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Complementary DNA cloning and expression of the papaya ringspot virus sequences encoding capsid protein and a nuclear inclusion-like protein

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Complementary DNA cloning and expression of the papaya ringspot virus sequences encoding capsid protein and a nuclear inclusion-like protein
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Papaya ringspot virus sequences encoding capsid protein and a nuclear inclusion-like protein, Complementary DNA cloning and expression
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Nagel, Julianne, 1957-
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vi, 49 leaves : ill. ; 28 cm.

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Capsid ( jstor )
Capsid proteins ( jstor )
Complementary DNA ( jstor )
DNA ( jstor )
Gels ( jstor )
Genomes ( jstor )
Nuclear inclusions ( jstor )
Plasmids ( jstor )
RNA ( jstor )
Virology ( jstor )
Dissertations, Academic -- Plant Pathology -- UF
Papaya -- Diseases and pests ( lcsh )
Plant Pathology thesis Ph. D
Plant genetic engineering ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Includes bibliographical references (leaves 45-48).
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Typescript.
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Vita.
Statement of Responsibility:
by Julianne Nagel.

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COMPLEMENTARY DNA CLONING AND EXPRESSION OF THE PAPAYA
RINGSPOT VIRUS SEQUENCES ENCODING CAPSID PROTEIN
AND A NUCLEAR INCLUSION-LIKE PROTEIN
By
Julianne Nagel
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA FOR
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1985


In memory of my father, Robert C. Nagel.


ACKNOWLEDGEMENTS
I would like to thank my family for their faith
encouragement. I am grateful to my major professor, Ernest
Hiebert, for allowing me great freedom in my research and
for always being available when I needed help. I would also
like to thank the members of my committee, D.R. Pring, D.C.
Loschke, F.W. Zettler, and L.C. Hannah, for sharing their
expertise and generously allowing me access to their
laboratories and supplies.
I am indebted to Maria de Mejia for convincing me to
work with PRSV instead of DMV. I appreciated John Payne's
help with the computers and his decency in not graduating
before me. Margarita Licha, Feiko Ferwerda, Kris Figura,
and Gene Crawford are thanked for their friendship and for
preventing my starvation by supplying me with countless
lunches and dinners. Finally, I thank Randy Ploetz for his
love and encouragement.
in


TABLE OF CONTENTS
Page
DEDICATION ii
ACKNOWLEDGEMENTS iii
ABSTRACT V
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: COMPLEMENTARY DNA CLONING AND EXPRESSION
OF THE CAPSID AND A NUCLEAR INCLUSION-LIKE
PROTEIN OF PAPAYA RINGSPOT VIRUS IN E. COLI
Introduction 5
Materials and Methods 5
Results 9
Discussion 20
CHAPTER 3: COMPLEMENTARY DNA CLONING OF PAPAYA RINGSPOT
VIRUS BY AN RNA:CDNA HYBRID METHOD
Introduction 25
Materials and Methods 26
Results 27
Discussion 32
CHAPTER 4: CONCLUSIONS 34
APPENDIX A: RECIPES FOR HEIDECKER AND MESSING CLONING
PROCEDURE 36
B: RECIPES FOR CANN ET AL. CLONING PROCEDURE... 41
C: VARIOUS OTHER RECIPES 43
LITERATURE CITED 45
BIOGRAPHICAL SKETCH 49
IV


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of
the Requirements for the Degree of Doctor of Philosophy
COMPLEMENTARY DNA CLONING AND EXPRESSION OF THE PAPAYA
RINGSPOT VIRUS SEQUENCES ENCODING CAPSID PROTEIN
AND A NUCLEAR INCLUSION-LIKE PROTEIN
By
Julianne Nagel
May, 1985
Chairman: Ernest Hiebert
Major Department: Plant Pathology
Papaya ringspot virus type W (PRSV-W), formerly
watermelon mosaic virus 1, is a member of the potyvirus
group of plant viruses. The potyviral genome consists of a
single plus-stranded RNA, about 10,000 nucleotide residues
in molecular size, with a small protein attached to the 5'
end and a poly (A) tail on the 3' end. In this research,
two complementary DNA (cDNA) cloning methods were used to
clone portions of the PRSV-W genome. Poly (T) was used to
prime cDNA synthesis in both procedures. In the Heidecker
and Messing procedure (Nucl. Acids Res. 11,4891-4906) the
vector was pUC9 and the viral RNA was copied into double-
stranded DNA before transforming Escherichia co 1 i JM83. In
the Cann et al. procedure (Nucl. Acids Res. 11,1267-1281) an
RNA:cDNA hybrid inserted into pUCl9 was used to transform EL
co1 i JM83. The Cann method was more efficient than the
Heidecker and Messing procedure, both in the number of
v


clones produced, and in the length of time required to
complete the procedure. The clones produced by either
procedure represented approximately the same area of the
genome.
Three cDNA clones that expressed viral gene products in
E. co1i were characterized. The expressed polypeptides
were fusion products with the amino terminus of the oC-donor
fragment of B-galactosidase. Clones Wl-77 and W2-1 were
1480 and 630 base pairs (bp) in size and expressed fusion
products with apparent molecular weights of 40,000 and
14,000, respectively, which were serologically related to
PRSV capsid protein. Clone Wl-18 was 1330 bp in size and
produced a 52K product that was serologically related to a
54K nuclear inclusion protein of tobacco etch virus. The
sequences encoding the capsid and 57K nuclear inclusion-1 ike
proteins of PRSV-W were physically mapped to adjacent
positions through Southern blot analyses of clones Wl-77 and
Wl-18 and are presumed to be located at the 3' end of the
viral genome.
vi


