972 R. Hernandez et al. Insect Biochemistry and Molecular Biology 30 2000 969–977
Table 2 with the PCR DIG Probe Synthesis Kit Boehringer Mannheim, Indianapolis, IN, and using
Gonzalez cDNA as the template. PCR conditions included denaturation at 95
° C for 5 min, then 5 cycles
of 94 °
C for 30 s and 72 °
C for 4 min, 5 cycles of 94 °
C for 30 s and 70
° C for 4 min, 35 cycles of 94
° C for 20
s and 68 °
C for 4 min, and a final extension of 68 °
C for 5 min. Hybridization and signal detection were carried
out using the DIG High Prime DNA Labeling and Detec- tion Starter kit II Boehringer-Mannheim. Briefly, the
baked Nylon membrane was prehybridized with a stan- dard hybridization buffer containing formamide for 30
min at 48
° C; the probe was denatured by boiling for 5
min, added to the prehybridization solution and incu- bated in a hybridization oven for 12 h at 48
° C with
gentle agitation. High stringency conditions included 2 post-hybridization washes of 5 min each in 2
× SSC and
0.1 SDS at room temperature and 2 washes of 15 min each in 0.5
× SSC and 0.1 SDS at 68
° C under constant
agitation. Finally the labeled DNA bands were detected following the manufacturer’s protocol, after exposing an
X-ray film BioMax MR film, Eastman Kodak Com- pany, Rochester, N.Y. to the membrane for 18 h.
3. Results
A single DNA band of 300 bp obtained by using primers Acel and Ace3 in PCR was isolated and cloned.
Twenty clones were selected, seven of which were sequenced and three different sequences showing a 330
bp fragment were obtained. These three sequences were compared with published sequences in the GenBank
showing similarity to known AChEs. We have reported one sequence that encodes a putative acetylcholinester-
ase Hernandez et al., 1999. By using gene specific pri- mers obtained from the other two sequences in the 5
9- and 3
9-RACE and subsequent primer walking, we obtained the entire sequence of two esterase cDNAs. The
cDNAs had an open reading frame of 1629 bp for clone 8 Fig. 1 and 1632 bp for clone 13 Fig. 2 encoding
proteins of 543 and 544 amino acids respectively. Con- served amino acids in the cholinesterase family present
in Torpedo marmorata Bon et al., 1986 were also found at the corresponding positions in our sequences
from B. microplus: Ser 224, His 464, Glu 351, 3 pairs of Cys residues 91-118, 278-289, and 426-545, Asp
196, Asp 421, Arg 173, and Arg 541 Fig. 3. However, tryptophan 108 reported in T. marmorata as a critical
component of the choline-binding site is absent in both sequences reported here.
Analysis of the deduced amino acid sequences from both clones using the program GenomeNet www.server
on the Internet , showed motifs for protein kinase and casein kinase phosphorylation sites, as well as N-myris-
toylation and N-glycosylation sites. Motifs were present indicating that both proteins are type-B carboxylester-
ases. The hydropathy index analysis revealed that clone 13 codes for a membrane protein that has one transmem-
brane helix in the N-terminal region. In contrast, clone 8 was identified as a soluble protein using the BCM Search
Launcher: Protein Secondary Structure Prediction on the Internet :9331seq-searchstruc-predict.html.
cDNA synthesized from the Tuxpan strain was sequenced using specific primers obtained for the Gon-
zalez susceptible strain. Alignment of cDNA from Gon- zalez and Tuxpan strains showed identical nucleotide
sequences in clone 8, but two point mutations were detected when sequences derived from clone 13 were
compared. One is a silent mutation that does not change the amino acid sequence nucleotide 1458 T→C,
whereas the other nucleotide 1120 G→A changes an aspartate to an asparagine, substituting a polar uncharged
amino acid for a negative charged Figs. 2 and 3. When genomic DNA was amplified using the specific primer
pairs GS138CR-MU1G or GS138CR-MU1A, which amplify wild type G or mutant A, respectively, both
alleles were found in all strains examined in this study except the Coatzacoalcos strain, which showed only the
mutant allele Fig. 4.
Two digoxigenin-labeled probes derived from clones 8 and 13 were used to analyze genomic DNA digested
with restriction endonucleases. When restriction frag- ments of digestion with 2 nucleases EcoRI and NsiI
were probed with a 263 bp oligonucleotide derived from clone 8, a single band of the same size was detected in
all 6 strains Fig. 5. When EcoRI digested DNA was probed with a 279 bp oligonucleotide derived from clone
13, two different restriction patterns were revealed. The Gonzalez, Ramiren˜o, Tuxpan, San Felipe and Corrales
strains showed two fragments between 10 and 20 kb, whereas the Coatzacoalcos strain showed a single frag-
ment 10 kb, with a stronger hybridization signal than the other strains Fig. 6. The strong signal was con-
served in the Coatzacoalcos strain when hybridization was carried out with DNA digested with two restric-
tion enzymes.
Genomic DNA from the six strains was amplified by PCR with the same primers used to produce probes 8
GS81-GS82 and 13 GS131-GS132. These genomic DNA fragments maintained the same size as the cDNA
fragments indicating that there were no intronic sequences in these specific regions data not shown.
4. Discussion
