550
A 550
2000
200(
1500
~
J
p525cat 15@
pP5cuf
pSVcaf
1000
1000
500
500
0
B
4
8
12
~
0
4
8
ZI
12
Fig. 4.
A comparison of the growth curves of E.
co2i transformed with various plasmids on LB medium
(Gibco) to w h i c h chloramphenicol was added at a
concentration of 5 ~g/ml (A) and 12.5 pg/ml (B).
N u m b e r s on the a b s c i s s a represent hours of the
growth.
promoter (pBR325). The very long lag period in bacteria carrying the
pSV2-cat plasmid indicates a very low efficiency of CAT expression
when controlled by the SV40 promoter and this low activity may, in
fact, be due to a second enzyme, chloramphenicol hydrolase ( l g ) ,
which a c t s v e r y slowly on CM and is not usually considered as
conferring CM resistance.
These results were confirmed by direct
measurement of CAT activity in the crude cell extracts. As Seen in
Fig. 5, cells transformed with control plasmid, pSV2-cat, do not show
CAT activity even when larger aliquots of c e l l e x t r a c t are used while
cells with pPS-cat show easily detectable activity.
The cells transformed with p525-cat show a much higher activity than pP5-cat even
in aliquots diluted 5 times; however, this activity is lower than when
the CAT gene is under the control of its natural promoter in pBR325.
The observation that the protamine promoter region shortened by
c u t t i n g with Aat II at position 295 (Fig. 2) and incorporated in
p525-cat is more efficient in causing expression of the CAT than the
full-length promoter in pPS-cat, suggests that there must be a strong
prokaryotic promoter region upstream (5') of the Aat II site which is
brought into a favourable orientation with regard to the CAT gene in
p525-cat.
In contrast, in pPS-cat, this promoter region is too far
upstream to be effective and a second region which is in the correct
position in pP5-cat has a much lower prokaryotic promoter activity.
We can tentatively identify the strong promoter region as being about
TROUT PROTAMINE PROMOTER
ACTIVITY IN E. COLI
439
A412 nm
1500
pBR325 (10~ll)
1000
500
pP5 cat (501J[)
pP5 oaf (10~J[)
pSV cur (50~t)
0
10
20
40
60
T rain
Fig. 5.
A comparison of the CAT activity in crude
e x t r a c t s from the cells transformed with various
plasmids.
150 bp upstream of the eukaryotic TATA box between positions 206
and 233 in Fig, 2. The weaker promoter that is active in pPS-cat is
27 bp upstream from the protamine TATA box between positions 323
and 336 in Fig. 2.
Detailed studies have been made of consensus sequence feat ures of
prokaryotic promoters (3,g,13,19).
Two regions are important, one at
-10 (the initiation site is +1) in which a sequence TATAAT (3,8) or
T A T A T T ( 19) is c o n s e r v e d , and a second at -33, of the form
TTGACA.
The distance between these two promoter elements is also
critically important and is optimally 17 + 1 bp.
In Fig. 6 t h e DNA s e q u e n c e s of the t hree plasmids pBR323
(containing the native CAT gene plus its wild-type promoter)~ pP3-cat
and p 3 2 3 - c a t a r e shown and compared with prokaryotic prom ot er
consensus sequences.
The e f f i c i e n c y of each promoter region is
proportional to its degree of similarity to the consensus sequences and
to the spacing between the two sequences.
This l a t t e r p a r a m e t e r is
of critical importance and a change of even 1 bp has a large e f f e c t
(3).
The lower a c t i vi t y of the promoter in pP3-cat compared with
the natural CAT pr om ot e r in pBR323 is t h e r e f o r e probably due to the
f a c t that the two promoter regions are separated by 19 rather than
the optimum 17 bp.
It is also possible in pP3-cat that although the
stronger promoter region is present, it is too far upstream and is not
in phase with an appropriate ATG initiation codon to allow transcription of a functional CAT mRNA.
In addition, its presence might
have an inhibitory e f f e c t by sequestering RNA polymerase in nonproductive initiation complexes.
460
3ANKOWSKI ET AL.
-35
CoQsensHs
cat
pP5cat
p525cat
17bp
-IO
§
CAACTTTCAT~AA'I
CGGCAC TAAG $GTTC
3AAATAAGATCA
320
CTGTC% TAAC 2TTATTGCATCATTTATCTATAA~ ]ACATCACTCCA
2OO
CGTCAT TAAC ?TCAC
ACACAGATCAC~ATTT~ %AATGACAAAAT
Fig. 6.
A comparison of putative prokaryotic
regulatory
signals
p r e s e n t within the trout
protamine
gene promoter with the prokaryotic
promoter consensus and the cat gene promoter (8).
The presence of functional prokaryotic prom ot er el em ent s in the
eukaryotic promoter region of the trout protamine gene would suggest
th at t h e r e could be expression of the intact cloned protamine gene in
E. c o l i .
We have grown several such plasmids, e.g. p3P22 (compare
r e f e r e n c e 10) in good yield with no apparent deleterious e f f e c t on the
growth of the bacterial host.
However we have no evidence that a
functional protamine mRNA is made or t hat such an mRNA could be
translated in E. c o l i .
It is d i f f i c u l t to evaluate the significance of the presence of
p r o k a r y o t i c p r o m o t e r e l e m e n t s in a e u k a r y o t i c promoter.
Two
possibilities are evident:
one is t h a t they represent t r a c e s of
prokaryotic ancestry which might occur in many present-day eukaryotic
genes, and the second is t hat the protamine genes are more r e c e n t l y
derived from prokaryotic ancestors.
In this connection, it has been
noted previously that strong similarities exist between trout protamine
gene sequences and an open reading frame present 3' t o. the tyrosine
tRNA gene o f E. c o l i ( 1 ) .
Acknowledgements
3.M.3. holds an Alberta Heritage Foundation for Medical Research
P o s t- d o cto ra l Fellowship and the work has been generously supported
by a Medical Research Council of Canada Term Operating Grant to
G.H.D.
The expert assistance of Marnie Cudmore in preparing the
manuscript is acknowledged.
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ACTIVITY IN E. COLI
461
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\