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and serve as a stock
4. Prepare cosmid mimprep DNA from the 48-96 inoculated 2-mL cultures, as
described m Section 3.1 9.
5. Mix m a microfuge tube: 5 pL mimprep DNA, 2 pL of the appropriate digestion
buffer, 5 U of a diagnostic restriction enzyme, and distilled water to 20 pL. As a
control, digest the unmutagenized cosmid clone Incubate the digestions for 2 h
at the appropriate temperature.
Load all digestions on a 0.8% agarosegel, togetherwith a HzndIII digestofphage
6.
lambda DNA as a marker (see Note 16) and electrophorese until the bromophe-
no1 blue dye has migrated at least 10 cm
7. Thoroughly compare the digests of the mutagemzed cosmld clones with the digest
72 de Wind, van Zpl, and Berns
of the nonmutagemzed cosmid clone This enables monitoring for deletions or
rearrangements Discard aberrant cosmid clones
8 This selected group of clones IS further analyzed for the site of Insertion of the
mutagenic ollgonucleotlde. For this purpose, repeat steps 5 and 6, now performing
two digests per clone, using two enzymes per digest. The first digest contains the
diagnostic enzyme and the enzyme that cleaves the oligonucleotlde, the second
digest contains the enzyme that cleaves the clomng linker and the ohgonucle-
otide (see Note 28).
9. Indicate on a physical map of the cosmid, the sites of insertion of the oligonucle-
otlde in the various characterized mutant cosmlds.
10 Repeat steps 4-9, maculating cultures from the frozen mlcrotlter stocks, until a
satisfactory density of mutants 1sachieved (see Note 42)
3.2.4. Generatlon of Virus Mutants by Overlap Recombination
Including a Oligonucleotide-Containing Fragment
Once a set of derivatives of a cosmld clone has been generated (each denva-
tlve bearing the mutagenic ohgonucleotide at a different site), oligonucleotrde-
bearing virus can simply be generated. This 1s done by overlap recombmatlon
using one of the mutagemzed cosmid derivative plus three (if a set of cosmid
clones for overlap recombination exists of in total four clones) wild-type
cosmid clones (see Fig. 3 and Note 43).
For the generation of a saturating set of virus mutants by overlap recombl-
nation, we mitlally select a panel of mutant cosmid derivatives with a distance
of, on the average, 1 kbp between two subsequent ohgonucleotide integration
sites. This distance 1s a compromise between practical feasibility and the
assumption that we will find mutants for almost any gene encoded on the
cosmid. The latter assumption 1s based on the followmg parameters, derived
for the sequenced UL region of the HSV- 1 genome (20): (1) The average size
of the HSV- I genes is 1.9 kbp-73% of the open reading frames are larger than
1 kbp; and (2) 89% of the genome 1s protein-encodmg. For a cosmld with an
insert size of, e.g., 40 kbp, this will amount to 40 different overlap recombma-
tlons. If, at a later stage, one needs additional mutants, one can refer to the set
of characterized mutant cosmld derivatives that have not been chosen for mi-
teal overlap recombination (see Note 42). If, after transfection, no regeneration
of viable vnus 1s found after a single overlap recombmatlon, we repeat the
overlap recombination up to three times before we assume that the oligonucle-
otlde is likely inserted in a gene that is essential for W-US growth (see Note 44).
The protocols for preparation of the DNAs for overlap recombmatlon and
for transfection itself are almost the same to those described before; we will
therefore refer to the appropriate sections m this chapter and elaborate only
here on differences we introduced to faclhtate the simultaneous performance
of a large series of overlap recombmatlons. The preparation of all mutant
73
Characterization of a Herpesvirus Genome
cosmid derrvatives for transfectron and the repeated transfections make thus
protocol the most lengthy of this chapter.
To keep the work manageable, we advlse that It be divided over mul-
tiple portions.
1 Usmg the frozen stocks m microtiter plates, maculate flasks containing 50 mL
LB + ampicillin with all mutant cosmid clone derivatives selected for overlap
recombinatron
2. Perform a 20-fold scaled-up rmmprep protocol (see Section 3 1 9.) m 50-mL com-
cal tubes to isolate the cosmids Dissolve the Isolated DNA by mcubation for 15
min at 65°C or overnight at 4°C m 50 pL TE
3. To estimate the yield and to verify the identity and integrity of the isolated DNAs,
perform triple digestions with the dragnosttc plus the ohgonucleotide-cleaving
plus the clonmg linker cleaving enzymes, on all preps. Mix m a mtcrofuge tube
1 pL midi prep DNA, 2 uL 10X digestion buffer (see Note 28), 5 U of each of
the three enzymes, and distilled HZ0 to 20 pL Incubate for 2 h at the appropriate
temperature.
