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K,KpnI, S, Scar. Arrows indicate the positton of the msertton of the ohgonucleottde m
the different mutant derivatives of c-448 that were used m overlap recombination V.
the specific mutant yielded a viable vuus mutant after overlap recombmation. Bottom
panel Agarose gel of BamHI + EcoRI double digests of most of the oligonucleottde
contammg mutant PRV strains indicated on the top panel (C) Note that EcoRI only
cleaves the inserted ohgonucleotide. Mutant BA420-114 contams a deletion of 0 2
kbp m the thymidme kmase gene, between the ohgonucleottde insertton sites of mu-
tants 420 and 114, that is introduced by exchanging EcoRI-BgZII fragment of mutant
420 for the correspondmg fragment of mutant 114 Left are indicated the BamHl frag-
ments of the virus (see top panel [A]) Right. Sizes of the marker fragments (see also
Note 16). Sizes of the BamHI fragments after cleavage of the Inserted ohgonucleotlde
by EcoRI, m the various mutant PRV strains.
New fragments
generated by EcolRI
Mutagemzed fragment Mutant number digestion, kbp
BamHJ fragment 2 (18 1 kbp) B3 7 5,10.6
BamHl fragment 2 (18 1 kbp) B4 91,90
BamHl fragment 2 (18 1 kbp) B23 100, 8 1
BamHI fragment 2 (18.1 kbp) Bl 10.9, 7 2
BamHI fragment 2 (18 1 kbp) B6 12.0, 6 1
BamHl fragment 2 (18 1 kbp) B14 14 0, 4.1
BamHl fragment 2 (18 1 kbp) B31 14 6, 3 5
BumHI fragment 2 (18.1 kbp) B17 164, 15
BarnHI fragment 2 (18.1 kbp) B25 174, 07
BumHI fragment 2 (18.1 kbp) BA420- 114 2.8, 0.2
B15
BumHI fragment 2 (18.1 kbp) 0.6, 0.2
BumHI fragment 2 (18.1 kbp) B35 8.5, 0 9
BamHl fragment 2 (18.1 kbp) B9 7.8, 1 6
BumHI fragment 2 (18 1 kbp) B59 7 5, 1.9
fragment 2 (18.1 kbp)
BamHI B267 70, 24
BamHI fragment 2 (18.1 kbp) B26 13 7, 2.3
BanzHI fragment 2 (18 1 kbp) B24 11 2, 4.8
fragment 2 (18.1 kbp) B5 102, 58
BamHI
fragment 2 (18 1 kbp) B34 95, 65
BamHI
BumHI fragment 2 (18.1 kbp) B37 83, 77
Characterization of a Herpesvirus Genome 67




c-179
C-448
˜...,, C-443
“... ,., ... pN3HB
“.... .........
..
V”
” vvi v v v vv v vvvvv oligo-
V vvvv
8” ---.Pr.-m
N&r-
OOPF2™a.Nd GiTZ % &aww
2 X&M:: t-W k Ff V) 4 $˜0 insert.
IIIIlllllll IlIllII111111III1111 IIllllll
c
5kbp. b




1
231
5:

