<<

. 10
( 61 .)



>>

de Wind, van Z@, and Berns
60
3 Decant the supernatants, tapping off the last drops
4. Resuspend the cell pellets by vortexmg m 0 2 mL cold solution 1 Incubate for 5
mm on ice
5. Add 0 4 mL solution 2, mix by shaking, and incubate for 5 mm on ice.
6. Add 0.3 mL solution 3, mtx by shaking, and mcubate for 5 mm on Ice
7 Spin for 5 min at maximum speed
8 Carefully transfer the supernatants to new mtcrofuge tubes contammg 2 pL RNase
A stock solution Mix and incubate for 15 mm at 37°C
9. Add 0 4 mL phenol,chloroform.isoamyl alcohol. Vortex for 30 s, centrifuge for 2
mm at maximum speed, and transfer the supernatant to new tubes
IO. Add 0 7 mL isopropanol, mix, and centrifuge for 5 mm at maximum speed. Discard
the supernatants, centrtfuge briefly, and remove the last drops usmg a micropipet
1 I Dissolve the pellets in 50 pL TE. The plasmid preps may be stored at 4°C or at -2O™C.

3.1.10. Cleavage and Phosphatase Treatment of the Cosmld Vector
Prior to lrgatron to the virus DNA fragments, the vector, that now con-
tains the approprtate clonmg site, IS lmeartzed. Subsequently, the 5™ phosphate
moieties are removed by treatment with calf intestinal phosphatase (CIP). This
prevents recirculation of the vector
1, In a microfuge tube, mix the following 2 pg cosmid vector (approx 20 PL of a
mintprep), 4 pL of the appropriate 10X restrtction enzyme dtgestton buffer, 10 U
restriction enzyme that cleaves the cloning linker, and distilled water to 40 pL
Incubate for 2 h at 37°C.
2 Add 80 pL TE and one vol phenol chloroform tsoamyl alcohol Vortex for 30 s,
centrifuge for 2 mm at maximum speed, and transfer the supernatant to a clean
microfuge tube Subsequently, precipitate the DNA by addttion of 0.1 vol 3M
sodmm acetate pH 5.2 and 3 vol ethanol Mix and store on ice for 15 mm, and
centrifuge for 10 mm at maximum speed. Wash the pellet with 1 mL 70% ethanol
and take the DNA up m 48 pL distilled water Take apart a sample of 5 pL (I e ,
approx 0.2 pg) as a control for the effctency of the CIP treatment (step 6).
3. To the rest of the DNA, add 5 pL 10X CIP buffer and 1 U CIP Incubate for 30
mm at 37°C add again 1 U CIP, and remcubate for 30 mm at 37°C (see Note 25).
4. Add 50 pL TE and 1 vol phenol. chloroform. isoamyl alcohol. Vortex for 30 s,
centrifuge for 2 mm at maxtmum speed, and transfer the supematant to a clean
mtcrofuge tube. Repeat the extraction twtce, both times using 50 pL chloroform,
Subsequently, precipitate the DNA by addition of 0.1 vol 3M sodium acetate pH
5.2 and 3 vol ethanol. MIX and store on Ice for 15 min and centrifuge for 10 min
at maximum speed Wash the pellet with 1 mL 70% ethanol and dissolve the
DNA m 10 pL TE. The DNA can be stored at -2O™C.
5 To verify cleavage and yield of the vector, electrophorese 1 pL on a 0 8% agarose
mmigel Also load 0 2 pg uncleaved cosmid vector DNA as a marker
6. To verify the efficiency of the CIP treatment, mix 0 1 pg of the dephosphorylated
vector wtth 1 mL 10X hgase buffer, 1 (Werss) Umt T4 ligase and distilled water
67
Characterization of a Herpesvirus Genome
to 10 nL As a control, perform the sameligation using 0.1 ng of the untreated
cleaved DNA, set apart in step 2. Incubate for 4 h to overnight at 15°C and
transform E. colzwith 2 nL of both ligation mixtures, as described m Section
3.1.8. The CIP-treated DNA should yield at least lo-fold less colonies than the
untreated DNA
3.1.11 Cloning of Virus DNA Fragments into the Cosmid Vector
The vn-us DNA fragments are ligated to the linearized dephosphorylated
vector fragments under condttions that favor the formation of linear concate-
mers (see Fig. 2). Since the vector does not self-hgate owing to the CIP treat-
ment, an excessof vector IS used to ensure the formation of vector-insert hetero
concatemers. Subsequently, a monomer is packaged mto empty phage lambda
heads using a cornmerctally available packagmg kit. During the latter process,
the concatemers are cleaved at the cos sites present in the vector molecules; the
virus DNA fragment between two subsequent cleaved cos sites is then mter-