4. Load the digests on a 0 8% agarose gel, also loading 1 ng of a HrndIII digest of
lambda DNA as a marker (see also Note 16) Electrophorese until the bromo-
phenol blue dye has migrated 10 cm. Carefully scrutinize the gel to ascertam the
intactness and identity of the isolated cosmids (see Note 45)
5 If all mutant cosmids are found to be correct, the cosmids are digested with the
restrictron enzyme for which the clonmg linker has a site, to liberate the mutant
vnus genomic fragment from the cosmid vector. To this end, mix m a microfuge
tube: 5 pg DNA, 5 pL 10X digestion buffer, 25 U restriction enzyme, and dis-
trlled water to 50 pL Incubate for 2 h at the appropriate temperature
6. Load the digests on 0 5% preparative ultrapure agarose gels Electrophorese until
the bromophenol blue dye has migrated approx 5 cm
7 Cut out all of the insert bands. Elute each of the DNAs using an electroelution
apparatus
8. After electroelution, transfer the eluted DNAs each to a microfuge tube, add one
volume phenol:chloroforn-risoamyl alcohol, vortex gently for 30 s, centrifuge for
2 mm at maximum speed, and transfer the supernatant to a clean microfuge tube.
Repeat the extraction If necessary. Subsequently, precipitate the DNA by addi-
tion of 0 1 vol3Msodmm acetate pH 5.2 and 2 vol ethanol. Mix by mversron and
store for 1.5min on ice. Centrifuge for 5 mm at maximum speed and drscard the
supernatant. Wash the pellets with 1 mL 70% ethanol, dry briefly, and dissolve
the DNA in IO pL sterile 0 1X TE.
9. Electrophorese 1 ltL of all gel purified DNAs on a 0.8% agarose gel to estimate
mtegrity and concentratton.
10. Prepare 50 pg of each of the other three (nonmutagemzed) cosmrds to be used for
overlap recombmation, using a fivefold scaled-up version of the protocol as
described in Section 3 1 15
11. For every overlap recombmation to be performed, seed the day prior to trans-
74 de Wind, van Zijl, and Berns
fectlon two 3.5-cm wells or dishes with permisslve cells so that cell densrty ˜111
be 25-40% at the time of transfectlon
12. Perform overlap recombmatlon using a lo-fold scaled-down version of the pro-
tocol described m Section 3 1 16. (see Note 46) Note: Great care should be taken
during these steps to avoid crosscontammation between mutants. To obtain sepa-
rate plaques for every overlap recombmatlon, add 10 pL of every CaPO, preclpl-
tate to one well, and 90 PL to the other well After the glycerol shock, overlay the
cells with medium containing 1% methylcellulose to obtain separate plaques As
a positive control for the overlap recombmatlon, perform overlap recombmatlon
using unmutagemzed fragments only (see Note 47)
13 Using a mlcroplpet equipped with a yellow tip, pick six separate plaques of every
overlap recombmatlon Resuspend the virus from every plaque m a sterile
mlcrofuge tube contaimng 100 PL tissue culture medmm The tubes may be fro-
zen and stored at -80°C.
3.2.5. Plaque Purification, Expansion,
and Restriction Digest Analysis of Virus Mutants
Once a set of viable vu-us mutants has been obtamed, mdlvldual mutants are
plaque purified and expanded to obtain a pure stock each of the virus mutants
Note: Great care should be taken durmg these steps to avoid crosscontaminatlon
between mutants. Especially slow-growing mutants will easily be overgrown,
and lost, after infection with a faster growing mutant (or weld-type) virus. We
do not provide a protocol for plaque purification and expansion of the mutants
here; these protocols may be found in Chapter 4 of this volume. Next, DNA of
plaque-purified mutants IS isolated and analyzed by restriction enzyme analy-
SIS and gel electrophoresis. Virus mutants of which gel patterns of the restnc-
tlon enzyme digests are m accordance with the expected patterns are amenable
to phenotypic analysis. We do not provide protocols for this, instead, we refer
to other chapters m this volume.
1 Perform three rounds of plaque purification on three of the plaques that are plcked
for every overlap recombmation, as described m Chapter 4 of this volume.
