AtHXK
1
and AtHXK
2
and potato StHXK
1
and StHXK
2
, one from tobacco and one from spinach [1,6,7] and accession numbers X94302;
AF118133. The Arabidopsis HXK genes were pro- posed to be sugar sensors based on results ob-
tained with
transgenic Arabidopsis
plants overexpressing AtHXK [1]. Similar results were
obtained with transgenic tomato plants overex- pressing AtHXK suggesting that the sugar signal-
ing pathways in tomato might also be mediated by hexokinase [8]. To analyze the role of tomato
hexokinase in tomato plants it was important to isolate and characterize tomato HXKs. In this
paper, we describe the isolation of two different partial sequences of tomato HXKs that were used
to demonstrate the existence of two divergent HXK genes in tomato plants. A full-length cDNA
encoding one of the two HXKs was cloned and designated LeHXK
2
based on its higher homology to StHXK
2
. Kinetic analyses of LeHXK2 follow- ing expression in yeast cells suggest that LeHXK2
is not the GLK previously reported by Martinez- Barajas and Randall [9]. Expression analysis of
LeHXK
2
indicates that LeHXK
2
is expressed in a large spectrum of tissues and organs and at differ-
ent stages of fruit development with the lowest expression in leaves and the highest in flowers.
2. Materials and methods
2
.
1
. PCR amplification of cDNA and sequencing Alignment of the predicted amino acid se-
quences of three plant HXK genes Arabidopsis: U28214, U28215 and Solanum tuberosum X94302
identified several conserved domains. The domains corresponding to amino acids MTVEMHA and
EMVINMEW were used to design the forward 5- ATG ACI GTI GAR ATG CAY GC -3 and
the reverse 5- CCA YTC CAT RTT DAT NAC CAT YT -3 degenerate primers, where I =
inosine, N = all the four nucleotides, D = AGT, R = AG, and Y = CT. PCR was carried out with
cDNA from fruit 10 DAA. Amplifications were for 30 cycles, each consisting of 1 min at 95°C, 30
s at 50°C and 1 min 30 s at 72°C. The resulting approximately 700 bp fragment was purified on a
S-400 MicrospinTM Column Pharmacia Biotech and cloned into the pGEM
®
-T plasmid Promega. Four PCR products were partially sequenced us-
ing the Thermo Sequenase Cycle Sequencing Kit Amersham Life Science, revealing two different
sequences that encoded putative HXK proteins.
2
.
2
. cDNA isolation and characterization An oligo dT-primed tomato fruit cDNA library
constructed in l Uni-ZAP XR [10] was separately screened with the two partial HXK sequences. All
the positive clones obtained corresponded to only one of the two partial sequences. The longest
insert was commercially sequenced MWG-Bio- tech, Germany and the deduced amino acid se-
quence aligned with other HXK sequences using ClustalWebi.ac.uk.
2
.
3
. Plasmids and yeast transformation A yeast shuttle vector, pFL61, containing the
URA
3
gene as a selective marker and the constitu- tive phosphoglycerate kinase PGK promoter and
terminator [11] was used to express the tomato HXK cDNA clone in yeast cells. The cDNA was
inserted as a NotIEcoRI fragment between the NotIEcoRI sites of pFL61; after verification, the
new plasmid was named pFL61-LeHXK
2
. The yeast Saccharomyces cere6isiae strain used was
DFY632 —
MATa, ura
3
-
52
, hxk
1
::LEU
2
, hxk
2
::LEU
2
, glk
1
::LEU
2
, lys
1
-
1
, leu
2
-
1
[12]. Yeast transformations were carried out by grow-
ing yeast cells in YEPG liquid medium, consisting of 1 yeast extract Difco, 2 Bacto Peptone
Difco and 2 Gal, to mid-logarithmic phase, treating the cells with lithium acetate according to
Ito et al. [13] and selecting for transformants on −
Ura + Gal plates. Selective medium for uracil auxotrophic growth − Ura + sugar contained
0.5 ammonium sulfate, 0.17 yeast nitrogen base without amino acids Difco, 0.2 casamino
acids Difco or Sigma, 0.004 adenine Sigma, 0.008 Tryptophane Sigma and 2 Gal, Fru or
Glc.
2
.
4
. Protein purification of yeast expressed LeHXK
2
and kinetic characterization DFY632 yeast cells transformed with either
pFL61 or pFL-LeHXK
2
were grown in minimal medium supplemented with Gal to the logarithmic
phase, harvested by centrifugation and washed with water. Proteins were extracted as described
by Kanayama et al. [4]. For the kinetic character- ization the 80 ammonium sulfate precipitate
was collected, resuspended in extraction buffer and desalted on Sephadex G-25. The protein was
applied to MonoQ Pharmacia, 5 ml preequili- brated with the extraction buffer but containing
only 20 mM HEPES pH 7.0. Unbound protein was eluted with the same buffer, followed by a
0 – 0.5 M KCl salt gradient. Fractions of 0.5 ml were collected and Glc phosphorylating activities
were measured as described below. One major peak of Glc phosphorylating activity was ob-
served and the most active fractions 1 ml were used as the partially purified enzyme for charac-
terization.