nalized into the lambda heads, usmg the packagmg signal present in the cos
sites. After packaging, E colz IS infected with the phages; this is followed by
selection on ampictllm containing LB plates (Fig. 2). Transformant E. cob colo-
nies contam the recircularized cosmid containing the Inserted virus DNA frag-
ment. We do not provide a protocol here for packaging and mfection smce the
procedure varies somewhat depending on the packaging kit used; a detailed
protocol comes with each commerctal kit.
1. Mix 1 pg of the prepared shearedvirus DNA, 1 pg dephosphorylatedcosmid
vector DNA, 1pL 10X hgasebuffer, 1(Weiss)Unit T4 ligase,anddistilled water
to 10mL. Incubate for 4 h overnight at 15°C
2. Package0.5 pg of the concatemers phagelambda headsand infect (and plate
in
out) E co11 describedin the manual of the packagingkit.
as
3.7.12. Analysis of Cosmid C/ones
Cosmid clones containing virus DNA must be thoroughly analyzed to
fulfil the following criteria: (1) At least one (see Note 8) set of clones,
containing all of the genomic information of the virus (see Note 14) and hav-
rng overlaps of the optimal size, has to be selected (see Note 13); and (2) it
should be verified that the selected cloned virus DNA fragments do not contain
deletions or rearrangements.
To these purposes, miniprep DNA of the cloned vn-us DNA fragment 1s
cleaved with mixtures of two restriction enzymes: the enzyme that cleaves the
cloning site for the virus DNA (this liberates the insert from the vector),
together with a dragnostic enzyme, for which a prectse restriction map of the
virus genome is available. On an agarose gel, size, overlaps, and integrity of
the cloned virus DNA fragments can then be analyzed.
de Wind, van Z(il, and Berns
62
1 Using wooden toothpicks, inoculate, with fresh single transformant colonies, all wells
of two stenle marked 96-well mtcrottter plates containing 100 pL LB contammg 15%
glycerol as well as amptctlhn, per well. Use the first 48-96 toothpicks to also mocu-
late numbered tubes with 2 mL LB contammg amptctllm (see Note 26) Cover the
well plates and incubate the plates overnight at 37™C m a standing incubator (see
Note 27). Incubate the tubes overmght at 37°C m a shakmg mcubator.
2 Freeze the well plates m a -80°C freezer These mmicultures are stable and
serve as a stock
3. Prepare cosmrd mmtprep DNA from the 48-96 maculated 2-mL cultures, as
described m Section 3 1 9
4 Mix in a nncrofuge tube 5 ˜.ILminiprep DNA, 2 pL of the appropriate digestion buffer
(see Note 28), 5 U each of the diagnostic enzyme and of the enzyme that cleaves the
cloning linker, distilled water to 20 pL In addition, cleave 4 pg of total purified virus
DNA, in a volume of 40 pL, with 20 U of the diagnostic enzyme (this is sufficient for
two gel loadings). Incubate the digestions for 2 h at the appropriate temperature
5 Load all digestions on a 0 8% agarose gel, together with a HzndIII digest of phage
lambda DNA as a marker (see Note 16) and electrophorese until the bromophe-
no1 blue dye has migrated at least 10 cm
6 Compare the digests of the vartous cosmtds with that of the cleaved total vnus
DNA This enables one to determine the locatton of the cloned fragments on the
vtrus restriction map In addition thts enables screenmg for clones that have over-
laps of the desired size. Moreover, large deletions or rearrangements are easily
detected. If no complete set for overlap recombmatton can be constttuted usmg
the first 48-96 analyzed clones, inoculate another 48-96 clones from the frozen
stocks, and repeat the analysis. Repeat the procedure until suffictent, apparently
intact, clones have been found to constitute one or more sets of clones for overlap
recombmatron. Include ample backup clones since minor rearrangements may be
found durmg more detailed analysis
7 Once a set of clones to be tested in overlap recombination IS selected, proceed by
growing up a 500-mL LB + amptctllm culture of each clone in 2-L flasks Start
from the frozen stocks
8. Isolate and purify cosmid DNA by a scaled-up alkaline lysts and Cesmm chloride-
ethtdmm bromide density gradient centrrfugation, as described in Section 3 1.13

