Supplementary Material
Common materials and methods
All polymerase chain reactions (PCRs) were carried out in a final volume of 50 μL in 1X
Phusion™ buffer GC with 0.2 mM of each dNTP, 1 μM of each primer (see Supplementary
Table 1 for oligonucleotide sequences), 1 M betaine, 0.6 U of Phusion™ DNA polymerase (New
England Biolabs, Ipswich, MA) and 1 μL of template, except where indicated otherwise. All
PCRs were carried out in a Mastercycler® ep gradient S thermal cycler (Eppendorf, Foster City,
CA). DNA extraction from agarose gel fragments was carried out using the QiaQuick™ Gel
Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions with 50 μL
of elution buffer.
Sequences obtained from degenerate and semi-degenerate PCRs, TOPO® clones and
RACE reactions were analyzed and assembled into full-length mRNA and genomic DNA
sequences using ContigExpress, a component of Invitrogen Vector NTI 10.3.0.
Molecular cloning of the T. ni fdl gene
Degenerate PCR: Genomic DNA was isolated from Tn-4h cells1 according to the method of
Laird et al.2 Degenerate PCRs were carried out using T. ni genomic DNA with the primers
ASPDEG and SPDEG (Supplementary Table 1) as described previously.3 The spent reactions
were separated on a 1.2% agarose gel and amplification products of the expected size (about 420
bp) were recovered from the gel, purified, and directly sequenced with the same primers as were
used in the PCRs.
1
Semi-degenerate PCR: Semi-degenerate PCRs were carried out using T. ni genomic DNA to
extend the sequence of the degenerate fragment in both the 3′- and 5′- directions. Degenerate
primers were designed using amino acid sequences highly conserved between conceptual
translations of the Sf-fdl and putative Bm-fdl genes (Fig. 4). To obtain a portion of the 5′-end of
the Tn-fdl gene, a semi-degenerate PCR was carried out using primers TnFDL ASP2 and TnFDL
SP4DEG (Supplementary Table 1). The PCR was incubated for 20 sec at 98ºC and then cycled
25 times for (i) 10 s at 98ºC, (ii) 15 s at 72 to 60ºC (with a decreasing temperature of 0.5ºC per
cycle), and (iii) 60 s at 72ºC. The reaction was cycled another 30 times for (i) 10 s at 98ºC, (ii) 15
s at 60ºC, and (iii) 60 s at 72ºC, and finally incubated for 2 min at 72ºC. The spent reaction was
separated on a 1.4% agarose gel and an amplification product of about the expected size (1100
bp) was recovered from the gel and purified. This DNA fragment was re-amplified using the
primers TnFDL ASP3 and TnFDL SP4DEG (Supplementary Table 1) under the same cycling
conditions described above, gel purified, and directly sequenced using the same primers as were
used in the PCR.
To obtain a portion of the 3′-end of the Tn-fdl gene, a semi-degenerate PCR was carried
out using primers TnFDL SP1 and TnFDL ASP6DEG (Supplementary Table 1) under the same
cycling conditions as described above. The spent reaction was separated on a 1.4% agarose gel
and an amplification product of the expected size (about 730 bp) was recovered from the gel and
purified. This fragment was cloned into pCR®2.1-TOPO® (Invitrogen, Carlsbad, PA) according
to the manufacturer’s instructions and three independent clones were sequenced to obtain a
consensus.
