rabbit reticulocyte system, which was not the case in the wheat germ system, suggesting a possible
difference in the mechanism of translation initia- tion between plants and animals. Systematic muta-
genesis
experiments with
the vertebrate
preproinsulin gene expressed in transfected animal cells confirmed the importance of purine at posi-
tion − 3, as well as the importance of C at posi- tions − 2 and − 1 and G at position + 4 [12,13].
More recent in vitro and in vivo studies with influenza and parainfluenza viral RNA also sug-
gested the importance of positions + 4, + 5 and +
6 [14,15]. However, the role of the + 5 and + 6 positions was questioned recently by Kozak [16].
Experimental data also supported the importance of A at position − 3 in Saccharomyces cere6isiae
[17]. Transient expression in tobacco mesophyll protoplasts of the b-glucuronidase reporter with
two different AUG contexts: CCACC AUG G as an ‘optimal’ sequence for the rat preproinsuline
gene in COS cells and AACA AUG G as a plant consensus context did not show any significant
difference in translation efficiency between the two constructs [18]. Luehrsen and Walbot [19] studied
the effect of an upstream out-of-frame AUG codon that severely affected expression of the re-
porter gene, even though it was surrounded by a poor context, suggesting a lesser role for AUG
context in plants than in animals. On the other hand, the importance of AUG context in plants
was supported by a study showing a 4-fold im- provement of translation of a chitinase protein
when positions − 3 and + 4 were modified into A and G, respectively [20]. Similar results were ob-
served for two viral RNA [21,22] and the GUS reporter gene [23].
Much less is known about the impact of other positions and the interaction between them. Thus,
it is impossible to predict how efficiently a tran- script with an AUG initiation context different
from the consensus will be translated. Another interesting issue is how the consensus between
organisms diverged during evolution. For in- stance, does the distinct consensus determined for
the dicot and monocot plants reflect a modifica- tion in the translational machinery? In this study,
we have addressed these issues by comparing luci- ferase LUC expression produced from 16 gene
constructs with distinct AUG contexts introduced in Nicotiana tabacum, Zea mays and Picea abies
cells.
2. Methods
2
.
1
. AUG context database research In April 1996, we retrieved sequences from the
GenBank-databases. The sequence description was limited to definition, accession number, references,
expression information developmental stage and tissue type, leader length and sequence plus six
nucleotides after the AUG, considered by submit- ting authors as the initiator. Redundancies for
example cDNA and genomic sequence were elimi- nated by looking for sequences with the last 16
nucleotides identical. The final database of 2190 genes was subdivided into three databases contain-
ing dicot 1533, monocot 624 and others, mainly conifer, 33 plant genes.
We retrieved records from sequence databases by key word searches. We used the following
query: plantae [src] NOT thallobionta AND mRNA
[fea] OR CDS [fea] OR UTR [fea] NOT chloro- plasts OR mitochondri
A total of 7861 sequences were retrieved. The sequences were edited using Word for Windows
6.0. Several macro definitions were written in WordBasic in order to compile and analyse the
sequences. Entries without the AUG initiation codon, and not preceded by at least one nucleotide
were deleted. We used the Cavener 5075 rule [4] to describe the consensus.
2
.
2
. DNA contructs Using as template the luc gene [24] cloned into
the HindIII-SacI restriction sites of plasmid Blue- script SK + , we amplified the 5 region of luc with
the primers
5 CCGAAGCTTGGATCCCTC-
GAGGAAGACGCCAAAAAC 3 and 5 TG- GATAGAATGGCGCCG 3. The first primer
introduced the HindIII, BamHI and XhoI sites in front of the coding sequence of luc and removed
the ATG initiation codon. The second primer replaced T, at position + 48, by A in order to
remove the XbaI site at this position without changing the corresponding amino-acid sequence.
The amplified fragment was cut with HindIII 5 end and cloned in the template plasmid cut with
XbaI, filled in with Klenow and cut with HindIII.
The HindIII-SacI
fragment containing
the BamHI and XhoI restriction sites as well as the
whole luc coding sequence without the ATG initia- tion codon was then substituted for the gusA gene
in the 35SGUS construct described in Lukaszewicz et al. [25] and cut with BamHI and SacI. This
construct, thus containing the luciferase gene with- out ATG initiation codon and under the control
of the CaMV 35S promoter and nos terminator, was used as a negative control construct 17.