4 9.4
5,5™,6
6.6
7

8,8
4.4
9
10
11
12
23
20

13
14,14™
15




Fig. 5

bands do not light up sufficiently above the background smear, they can be visu-
alized by Southern blotting of the gel, followed by hybridization to a labeled probe
consisting of total purified virus DNA, or of cloned restriction fragments of the virus
(this method of analysis was done by us in initial experiments [2]). We will provide
here a protocol for the infection of cells by PRV, of the isolation of DNA from these
cells, and of restriction digestion of this DNA.
1. Seed a IO-cm dish with permissive cells 1 d prior to infection, so that cells are
subconfluent at the time of infection.
de Wind, van ZI$ and Berns
68
2 The next day, asplre the medium and overlay the cells with 2 mL of a high-titered
virus suspension (a multlphcity of infection of at least 10, e.g , a culture super-
natant) Note that, m parallel, DNA should be prepared and assayed from both
reconstituted as well as nonmampulated vnus Incubate for 1 h at 37”C, wash the
cells twice with 10 mL PBS, and add 10 mL medmm. Incubate for the length of
an infection cycle (which 1s 8 h for PRV).
3. Wash the cells with 10 mL cold PBS and lyse the cells by the addition of 2 mL
lysls buffer
4. Scrape off the lysate and transfer it to a 15-mL conical tube Incubate the tube for
2 h at 55°C or overnight at 37°C
alcohol Mix by shaking gently for 5 mm,
5. Add 1 mL phenol.chloroform:lsoamyl
centrifuge for 5 mm at 5OOOg,and transfer the supernatant to a clean tube Subse-
quently, precipitate the DNA by addition of 2 vol ethanol After inversion of the
tube, a cloudy DNA precipitate should appear
6 Fish out the DNA precipitate using a drawn-out Pasteur plpet, wash the DNA in
70% ethanol, and transfer to a mlcrofuge tube. Centrifuge briefly, plpet off the
ethanol, let the remaining ethanol evaporate and let the DNA dissolve overnight
at room temperature (or 15 mm at 65°C) m 200 pL TE. Store the DNA at 4°C
7 Using a yellow tip with cut-off end for pipeting the DNA, which should be VIS-
COUS,run 1 pL on an agarose mmlgel together with a known quantity of phage
lambda DNA to estimate the DNA concentration.
8 Digest DNA of the isolates of both the reconstituted and nonmampulated viruses
with the dlagnostlc restrlctlon enzyme(s)and preferably also with a number of
other restriction enzymes as follows* Mix in a mlcrofuge tube 2-4 pg DNA, 2 pL
of the appropriate 10X digestion buffer, 10 U restriction enzyme, and distilled water
to 20 pL Incubate for 2-4 h at the appropriate temperature for the enzyme. Ana-
lyze the digests by running side to side the different digests of DNAs from recon-
stituted and nonmanipulated viruses on a large 0 8% agarose gel. If virus bands
are not clearly visible, blot the gel and analyze by hybridization with vn-ns probes
The digests of both vu-uspreparationsshould be identical (seeNote 36)
3.2. Oligonucleotide Insertion Mutagenesis
of individual Cosmid Clones
Once the set of cosmid clones for overlap recombmatlon has been validated,
individual clones can be used for modification m vitro. We will describe here the
generation of a large series of mutant derivatives of a single cosmid (i.e., one of
the cosmlds generated for overlap recombmatlon). Each of these mutant cosmld
derivatives lacks the expression of (at least a part) of a single gene. This IS
achieved by the insertion of a mutagenic ohgonucleotlde, at a random site, wlthm