5′-RACE: 3’- and 5’- RACE were used to complete the sequence of the putative Tn-fdl
gene. Total RNA was isolated from a mid-log culture of Tn-4h cells with the RNeasy® Plus
2
Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and used to
prepare 5′-RACE-ready RNA with the GeneRacer™ kit (Invitrogen) according to the
manufacturer’s instructions. This RNA was then reverse transcribed using Thermo-X™ reverse
transcriptase (Invitrogen) with the TnFDL ASP1 primer (Supplementary Table 1) according to
the manufacturer’s instructions and incubated for (i) 5 min at 50ºC, (ii) 15 min at 55ºC, (iii) 30
min at 60ºC, and (iv) 15 min at 60ºC and then diluted with of 40 μL of TE buffer and stored at 20ºC. A sample of the cDNA was then used for a 5′-RACE reaction with the TnFDL ASP6 and
GeneRacer™ 5′ primers (Supplementary Table 1). The reaction was incubated for 30 s at 96ºC,
cycled 5 times for (i) 20 s at 96ºC and (ii) 60 s at 72ºC, and then cycled 13 times for (i) 20 s at
96ºC, (ii) 20 s at 72 to 60ºC (with a decreasing temperature of 1ºC per cycle), and (iii) 40 s at
72ºC. The reaction was then cycled another 30 times for (i) 20 s at 90ºC, (ii) 20 s at 60ºC, and
(iii) 40 s at 72ºC, separated on a 1.4% agarose gel, and an amplification product of about 520 bps
was recovered and purified. This DNA fragment was re-amplified using the GeneRacer ™ 5′
nested primer and either the TnFDL ASP6 or ASP7 primers (Supplementary Table 1). These
reactions were incubated for 30 s at 96ºC, cycled 5 times for (i) 20 s at 96ºC and (ii) 45 s at 72ºC,
and then the reactions were cycled 13 times for (i) 20 s at 96ºC, (ii) 15 s at 72 to 60ºC (with a
decreasing temperature of 1ºC per cycle), and (iii) 30 s at 72ºC. Finally, these reactions were
cycled another 30 times for (i) 20 s at 96ºC, (ii) 15 s at 60ºC, and (iii) 30 s at 72ºC, separated on
a 1.4% agarose gel, and amplification products of about 520 and 500 bps were recovered and
directly sequenced using the TnFDL ASP6 or ASP7 primers, respectively.
3′-RACE:
3′-RACE-ready cDNA was prepared from T. ni total RNA isolated as
described above. The reverse transcription step was carried out using Thermo-X™ reverse
transcriptase with the GeneRacer™ Oligo dT primer (Supplementary Table 1). The reaction was
3
set up according to the manufacturer’s instructions, incubated under the same conditions as for
5′-RACE, and then diluted with 40 μL of TE buffer and stored at -20ºC. A sample of the cDNA
was then used for a 3′-RACE reaction with the TnFDL SP4 and GeneRacer™ 3′ primers
(Supplementary Table 1). The reaction was incubated for 30 s at 96ºC, cycled 5 times for (i) 20 s
at 96ºC and (ii) 45 s at 72ºC, and then cycled 13 times for (i) 20 s at 96ºC, (ii) 15 s at 72 to 60ºC
(with a decreasing gradient of 1ºC per cycle), and (iii) 30 s at 72ºC. Finally, the reaction was
cycled another 30 times for (i) 20 s at 96ºC, (ii) 15 s at 60ºC, and (iii) 30 s at 72ºC, separated on
a 1.4% agarose gel, and an amplification product of approximately 600 bps was recovered and
purified. This DNA fragment was then re-amplified using the TnFDL SP5 and GeneRacer™ 3′
nested primers (Supplementary Table 1). This PCR was incubated for 15 sec at 96ºC, cycled 5
times for (i) 15 s at 96°C, (ii) 35 s at 72°C, and then cycled 13 times for (i) 15 s at 96°C, (ii) 15 s
at 72 to 60°C (with a decreasing temperature of 1°C per cycle), and (iii) 20 s at 72 °C. The
reaction was finally cycled another 30 times for (i) 15 s at 96°C, (ii) 15 s at 60°C, and (iii) 20 s at
72°C, separated on a 1.4% agarose gel, and an amplification product of 500 bps was recovered,
purified and directly sequenced using the TnFDL SP5 primer (Supplementary Table 1).