3.1.13. Large-Scale Plasmid Isolation by Alkaline Lysis, Followed
by Cesium Chloride- Ethidium Bromide Density Gradient Cen trifuga fion
The first part of this procedure (steps l-6) is essentially the same as the
mimprep procedure described m Section 3.1.9.
1 Centrifuge the cultures for 15 mm at 5OOOg.
2 Decant the supernatants, shaking off the last drops
3. Resuspend the cell pellets by vortexmg in 8 mL cold solution 1 Transfer the cells
to 50-mL conical tubes and incubate for 5 mm on me.
4 Add 16 mL solutron 2, mix thoroughly by mversion, and Incubate for 5 mm on tee
63
Characterization of a Herpesvirus Genome
5. Add 12 mL solution 3, mix thoroughly by inverston, and incubate for 20 mm on Ice.
6 Centrifuge 30 mm at 5OOOg,4°C
7. Carefully transfer the supernatants to new 50-n& conical tubes, taking care not to
transfer any of the loose pellet. Precipitate the nucleic acid by adding 0 6 vol rsopro-
panol, mix, and immediately centrifuge 15 min at 5OOOg,room temperature. Discard
the supematant, centrifuge briefly, and remove the last drops using a mrcroprpet
8 Dissolve the pellets in 3.7 mL TE Add 4 3 g CsCl, dissolve, and add 0 6 mL 10
mg/mL ethidmm bromide solution (see Note 29). MIX and centrifuge for 15 mm
at 5000g at room temperature to remove the precipitated protein
9. Fill wrth the supematant 5 mL quick seal (0.5 x 2 m.) centrifuge tubes, equrhbrate,
seal, and centrifuge for 8-16 h at 65,000 rpm at 15™C m a VT1 80 vertical rotor
10. Pierce the top of the tubes with a 25-gage needle Under long-wave UV light, two
bands are vrsrble. The upper one consists of bacterial DNA and relaxed cosmrd
DNA, the bottom one consists of supercoiled cosmrd DNA Pierce the tubes just
below the lower band with a 25-gage needle attached to a 1-mL syringe and dram
the supercoiled (lower) cosmid band. Transfer the DNAs to 15-mL conical tubes
taking care not to shear the DNA by squeezing the syringe too hard
11 Add 3 vol TE and 2-3 (end) vol ethanol. MIX and leave for 15 mm on Ice Centrr-
fuge the tubes for I5 mm at 5000g at 4°C Discard the supernatant and remove
the last drops usmg a mrcroplpet after recentrrfugmg briefly. Dissolve the pellets
in 400 pL TE and transfer the solution to microfuge tubes
12 To remove the ethrdmm bromide, extract the DNA by vortexmg gently for 30 s
with 1 vol phenol:chloroform:isoamyl alcohol. Centrifuge for 5 min at 5000g and
transfer the supernatant to a new tube Repeat the extractron until the supernatant
does not fluoresce any more when kept m front of a long-wave UV lamp. Precipr-
tate the DNA by adding 0 1 vol3Msodmm acetate and 2 vol ethanol After mver-
sron of the tubes, a cloudy DNA precipitate should appear
13. Frsh out the DNA precipitate using a drawn-out Pasteur prpet, wash the DNA m
70% ethanol, and transfer to a new mtcrofuge tube. Centrifuge briefly, prpet off
the ethanol, let the remammg ethanol evaporate, and dissolve the DNA overnight
at 4°C (or 15 mm at 65°C) m 200 pL TE. Store the DNA at 4°C.
14. Determine the DNA purity and concentration by measurement of the OD260/OD280
ratio (see Note 30)
15. Verify the tdenttty and integrity of the cosmids by digesting of 1 pg DNA with
appropriate diagnostic restrrctron enzymes, followed by gel electrophoresrs, as
described m the previous section (steps 4 and 5).
3.1.74. Regeneration of Virus by Overlap Recombination
of Cloned Subgenomic DNA fragments
Once a set of overlappmg cloned subgenomic cloned fragments of the virus,
that meets the requested criteria, has been selected and expanded, the frag-
ments will be used to test regeneration of viable vnus after cotransfectlon of
permisslve cells. For efficient recombmatlon, the cloned inserts have to be
excised from the cosmrd vector by restriction enzyme digestion, using the
de Wind, van ZJI, and Berns