HXK activity was measured according to Schaffer and Petreikov [14] for kinetic measure-
ments or Bouny and Saglio [15] for inhibitor studies. Assays for kinetic measurements con-
tained, in a total volume of 1 ml, 30 mM Hepes – NaOH pH 7.5, 1 mM MgCl
2
, 0.6 mM EDTA, 9 mM KCl, 1 mM NAD, 1 mM ATP, 2 U NAD-
dependent Glc 6-P-dehydrogenase G6PDH, Leu- conostoc. For the assay of Glc phosphorylation,
the reaction was initiated with 2 mM Glc; for the assay of Fru phosphorylation, 2 U of PGI were
added and the reaction was initiated with Fru. Reactions were carried out at 37°C and the A
340
was monitored continuously. Inhibitor studies were carried out in a similar fashion but con-
tained 50 mM glucosamine or mannoheptulose or 5 mM ADP in 100 mM Tricine pH 7.5, 2 mM
MgCl
2
, 3 mM DTT, 2 mM NAD, 2 mM ATP, 4 U G6PDH Leuconostoc. Assays for Glc 6-P
inhibition contained 5 mM Glc 6-P in 100 mM Tricine pH 7.5, 2 mM MgCl
2
, 3 mM DTT, 1.5 mM ATP, 150 mM NADH, 1.5 mM PEP, 3.6 U
PK, 16.6 U LDH. Reactions were initiated with 1 mM Glc and were carried out at 25°C.
2
.
5
. DNA extraction and Southern blot analysis Genomic DNA was extracted from green
leaves of tomato plants according to Dellaporta et al. [16]. DNA 10 mg were totally digested
with different restriction enzymes, separated on 1.2 agarose gels, then transferred to nylon
membranes in 20 × SSC according to standard methods. Radiolabeling of cDNA probes was
carried out by the random priming method using the Prime-It kit Stratagene. Prehybridization
and hybridization
reactions were
carried out at 65°C overnight in 6.6 × SSC, 0.1 SDS,
5 × Denhardt’s solution, 0.1 SDS at 65°C for 30 min.
2
.
6
. RT-PCR expression analysis Tissues were collected from plants grown under
controlled conditions 12 h day at 25°C12 h night at 20°C, frozen in liquid N
2
and stored at −
80°C. Before extraction, tissues were reduced to a powder under liquid N
2
. Total RNA was then isolated by the hot phenol method described
by Verwoerd et al. [17]. Total RNA 2 mg was reverse transcribed and amplified using the Ac-
cess RT-PCR System Promega, France and the appropriate primers. To amplify LeHXK
2
, the primers were S2 5- T AGC TAT GTA GAC
AAT CTC CCT- 3 corresponding to amino acids SYVDNL and AS1 Fig. 2. To amplify
Actin Tom52 gene product, Genebank Accession U60482 the primers were ActinS 5- TGG CAT
CAT ACC TTT TAC AAT GAA- 3 and Acti- nAS 5- CCT GAT ATC AAC GTC ACA CTT
CAT- 3, corresponding to amino acids WHHT- FYNE and MKCDVDIR. The positions of
HXK primers are shown on the deduced amino acid sequences of Fig. 2. First strand cDNA reac-
tions in which RT was inactivated by heating at 94°C for 2 min did not yield amplification prod-
ucts.
The relative amounts of LeHXK
2
mRNA were determined via semi-quantitative RT-PCR in two
independent experiments. The first strand cDNA synthesis reactions were performed on 2 mg total
RNA which had been treated with RQ1 RNase- Free DNase Promega using an oligo-dT primer
and MMLV RT as described in Moing et al. [18]. cDNA corresponding to 200 ng of total RNA
was then PCR-amplified using the sense and anti- sense primers described above. Reactions were
run on a Gene AmpTM PCR System 9600 Perkin Elmers Applied Biosystems, France for
30 cycles of 94°C 30 s, 50°C 30 s, 68°C 1 min for HXK primers and 20 cycles for actin
primers. These conditions were chosen so that amplification occurred in the linear range. PCR
products were separated on 1 agarose gels and blotted onto nylon membranes which were then
hybridized under stringent conditions with the LeHXK
2
sequence shown in Fig. 1. Signals were quantified with an Instant Imager Packard.
3. Results