2 Using high-tltered stocks of all plaque-purified vn-us mutants and also of wdd-
type virus, infect IO-cm dishes with permissive cells. Isolate DNA of the infected
cells as described m Section 3.1 lg., steps l-7
3. The DNA preparations are digested with a diagnostic enzyme, to verify that no
(visible) genomic alterations have occurred, and the same diagnostic enzyme plus
the enzyme that cleaves the inserted ohgonucleotlde, to verify the identity of the
mutant Perform the digestions as follows: MIX m a mlcrofuge tube: 2-4 pg DNA,
2 pL of the appropriate 10X digestion buffer (see Note 28), 10 U of each restric-
tion enzyme, and distilled water to 20 PL Incubate for 2-4 h at the appropriate
temperature for the enzymes
4. Analyze the digests by running side to side the different digests of DNAs from
reconstituted and nonmampulated vn-uses on a large 0.8% agarose gel. Also, load
75
Characterization of a Herpeswrus Genome
1 l.tg HzndIII digested lambda DNA as a marker. If vu-us bands are not clearly
visible, blot the gel and analyze by hybridization with virus probes The digests
with the diagnostic enzyme should be identical for all vuus preparations. The
double digests should have the diagnostic fragment cleaved mto two fragments,
with sizes depending of the site of msertron of the ohgonucleotide An agarose
gel exemplifying this is shown m Fig. 5
5 Of every mutant vu-us, choose one plaque-pure strain that 1s found correct by
restriction enzyme analysis for further experiments. If no strain is found that
meets these criteria, repeat this protocol using the three additionally picked, fro-
zen plaques from the overlap recombination.
3.2.6. Other Uses for Cosmd Clones
Bearing a Oligonucleotide Containing a Unique Restriction Site
We will mention here two mformatrve examples showmg how the presence
of an inserted unique restriction site, combined with overlap recombination,
has further expanded the possibilities to modify the virus genome
Although the ohgonucleotide abolishes expresston of the gene it is Inserted
m, for the production of a safe vaccine strain of a herpesvirus, one prefers to
entirely delete a gene that is found to be a determinant of the virulence of the
virus. This deletion can easily be generated using a cosmid bearmg an olrgo-
nucleotrde at the 5™ end of the gene of interest and a cosmid bearmg an ohgo-
nucleotide 3™ of the first ohgonucleotide msertion m the same gene. A novel
cosmid having a deletion between the ohgonucleotides m both mutants IS
obtained by ligating the cosmid insert fragment left of the 5™ inserted ohgo-
nucleotrde to that right of the more 3™ inserted ohgonucleotrde. After overlap
recombmation, the resulting virus strain, that now contams a specified dele-
tion, can be used as a safe vaccine strain. The stram BA420- 114, as depicted in
Ftg. 5, bearing a deletion in the thymidme kmase gene, IS an example of an
avirulent PRV strain that is generated rn this way (26).
The unique restriction site also provides opportumties for the development
of the virus as a vector for heterologous genes. In the latter case, the umque
site inserted in the cosmid serves as a socket to plug m cassettes contammg
the gene of interest. In a subsequent overlap recombmatron the, heterologous
gene containing, virus IS reconstituted (22).
3.3. The Generation and Use of Probes for the Study
of Layout, Expression, and Identity of Virus Encoded Genes
In the previous section (see Section 3.2.), we described the generation of a
large series derrvatrves of a cosmrd containing 30-40 kbp of the vnus genome.
The mutagemc oligonucleotide that is present m each of these mutant cosmrds
contains a restrictron site that is unique for the cosmrd. The latter enables one
to specifically subclone small virus genomic fragments that flank the inserted
de Wind, van Z# and Berns
76

oltgonucleottde m each mutant. This is done by simply digesting each mutant
cosmtd with this enzyme and a second enzyme that has a 4-bp specificity (and
thus frequently cleaves the vu-us genome; see Note 6) and gives a sticky over-
hang. The resulting digest contarns many short fragments; however, only two
of these (at both sides of the inserted oligonucleottde) contam a unique over-
hang at one end and the overhang of the 4-bp recogmzing enzyme at the other
end. This enables to subclone these fragments into a vector that IS predigested
with enzymes that yield the same overhangs as the two enzymes with which
the mutant cosmid has been cleaved (see also Fig. 1C). The resultmg subclones
are analyzed for insert size and the ortentation with respect to the mutagenic
ohgonucleotide (facultatively). After this characterization, the subclones may
be used for the followmg purposes:
1. Crosshybridizationstudieswith DNA from aprototype herpesvirusto gain mfor-
matlon on genestructureof the virus under study,
2. To map and to determine the classof transcrtpts in the region of the inserted
oligonucleotide; and
3 Direct DNA sequencingto identify (by comparing with the sequence of a proto-
type herpesvu-us) the virus gene that has been mutagemzed insertion of the
by
oligonucleotlde
Here, we will provide a detailed description of the subcloning procedure
Itself, we will only give a global description of the procedures for cross-
hybrtdization studres, transcript mapping, and DNA sequence analysis.