enzyme that cleaves the cloning linker (see also Note 2). The digested cosmid
clones may subsequently be used for transfection. Regenerated virus 1s
expanded for DNA analysts and, tf possible, for experimental infection of
model animals. If results of these assays do not show differences with control
assays using nonmanipulated virus, the set of cosmid clones is used as a basis
for the generation of large series of oligonucleotide insertion mutants, as
described in the following part of this chapter (see Section 3.2.).

3.1.7 5. Excision of Cloned Fragments from the Cosmid Vector
1 For every cosmtd clone to be used m overlap recombinatton, mtx m a microfuge
tube 10 ug CsCl purified cosmid DNA, 5 yL 10X digestion buffer for the
restriction enzyme that cleaves the clonmg linker, 50 U of the enzyme, and
distilled water to 50 pL Incubate for 2 h at the indicated temperature for the
enzyme. Electrophorese 1 pL of each digest on a 0 5-O 8% agarose mmtgel to
verify completeness of the digestions Load 0 2 yg of each uncleaved cosmid clone
as a control
2. Add 50 uL TE and 1 vol phenol*chloroform*tsoamyl alcohol Vortex gently for 30 s,
centrtfuge for 2 mm at maximum speed, and transfer the supernatant to a clean
mtcrofnge tube. Subsequently, precipitate the DNA by addttton of 0.1 vol3M sodmm
acetate pH 5.2 and 2 vol ethanol After mverston of the tubes, a cloudy DNA precipitate
should appear Fish out the DNA precipitate using a drawn-out Pasteur ptpet, wash the
DNA in 70% ethanol and transfer to a new micromge tube. Centrifuge briefly, ptpet off
the ethanol, let the remannng ethanol evaporate, and dissolve the DNA overnight at
4°C (or 15 min at 65°C) m 10 pL sterile 0 1X TE. The DNA may be stored at 4OC
3. I. 16. Calctum Phosphate Transfection
The most common method to transfect adherent cells with DNA 1s by cal-
cium phosphate-DNA coprectpitation (19). These coprecipttates are taken up
by the cell by pmocytosis. Intracellularly, the homologous ends of the
internalized DNA fragments recombine resulttng m the regeneration of an
Intact virus genome (see also Note 14). Ultimately, this will lead to the produc-
tion of virus particles that infect neighboring cells, thus resultmg in plaque
formatton. This protocol consumes 20 pg DNA (see Note 3 1). It is advised to
transfect, as a positive control, total purified virus DNA.
1 One day before transfection, seed a IO-cm dish with permtsstve cells so that the cell
density will be 2540% at the time of transfection. Add medium to a total of 10 mL
to the cells.
2. For every transfectton, mix m a sterile microfuge tube, m equimolar amounts, a
total of 20 pg of digested cosmtd clones Take up m 950 pL HBS buffer and
vortex gently for 10 s.
3. Add 50 mL 2 5M CaCl, and immediately vortex gently for 30 s. Leave the tube
for 20 mm at room temperature m the dark.
Characterization of a Herpesvirus Genome 65
4 Resuspendthe precipitate well by pipetmg a few ttmes up and down and add
the precipitate dropwise to the cells (see Note 32). Swirl the medium and uv.x-
bate the cells 4-5 h at 37°C in the CO2incubator (seeNote 33).
5. Aspire the medium,add 2 mL medium containing 15%glycerol andswirl gently.
