Journal of Virological Methods, 3 1 ( 199 1) 113- 118 Elsevier 113

VIRMET 01101

Direct double-stranded DNA sequencing with baculovirus genomes

Hwei-gene Heidi Wang* and M.J. Fraser

Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, U.S.A.

(Accepted 20 September 1990)

Summary

Double-stranded DNA sequencing with the modified T7 DNA polymerase (SequenaseTM) was performed directly with nuclear polyhedrosis virus DNA genomes. The conditions were optimized to allow for a rapid and unambiguous sequence analysis of nuclear polyhedrosis virus genomes.

Double-stranded DNA sequencing; SequenaseTM; Nuclear polyhedrosis virus

Nuclear polyhedrosis viruses (NPV) belong to Subgroup A of the family Baculoviridae (Matthews, 1982). Autographa californica nuclear polyhedrosis virus (AcMNPV) is the prototype baculovirus, having a double-stranded circular genome of approximately 128 kb (Smith and Summers, 1978, Miller and Dawes, 1979).

The AcMNPV genome acquires lepidopteran transposons after passage in Trichoplusia ni (TN-368) cells (Fraser et al., 1983, 1985; Miller and Miller, 1982; Carstens, 1987; Kumar and Miller, 1987; Cat-y et al., 1989). Two classes of these transposons are characterized extensively, the TFP3 element (Wang et al., 1989) and the IFP2 element (Car-y et al., 1989).

TFP3 transposons are approximately 750 bp in length and are flanked by 15 bp terminal inverted repeats (Wang et al., 1989). Short stretches of oligonucleotides homologous to the internal sequences near both termini are useful in sequencing

Correwondence to: M.J. Fraser. Dem. of Bioloeical Sciences. Universitv of Notre Dame, Notre Dame, IN 465’56, U.S.A.

B d

*Present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring U.S.A.

Harbor, NY 11724,

0168-8510/91/$03.500 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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newly isolated TFP3 elements (Wang, et al., 1989). These oligonucleotides are oriented outwardly to allow sequencing from TFP3 inverted repeats into the flanking disrupted virus or host sequences (Wang, et al., 1989), or inwardly to obtain the internal sequences of new or altered TFP3 elements (Wang 1990).

Although these oligonucleotide primers facilitate the characterization of newly isolated TFP3 elements at insertion sites within the AcMNPV genome, the overall sequencing process is still time consuming. Restriction enzyme fragments of mutant viral genomes containing intact TFP3 elements are first cloned into plasmid vectors, and the purified plasmid DNAs are then subjected to double- stranded DNA sequencing by the dideoxy chain termination method (Sanger et al., 1977; Chen and Seeburg, 1985; Zhang et al., 1988).

Since dideoxy chain termination DNA sequencing can be accomplished with double-stranded plasmid DNAs, we reasoned that the same feat could be per- formed using the double-stranded circular DNA genome of a baculovirus. This report details our attempts to expedite the process of analyzing insertion sites for lepidopteran transposons within baculovirus genomes through direct sequencing from mutant AcMNPV DNAs. The conditions described should be useful in determining the nucleotide sequence directly from any baculovirus genome.

Materials and Methods

Preparation of AcMNPV viral DNA

Suspension cultures of Spodoptera frugiperda IPLB-SF2 1AE cells (Vaughn, et al., 1976) were initiated by seeding a 100 ml spinner bottle (Bellco) containing EX-CELL 400 (JR Scientific) with 2.5 x 10’ cells. The suspension cultures were incubated with constant agitation and aeration at 28OC for 3 to 5 days until the density reached 3 x lo6 cells/ml. The cells were pelleted at 1000 t-pm for 5 min and were resuspended in 5 ml of virus inoculum at a multiplicity of 10 plaque forming units (PFU) per cell. The cells were incubated in the virus inoculum for 1 h with gentle agitation, and were then added to 100 ml of fresh medium in a spinner bottle.

After 5 days at 28OC extracellular virus (ECV) was purified from the suspen- sion culture supematants essentially as previously described (Fraser et al., 1983). Cells and debris were removed at 2800 r-pm for 10 min, and the clarified super- natants were transferred to SW28.1 rotor tubes (Beckman) and pelleted through a 5 ml, 35% w/w sucrose cushion at 25 000 rpm for 30 min. The virus pellet was resuspended in 1 x TE buffer (10 mM Tris-HCl + 1 mM EDTA, pH 7.5), transferred to 1.5 ml Eppendorf tubes, and pelleted in a microfuge for 15 min to remove residual sucrose. The washed ECV pellets were resuspended in 2 ml of 1 x TE buffer containing 0.1 M KCl, 1% SLS, and 0.1 mg/ml proteinase K (Sigma) and incubated at 65OC for 1 h. The DNA was extracted with phenol and chloroform:isoamyl alcohol (24:l) and precipitated with ethanol. The pre- cipitated viral DNA was pelleted, resuspended in an appropriate volume of 0.1

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x TE buffer, and stored at 4°C. Vial DNA prepared according to this method was suitable for DNA sequencing.