Amplification of the full-length Tn-fdl open reading frame for baculovirus expression
After the full-length sequence of the Tn-fdl open reading frame had been determined, it was
PCR-amplified from both cDNA primed with the GeneRacer ™ Oligo dT primer and genomic
DNA (including the intron) using the TnFDL FL SP2 BD and TnFDL ASP BD primers
(Supplementary Table 1). These reactions were incubated for 20 s at 98°C, then cycled 25 times
for (i) 15 s at 98°C, (ii) 10 s at 72 to 60°C (with a decreasing temperature of 0.5°C per cycle),
and (iii) 60 s at 72°C. The reactions were then cycled another 30 times for (i) 15 s at 98°C, (ii) 10
4
s at 60°C, and (iii) 60 s at 72°C, separated on a 1% agarose gel, and amplification products of the
expected sizes were recovered and purified. The DNA fragments obtained using cDNA or
genomic DNA as templates were cloned into the pENTR™/D-TOPO® vector (Invitrogen)
according to the manufacturer’s instructions to produce plasmids designated pENTR-TnFDL-C
and pENTR-TnFDL-G, respectively. Four independent clonal isolates of each plasmid were
sequenced and a clone with the consensus sequence of pENTR-TnFDL-C was used to produce a
recombinant baculovirus designated AcTn-FDL by the BaculoDirect™ method (Invitrogen)
according to the manufacturer’s instructions.
Molecular cloning of the B. mori fdl gene
Bombyx mori genomic database searches: A tBLASTn search4 of Bombyx mori genomic
sequences was carried out with the SfFDL conceptual translation as the query using the online
NCBI interface. This search yielded, among others, sequences with the accession numbers
BAAB01046610, BAAB01083831 and BAAB01153187. BAAB01046610 comprised a putative
5′-coding exon, including a potential translation initiation site (nts 25-200), and its conceptual
translation product was highly similar to the N-terminal amino acid sequence of SfFDL. The
other B. mori sequences, BAAB01083831 and BAAB01153187, were joined in silico to yield a
contig encoding the putative Bm-fdl 3’-coding exon including a translational termination site.
The conceptual translation product of this exon was highly similar to the C-terminal amino acid
sequence of SfFDL. The 5’- and 3’- coding exons were joined in silico using splice junctions
predicted with high probability by NetGene25 to obtain a contiguous open reading frame
encoding the putative Bm-fdl gene product.
5
Amplification of the predicted full-length Bm-fdl open reading frame for baculovirus
expression
After the full-length sequence of the Bm-fdl open reading had been determined as described
above, it was amplified from both reverse transcribed RNA and genomic DNA (including the
intron) isolated from B. mori larvae. Genomic DNA was prepared by a modification of the
method of Laird et al.2 from a stage 2 B. mori larva (Qiufeng/Baiyu hybrid). Briefly, the larva
was homogenized in lysis buffer supplemented with RNAse A and then the homogenate was
incubated at 55°C for 1 h. The lysate was centrifuged at 13,000 g to remove debris and the DNA
was precipitated by the addition of an equal volume of isopropyl alcohol. The DNA was
dissolved in 500 µL of TE buffer and subjected to one phenol-chloroform extraction. Total RNA
was isolated from a stage 2 B. mori larva (Qiufeng/Baiyu hybrid) using the RNeasy™ Mini Plus
kit (Qiagen). The larva was homogenized in Buffer RLT plus and then centrifuged at 13,000 g to
remove debris. Total RNA was subsequently isolated according to the manufacturer’s
instructions and used to prepare 5’-RACE-ready RNA with the GeneRacer™ kit (Invitrogen)
according to the manufacturer’s instructions. In two separate reactions, samples of the 5′-RACEready RNA and total RNA preparations were reverse transcribed with the BmFDL ASP1 and
GeneRacer™ Oligo dT primers (Supplementary Table 1), respectively, using Invitrogen
Thermoscript™ reverse transcriptase. The reaction was set up according to the manufacturer’s
instructions and incubated for (i) 5 min at 50°C, (ii) 15 at 55°C, (iii) 30 min at 60°C, and (iv) 15
min at 65°C and then diluted with 40µL of TE buffer and stored at -20°C.