After 90 s (seeNote 34), aspire the medium andwash the cells twice with 10mL
sterile PBS,Add 10mL medium andincubate at 37°C in the CO, incubator until
a generalizedcytopathrceffect is visible (seeNote 35).
3.7.17, Analysis of Reconstituted Virus
The virus obtained by overlap recombination must thoroughly be mvestr-
gated to ascertain that no mutations, that have escaped detection during the
analysis of the individual cosmid clones, have occurred during the process of
manipulation, cloning, and transfection of the virus DNA. Several genotypic
and phenotypic parameters may be analyzed to accomplish this. First, a thor-
ough comparison should be made between restrictron digests of the genomes
of both virus that is reconstituted by overlap recombination, and nonrecon-
stituted (1.e , nonmampulated) virus Although tt is helpful to posses a
restrtctton map for a number of diagnostic enzymes, this is not an absolute
requirement, Digests may also be performed “at random,” i.e., with an
enzyme for which cleavage sites are not known; this provides fingerprints of
both the nonmampulated and the mampulated vu-us genomes. It 1sclear that
both fingerprmts should be identrcal. We are unable to give state-of-the-art
protocols for these analyses smce protocols somewhat depend on the virus
under study, the enzymes used, and so on. We will however provide a proto-
col of the methodology as was applied for PRV. Next to this type of geno-
typic analysts, reconstituted virus should be compared with nonmarupulated
virus with respect to various phenotypic parameters as, e.g., plaque pheno-
type, growth in cultured cells and, ultimately, pathogenesis and virulence
m experrmentally infected ammals. Since again there is no exclusive proto-
col for this, and phenotypic analysrs of herpesvirus does not fall within the
scope of this chapter, we do not provide protocols for these experiments
here. However, one may find applicable protocols m other chapters of this
volume.
3.7.78. Genotypic Analysis of the Reconstituted Virus Genome
For PRV, we have been able to study the genome of reconstituted strains by
directly isolating total DNA from cells infected for 8 h (i.e., the duration of a com-
plete infection cycle) at high multrplicity. This DNA consists for a major part of
virus DNA; agarosegels of restriction enzyme digestions will show a foreground
of bands derived from the virus genome and a background smear consisting of
drgested cellular DNA. A photograph of such a gel is shown in Ftg. 5. If the vnus
de Wind, van Zijl, and Berns
66
Ftg. 5. (opposite) Example of a servesof oligonucleottde msertton mutants that are
generated m a cosmtd (named c-448) that carries 41 kbp of the PRV genome (6)
Top panel™ (A) Physical map of the PRV genome (see also Fig. 1A). Indicated are
the recognition sites of the diagnosttc enzyme BamHI The numbermg refers to the
respective BamHI fragments (B) The four PRV cloned subgenomtc regions used m
overlap recombmation, (C) PRV subgenomic region cloned m cosmid c-448. Dtag-
nostic enzymes used for mapping the sue of msertton were (B) BarnHI, Bg, BglII,

<<

. 10
( 61 .)



>>