the cosmld clone. Subsequently, virus mutants that harbor the oligonucleotide
are generated by overlap recombination using the modified cosmid together with
the setof overlapping cosmlds.The stepsinvolved m this procedure are described
in the Introduction to this chapter and are schematically depicted in Fig. 3.
69
Characterization of a Herpesvirus Genome
3.2.1. Linearization of a Cosmid Clone by Restriction Enzymes
with 4-bp Specificity in the Presence of fthidium Bromide
This hasproven to be an excellent way to linearize a large DNA clone of the size
of a cosmid, at quasi-random sites.The latter 1sessentialsmce the aim of the pro-
cedure is to generate a “saturated” seriesof virus mutants, i.e., for every gene en-
coded m the cosmld clone, amutant virus strain should be obtained (see also Notes
4,6, and 8). Here we provide the protocol that is ophmalized for the linearization of
PRV cosmids using the enzymes FnuDII, N&II, and RsaI. However, cosmld
clones containing virus DNA of lower GC content (the GC content of PRV is 73%)
may require slightly different dlgestion conditions (see also Note 37).
1 Prepare, on ice, three mlcrofuge tubes, labeled FnuDII, HaeIII, and RsaI, respec-
tively, each containing 25 pg DNA of the cosmld clone to be mutagemzed (purified
by Cesium chloride-ethldium bromide density gradient centrifugation, see Section
3.1 13 ) Add 12 5 pL of the appropriate ethidmm bromide-containing digestion
buffer (see Section 2 ) and add stenle distilled water to 125 pL. Then add, respectively,
2 U FnuDII, 1 U HaeIII, and 0 5 U RsaI to the tubes. Incubate for 15 mm at 37™C
2. Add 10 pL 10% SDS to inactivate the enzymes Load the digests on a large 0 5%
preparative agarose gel Include as a control a lane with the uncleaved cosmld Elec-
trophorese overmght until the bromophenol blue dye has well migrated off the gel.
3. The gel should look, under long-wave UV light, as m Fig. 4 Visible should be
(from top to bottom) uncleaved relaxed cosmld, uncleaved supercolled cosmld,
linearized full-length cosmid, and multiply cleaved cosmld (a smear)
4. Cut out the three linear cosmld DNA bands, taking great care to avoid taking along
any of the multiply cleaved DNA. Elute the DNA usmg an electroelution apparatus.
5 After electroelutlon, transfer the eluted DNAs each to a mlcrofuge tube, add one
volume phenol:chloroform.lsoamyl alcohol, and vortex gently for 30 s, centn-
fuge for 2 mm at maximum speed and transfer the supernatant to a clean
microfuge tube. Repeat if necessary. Subsequently, precipitate the DNA by addi-
tion of 0 1 vol 3M sodium acetate, pH 5.2, and 2 vol ethanol. After inversion of
the tube, a cloudy DNA precipitate should appear
6 Fish out the DNA precipitate using a drawn-out Pasteur pipet, wash the DNA m
70% ethanol, and transfer to a mlcrofuge tube. Centrifuge briefly, pipet off the
ethanol, let the remaining ethanol evaporate, and let the DNA dissolve overnight
at 4°C (or 15 mm at 65°C) m 20 mL TE Store the DNA at 4°C.
7. Load 1 pL of each of the three linear cosmid preparations on a 0 5% agarose mimgel to
estimate concentration. asmarker0.1,0.2,and0.4 pg uncleavedlambdaDNA.
its Use
3.2.2. Ligation of the Mutagenic Oligonucleotide
to the Linear Cosmid DNA
To the ends of the randomly linearized cosmid clone, the oligonucleotide is
ligated, after whtch the ohgonucleotide containing cosmld is recirculated for
high-efficiency transformation The addition of the oligonucleotide to the
de Wind, van Zijl, and Berns
70