The predicted Bm-fdl full-length open reading frame was amplified from either the
genomic DNA or cDNA produced using the GeneRacer™ Oligo dT primer with the BmFDL FL
SP2 and BmFDL ASP1CLO (Supplementary Table 1) primers under the same conditions as
6
described above for amplification of the full-length Tn-fdl open reading frame. The spent
reactions were separated on a 1% agarose gel, and amplification products of the expected sizes
were recovered, purified, and cloned into pENTR™/D-TOPO® (Invitrogen) according to the
manufacturer’s instructions to produce plasmids designated pENTR-BmFDL-G (genomic DNA
template) or pENTR-BmFDL-C (cDNA template). Four independent clonal isolates of each
plasmid were sequenced to determine the consensus sequences. One pENTR-BmFDL-C clone
was used to produce a recombinant baculovirus designated AcBm-FDL by the BaculoDirect™
method (Invitrogen) according to the manufacturer’s instructions.
5′-RACE: To obtain the 5’-untranslated region of the Bm-fdl gene, 5′-RACE was carried
out using the BmFDL ASP4 and GeneRacer™ 5′ primers (Supplementary Table 1) with 5’RACE-ready cDNA prepared as described above. The reaction was incubated for 30 s at 96°C,
cycled 5 times for (i) 15 s at 96°C and (ii) 45 s at 72°C, and then cycled 12 times for (i) 15 s at
96°C, (ii) 15 s at 72 to 61°C (with a decreasing temperature of 1°C per cycle), and (iii) 30 s at
72°C. The reaction was then cycled another 30 times for (i) 15 s at 96°C, (ii) 15 s at 61°C, and
(iii) 30 s at 72°C and finally incubated for 1 min at 72°C. The spent reaction was separated on a
1.2% agarose gel and an amplification product of about 570 bps was recovered, purified, and reamplified using the BmFDL ASP5 and the GeneRacer™ 5′ nested primers (Supplementary Table
1) under the same cycling conditions as described above. The nested 5′-RACE reactions were
separated on a 1.4% agarose gel and an amplification product of about 550 bps was excised,
purified, and sequenced using the BmFDL ASP5 primer.
3′-RACE: To obtain the 3’-untranslated region of the Bm-fdl gene, 3′-RACE was carried
out using the BmFDL SP4 and GeneRacer™ 3′ primers (Supplementary Table 1) with 3′-RACEready cDNA prepared as described above. The reactions were cycled using the same conditions
7
as described above for 5′-RACE. The spent reaction was separated on a 1.4% agarose gel and an
amplification product of about 450 bps was excised, purified, and used for nested 3′-RACE
reactions with the BmFDL SP5 and GeneRacer™ 3′ nested primers (Supplementary Table 1)
using the same cycling conditions. The spent reactions were separated on a 1.4% agarose gel and
an amplification product of about 420 bps was excised, purified, and sequenced using the
BmFDL SP5 primer.
8
Literature Cited
1.
Joshi L, Davis TR, Mattu TS, et al. Influence of baculovirus-host cell interactions on
complex N-linked glycosylation of a recombinant human protein. Biotechnol Prog. JulAug 2000;16:650-656.
2.
Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A. Simplified
mammalian DNA isolation procedure. Nucleic Acids Res. Aug 11 1991;19:4293.
3.
Geisler C, Aumiller JJ, Jarvis DL. A fused lobes gene encodes the processing β-Nacetylglucosaminidase in Sf9 cells. J Biol Chem. Apr 25 2008;283:11330-11339.
4.
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res. Sep 1 1997;25:33893402.
5.
Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S. Splice site
prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence
information. Nucleic Acids Res. Sep 1 1996;24:3439-3452.
9
Supplementary Tables
Supplementary Table 1. Sequences of oligonucleotide primers used in this study.
10