To prepare constructs 1 – 16, two primers were first
synthetised for
each context:
5GAATTCX
1
X
2
X
3
X
4
ATGX
5
X
6
G 3
and 5
GATCCY
6
Y
5
CATY
4
Y
3
Y
2
Y
1
G 3 where X
1 – 6
and Y
6 – 1
correspond to coding and non-coding strands of the chosen context. After hybridization, both
primers were introduced between the BamHI and XhoI restriction sites of construct 17. At least two
independent plasmid preparations for each con- struct were used.
2
.
3
. Transient gene expression in tobacco using protoplast electroporation
Protoplasts were prepared from young tobacco cv SR1 leaves and electroporated as described in
Lukaszewicz et al. [25].
2
.
4
. Transient gene expression using biolistic transformation
Five hundred microlitre aliquots of tobacco BY2 [26], maize BMS [27] or Norway spruce
embryogenic cells provided by Dr J.-L. Fourre´, Unite´ des Eaux et Foreˆts, Universite´ catholique de
Louvain, Louvain-la-Neuve were spread on solid MS medium [0.44 Murashige and Skoog [28]
salts ICN, 1 agar and 3 sucrose]. The pH was modified to 5.75 before autoclaving. Growth oc-
curred at 26°C in darkness for 24 h.
Biolistic transformation was performed using the PDS-1000He System BIO-RAD as described
by Lukazsewicz et al. [25].
2
.
5
. Luciferase assays After 16 h incubation, electroporated proto-
plasts were collected by centifugation at 700 rpm Sorvall HL-4 for 6 min and resuspended, as
described by Luehrsen and Walbot [29], in 500 ml of Cell Culture Lysis Reagent CCLR: 100 mM
potassium phosphate, pH 7.8, 1 mM EDTA, 7 mM b-mercaptoethanol, 10 glycerol and frozen
in liquid nitrogen. To perform firefly luciferase LUC and control Renilla luciferase RUC as-
says, samples were thawed and sonicated for 15 s Virsonic Cell Disrupter 16-850; Virtis, Gardiner,
NY, set at 35 power. After sonication, extracts were centrifuged for 15 min at 15 000 rpm, and the
supernatant tested for LUC and RUC activity.
Bombarded tobacco BY2 cells, maize BMS cells, and Norway spruce embryogenic cells, as
well as young tobacco SR1 leaves, were collected in microtubes and resuspended in 500 ml of CCLR
and ground. The extracts were centrifuged for 15 min at 15000 rpm, and the supernatant tested for
LUC and RUC activity.
LUC and RUC activity was measured by lumi- nometry on a Lumat LB 9501 Berthold GmbH
Co., Wildbad, Germany. To test LUC activity, extracts 10 ml were placed in luminometer tubes
to which 50 ml of Luciferase Assay Reagent LAR, Promega: 20 mM Tricine, pH 7.8, 5 mM MgCl
2
, 0.1 mM EDTA, 3.3 mM dithiothreitol, 270 mM
Coenzyme A, 500 mM luciferin, 500 mM ATP were automatically injected. Photons were counted
for 6, 2 s after injection. For RUC activity, ex- tracts 10 ml were placed in luminometer tubes to
which 50 ml of Matthews buffer 0.1 M Na
2
HPO
4
KH
2
PO
4,
pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.02 BSA containing coelenterazin 10 mM
were automatically injected. Photons were counted for 6 s, 2 s after injection.
We checked whether luciferase activity was lin- ear with the protein amount. The relative luci-
ferase activity was calculated as the ratio between the test LUC and the control RUC activity.
2
.
6
. Statistics The number of independent transformations
was as follows: seven for tobacco leaf protoplast electroporation, six for tobacco leaf bombard-
ment, eight for tobacco suspension cell bombard- ment, 12 for maize suspension cell bombardment
with additionnal ten bombardments for con- structs 12, 13 and 14 and finally ten for Norway
spruce suspension cell bombardment. Two enzyme assays were performed on each tobacco leaf proto-
plast
electroporation and
one for
each bombardment.
In each figure, the mean relative activity for each construct and its corresponding 95 confi-
dence interval are represented as a percentage of
the most expressed construct for each plant type studied. We also calculated the probability of the
values obtained being significantly different using the Student law. We considered as significantly
different those pairs of values 5 or less likely to share a same group.
3. Results