cosmtd DNA 1s essentially performed m the same way as the addition of the
cloning linker to the sheared virus DNA (Section 3.1 4.) Finally, the randomly
linearized cosmld, which now contams the oltgonucleottde added to the ends,
is relegated at low DNA concentratton, to favor the formatton of cn-cular cosmld
molecules (as opposed to concatemers; see Note 38).
To phosphorylate the ohgonucleottde, mix m a microfuge tube 1 pL 10X kmase
buffer, 2 pg nonphosphorylated ohgonucleottde, 1 pL 10 mM ATP, 10 U poly-
nucleottde kmase, distilled water to 10 yL Incubate for 1 h at 37°C After
kmasmg, heat the oltgonucleottde to 70°C and let tt cool down, over a period of
30 mm, to room temperature (see Note 39)
In a mtcrofuge tube, mtx 2 pg of each of the three lmeartzed cosmid preparations
Add to the microfuge tube 50-fold molar excess of phosphorylated mutagemc
ohgonucleotide (this will be approx 150 ng), 2 pL 10X ligation buffer, 5 (Weiss)
Umts T4 hgase, and distilled water to 20 pL Incubate overnight at 15°C
Add to each tube 80 pL TE and 100 mL phenol chlorofornrtsoamyl alcohol, shake
gently for 2 mm, centrifuge for 2 mm at maximum speed, and transfer the supematant
to a clean microfuge tube. Subsequently, prectpitate the DNA by addition of 0 1 vol
3M sodium acetate pH 5 2 and 2 vol ethanol Store on ice for 15 mm and centrifuge
for 5 mm at maximum speed Wash the pellet wtth 1 mL 70% ethanol and take the
DNA up m 10 pL TE Let the DNA dissolve overnight at 4°C (or 15 min at 65°C)
4. Add to the DNA. 10 pL of the appropriate 10X dtgestton buffer, 100 U of the
restriction enzyme for which the ohgonucleotide has a site, and dtsttlled water to
100 yL. Incubate for 4 h at the appropriate temperature for the enzyme.
5 Load the entire digestion mixture on a 0.5% preparattve agarose gel and electro-
phorese until the bromophenol blue marker has migrated 2 cm into the gel
Cut out the cosmid DNA band and elute usmg an electroelutton apparatus
6.
After electroelution, transfer the eluted DNA to a microfuge tube, add 1 vol
7
phenolchlorofornnisoamyl alcohol, vortex gently for 30 s, centrtfuge for 2 mm
at maximum speed, and transfer the supernatant to a clean microfuge tube. Repeat
if necessary. Subsequently, precipitate the DNA by addition of 0 1 vol3Msodmm
acetate pH 5 2 and 2 5 vol ethanol MIX and store on me for 15 mm, and centrt-
fuge for 5 mm at maxtmum speed. Wash the pellet with 1 mL 70% ethanol and
take up the DNA m 20 pL TE.
8 Load 1 yL of each of the oligonucleotide-containing linear cosmid fragments on
a 0 5% agarose mmtgel to estimate Its concentration. Use as marker 0 1,0.2, and
0.4 pg uncleaved lambda DNA
9 MIX m a mtcrofuge tube 0.1 l.tg of the cosmid fragments, 40 pL 10X hgase buffer,
2 (Weiss) Units T4 ligase, and d&led water to 400 pL. Incubate for 4 h to
overnight at 15°C
10 Prectpttate the DNA by adding 0 1 vol sodium acetate pH 5 2 and 2 5 vol ethanol
Mix by mverston and leave on Ice for 15 mm, and centrifuge at maxtmum speed
for 5 mm Wash the pellet with 70% ethanol and dry at room temperature until
the ethanol has evaporated and dissolve the pellet m 10 pL TE (overnight mcuba-
tion at 4™C or 15 mm mcubatton at 65°C)
71
Characterization of a Herpesvirus Genome
3.2.3. Transformation and Analysis of Mutagenlzed Cosmids
After addition of the oligonucleotide, E. coli 1s transformeq with the
recircularlzed mutant cosmid. Smce E co/i is not transformed very well by
large plasmids, we have used a high-efficiency protocol for transformation that
is originally developed by Hanahan (20). However, since preparation of these
competent cells is somewhat cumbersome and not always reproducible, we
recommend purchasing ultracompetent E coli cells. These are available from
many suppliers and have a competency of at least 5 x IO8colonies/pg transformed
test plasmld. A protocol for transformation comes with every batch of cells.
After transformation, a large amount of transformant colomes 1sstored and,
batch by batch, cosmld DNA 1s prepared of these transformants. To screen
against deletions and rearrangements, this DNA 1sfirst analyzed by restriction
enzyme digestion with the diagnostic enzyme plus the enzyme that cleaves the
cloning linker and gel electrophoresis. Subsequently, cosmld clones that appear
to be intact are further analyzed to determine the site of Insertion of the muta-
gemc oligonucleotide. This is done by performing minimally two double-
dtgestlons, followed by gel electrophoresls: (1) By the dlagnostlc enzyme and
the enzyme that cleaves the mutagenic ohgonucleotlde; and (2) by the enzyme that
cleaves the cloning linker and the enzyme that cleaves the oligonucleotide (see
Note 40). These digests should enable to map the site of insertion of the oligo-
nucleotide within a few hundred basepairs.
1 Transform ultra competentE. coli with 2-5 pL (seeNote 4 1)of the reclrcularized
mutagemzedcosmld preparation usmg the protocol provided by the vendor of
the cells
2 Using wooden toothpicks inoculate,with fresh single transformant colonies, all
wells of (at least)five sterilemarked96-well microtiter platesprefiiled with 100pL
LB contaming 15% glycerol and amplclllm per well (see Note 27). Use the first
48-96 toothpicks to also inoculate numbered tubes with 2 mL LB contammg
ampicillm. Cover the well plates and incubate the plates overnight at 37°C m a
standmg incubator Incubate the tubes overnight at 37°C m a shaking Incubator.
3. Freeze the well plates m a -80°C freezer These frozen mmlcultures are stable

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