Sequencing reaction

The sequencing protocol was a modification of the one provided with the TM Sequenase kit (United States Biochemical Corporation). The p20UT oligonu-

cleotide primer was used in all sequencing reactions (Wang, 1990). Two pug of the viral DNA template was combined with a six molar excess of primer (3 pmol) as compared to 0.5 pmol used for single-stranded sequencing. The template/primer mix was boiled at 1OOOC for 12 min to ensure complete denaturation, and the mix was placed immediately on ice for 15 min to allow reannealing between the template and the primer. Two ~1 of 32PdATP (3000 Ci/mol, ICN Radiochemical Co.) was used per sequencing reaction rather than the 1 ~1 specified for single stranded DNA sequencing. The extension reactions were carried out at room temperature and the termination reactions were incubated at 42OC. The rest of the procedures were as recommended by the SequenaseTM manual.

Results and Discussion

The USK5c23 mutant virus (Kumar and Miller, 1987) and the pTFF3/5c23 plasmid containing the HindIII-I fragment of USK5c23 with the TFP3 element insertion (Wang, 1990) were used as templates for optimizing the sequencing conditions. The USK5c23 viral DNA was purified from ECV (Materials and Methods) and the pTFPc/5c23 plasmid DNA was purified through a CsCl gradient (Sambrook et al., 1989). The oligonucleotide primer p20UT (Wang et al., 1989; Wang, 1990) was previously synthesized based on the sequence internal to the TFP3 TRR (right terminal inverted repeat) and was used in all sequencing reactions.

Two critical variables in a sequencing reaction include the concentration of the primer with respect to that of the DNA template and the time allowed for the template and primer to reanneal. Double-stranded DNA sequencing employing plasmid DNAs requires an excess of primer to permit optimal reannealing between the primer and the denatured strand of the DNA template. Sufficient time is required for the primer and the denatured template strands to reanneal, but excessive reannealing leads to renaturation of the separated DNA strands. These two criteria, primer ratio and reannealing time, were examined in an optimization test (Fig. 1).

While the concentration of the double stranded DNA template was main- tained at 2 pg, the pmol concentration varied depending on the size of the DNA template. The two concentrations of p20UT primer tested were 3 and 7.5 pmol. Reannealing of template and primer was carried out on ice for either 5 or 15 min. The rest of the extension reaction was performed as recommended by the man- ufacturer (United States Biochemicals) and the fragments were electrophoresed

(pmole)

TFP3/5c23 USK5c23

3 7.5 3 7.5

TFP3/5c23

3 7.5

USK5c23

3 7.5

reanneal for 5 min reanneal for 15 min

Fig. 1. Optimization of dideoxy sequencing reactions for AcMNPV viral DNA. Picomole concentrations of primer and reannealing times were varied for double-stranded extension reactions using the primer p20UT with the plasmid, TFP3/5c23, or the mutant intact viral genome, USK 5~23. Each set of sequencing reactions is organized GATC. The position of the TTAA target site, the right terminal

repeat (TRa) of the TFP3 insert, and flanking viral sequences (AC) are indicated at the right.

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on a 6.6% polyacrylamide/urea gel. Sequencing patterns with the least amount of background bands were generated

using 3 pmol of primer and a 15 min reannealing time (Fig. 1). As the primer concentration increased from 3 to 7.5 pmol so did the background bands. The increase in reannealing time did not add to the background but rather improved the efficiency of extension. The sharp and intense bands from both the plasmid and viral DNA template reactions were the result of a longer reannealing time (Fig. 1).

The sequence extending from the p20UT primer binding site read 5’ GGCACT- GAATGGG 3’ and represented the majority of the TFP3 TRR. This was followed by the TFP3 transposon target site ‘TTAA’. The bands beyond the ‘TTAA’ tetranucleotides constituted the flanking viral sequences at the insertion site (Fig. 1). While these features near the primer were easily discerned, increased back- ground was evident with further extension and reading beyond the first 100 bases was difficult.

‘This is the first demonstration of direct sequencing from an AcMNPV viral genome, and to our knowledge the first example of double stranded genomic se- quencing from such a large piece of DNA (128 kb). The background encountered with extension might be eliminated using end-labeled primers for sequencing rather than the conventional uniform labeling and extension-termination reaction (McGraw, 1984; Heiner et al., 1988). The protocol we describe allows elimina- tion of the cloning step in analyzing new transposon insertion sites within the baculovirus genome, or in sequencing regions of the viral genome. The proce- dure permits a rapid analysis of new sequences within 2 days of viral DNA preparation.

Acknowledgements

We thank Dr Lois Miller for sharing the USK virus examined in this analysis. This research was supported by Public Health Service Grant No. ROl AI 22610 to M.J.F.

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