3.3.1. Construction and Characterization
of Subclones Flanking Each Inserted Olrgonucleotide
Every mutant cosmid derivative is to be dtgested with two restriction
enzymes: the enzyme that specifically cleaves the inserted mutagenic ohgo-
nucleottde, together with an enzyme that has a 4-bp specificity and thus will
cleave m the vicmity of the inserted ohgonucleotide For the latter enzyme,
Sau3A is a good choice; this enzyme recognizes the sequence GATC, leaving
a 4-bp 5™ overhang that is compattble with the BarnHI, BglII, and BcZI over-
hangs (see Note 48). The vector for subcloning, containing a polylinker (see
Note 49), is therefore cleaved with, e.g., BamHI plus the enzyme that generates
an overhang that is compatible with the overhang, generated by cleavage of the
oligonucleotide (see Note 50).
After subcloning, the two different flanking subclones for every oligonucle-
otide msertton mutant are isolated and then sizes are determmed by releasing
the inserts from the vector using enzymes that cleave the polylinker of the
vector at either side of the insert, followed by agarose gel electrophorests. In
addition, the orientation of the subclones with respect to the ohgonucleotide
may be determined by hybridrzation with a blot of a gel containmg the respec-
77
Characterization of a Herpesvirus Genome
tlve mutant cosmld that IS dlgested with the diagnostic enzyme for the vuxs
plus the oltgonucleotlde cleaving enzyme (the blot may be generated m Sec-
tion 3.2.4.; see Note 45)
1 Mix m a mlcrofuge tube. 10 ug of the appropriate plasmid vector, 10 uL of the
appropriate 10X enzyme digestion buffer (see Note 28), 50 U of the enzyme
that cleaves the oltgonucleottde (or an enzyme giving compatible ends, see
Note 50), 50 U BumHI, and distilled water to 100 pL Incubate for 2 h at the
appropriate temperature
2. Load the digest on a 0 8% preparative agarose gel, load as markers (m a small
slot) 1 pg uncleaved plasmid, 1 pg lambda DNA digested with Hind111 (see Note
16) Electrophorese until the bromophenol blue dye has migrated 5 cm Cut out
the linear plasmtd band and isolate the DNA using the Geneclean (or similar) kit.
Take up the DNA m 100 uL TE
3. Mix m microfuge tubes* 1 ug of each ohgonucleotrde containing cosmtd for which
flanking probes are to be obtained, 2 pL of the approprtate 10X digestion buffer
(see Note 28), 5 U of the of the enzyme that cleaves the oligonucleottde (see Note
28), 5 U Sau3A, and distilled water to 20 uL As control, digest the unmutagenized
cosmrd with both enzymes. Incubate for 2 h at the appropriate temperature
4. Add 80 uL TE and one volume phenol*chloroform˜tsoamyl alcohol, Vortex for
30 s, centrifuge for 2 mm at maximum speed, and transfer the supernatant to a
clean mlcrofuge tube. Subsequently, precipitate the DNA by addition of 0 1 vol
3Msodium acetate pH 5 2 and 3 vol ethanol. Mix and store on ice for 30 mm and
centrifuge for 15 mm at maximum speed. Wash the pellets with 1 mL 70% etha-
nol and take the DNA up in 200 uL TE
5 For each set of two flanking probes, mix m a mtcrofuge tube: 1 pL digested gel
purified vector, 1 uL of a digested mutant cosmtd derivative (see Note 5 1), 5 uL
10X ligase buffer, 1 (Weiss) Unit T4 hgase, and distilled water to 50 uL Incu-
bate for 4 h to overnight at 15°C. As a negative control, perform a simultaneous
ligation containing the digested gel purified vector plus the digested unmuta-
genized cosmid.
6. Transform competent E toll wrth 10 uL of each ltgatton, as described m
Section 3 1.8.
7. Count the transformant colonies; the hgattons of digests of oligonucleotide-bear-
ing cosmrds should clearly give more colomes than the hgatton of the digest of
the unmutagenized cosmtd For every ligation, inoculate six colomes, using
wooden toothpicks, rn 2 mL LB + amprcillm. Incubate overmght in a shaking
incubator at 37°C
8 Perform minipreps on all cultures, as described m Section 3.1.9
9 Mix in mtcromge tubes 2 uL mmtprep DNA, 2 uL of the appropriate 1OX enzyme
digestion buffer (see Note 28), 5 U of each of two restriction enzymes that cleave

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