CHAPTER 1
INTRODUCTION
Papaya ringspot virus is a potyvirus which causes
destructive diseases of papaya and cucurbits. The virus
limits the production of papaya in Caribbean countries,
Florida, Hawaii, India, South America, and Taiwan (Yeh et
al., 1984; and references therein) and causes serious losses
in cucurbit crops worldwide (Purcifull et al., 1984). The
strain of PRSV used in this study was type W, which until
1984, was known as watermelon mosaic virus 1 (Purcifull et
al., 1984). The type W strain differs from the prototype,
PRSV-P, only in one host range characteristic; type W does
not infect papaya (Purcifull et al., 1984). In Florida
PRSV-W overwinters in the wild cucurbit hosts, Melothria
pendula and Momordica charantia, and is transmitted
nonpersistently by many species of aphids (Adlerz, 1972;
Purcifull et al., 1984).
The PRSV particle is a long flexuous rod approximately
780 nm in length and 12 nm in width. The coat is
constructed of identical, repeating subunits each with a
molecular weight of 36,000 (36K) (Purcifull et al., 1984).
The potyviral genome consists of a single, plus-stranded
RNA, ca. 10,000 nucleotide residues (lOkb) in size with a
genome-1 inked protein on the 5' end (Hari, 1981) and a poly
(A) tail on the 3' end (Hari et al., 1979). At least seven
proteins are thought to be encoded by the genome (de Mejia
1


et al., 1985b). Two of the proteins of PRSV aggregate
separately in the cytoplasm of host plant cells to form
cylindrical inclusions (Christie and Edwardson, 1977) and
amorphous inclusions (de Mejia et al., 1985a). The
cylindrical inclusions are composed of 70K protein subunits
which array themselves to form 'pinwheel' structures
attached to the plasma membrane, frequently over
p1asmodesmata (Christie, personal communication). Later in
infection, the cylindrical inclusions usually move away from
the plasmalemma and aggregate near the nucleus (Christie and
Edwardson, 1977). The amorphous inclusions consist of
aggregations of a 51K protein subunit along with associated,
uncharacterized RNA (de Mejia et al., 1985a). The 51K
protein is the first and only nonstructura1 protein of
potyviruses to which a function has been ascribed; it
appears to be a helper factor which enables potyviruses to
be transmitted by aphids (de Mejia et al., 1985b).
Certain potyviruses, such as tobacco etch virus (TEV),
form a third type of inclusion in the nucleus of host plant
cells. These nuclear inclusions are composed of an
equimolar mixture of two viral proteins, a 49K protein
subunit and a 54K protein subunit (Knutsen et al., 1974).
Other potyviruses, such as PRSV, that are not known to form
nuclear inclusions _in vivo have been demonstrated to form
products serologically related to the 49K and 54K proteins
of TEV during i_n vitro translations (de Mejia et al., 1985b;
Dougherty and Hiebert,l980 ) It is probable that these


3
protein subunits are also formed in infected host cells but
do not aggregate in the nucleus to form structures apparent
by light microscopy.
Over 25 potyviruses have been translated _i_n vitro
(Hiebert, unpublished) and their products have been
immunoprecipitated with various antisera including those to
the capsid, cylindrical inclusions, amorphous inclusions,
and the 2 nuclear inclusion proteins. Through analysis of
the immunoprecipitated products, gene maps have been
proposed to locate the protein coding regions on the viral
genome (Dougherty and Hiebert, 1980; Heilman et al., 1980;
Vance and Beachy, 1984; Xiong, 1985; de Mejia et al.,
1985b). Each of these gene maps differs to some degree; for
example, Heilman et al. (1980) and Vance and Beachy (1984)
report that the capsid protein coding region is located near
the 5' end of the genome while others define its position at
the 3' end (Dougherty and Hiebert, 1980; Xiong, 1985; de
Mejia et al., 1985b). At the present time the potyviral
gene map is controversial. The map which was used as a
reference in this study is by Xiong (1985), and when adapted
for the molecular weights of the PRSV proteins is 5' end;
60K 'unknown' protein; 51K amorphous inclusion protein; 40K
'unknown' protein; 70K cylindrical inclusion protein; 49K
nuclear inclusion protein; 57K nuclear inclusion protein;
36K capsid protein; 3' end.
The primary objective of this research was to develop a
system to clone portions of a potyviral genome and obtain


4
the transcription and translation of these portions in
Escherichia co1i. By serologically identifying the
polypeptides expressed in E. co1i and correlating them with
specific areas of the viral genome, the gene map may then be
verified. This approach was taken by Daubert et al. (1982)
to map the location of the capsid protein gene of
cauliflower mosaic virus on the viral genome.
Also through such an expression system, polypeptides
could potentially be obtained for the two 'unknown' coding
regions of the potyviral genome. The products of these
coding regions are not known to aggregate in host cells and
have not been purified or used to produce antisera; their
existence has been demonstrated only in _in vitro translation
products. Saito et al. (1984) cloned cDNA fragments coding
for parts of two tobacco mosaic virus nonstructura1 proteins
and expressed the sequences in E. co1 i. These nonstructura1
proteins are normally extremely difficult to isolate from
infected plants, but by expressing the proteins in E. co1i,
sufficient amounts were obtained for characterization and
antibody production. Other examples of plant viral proteins
expressed in E. co1i include the capsid protein of satellite
tobacco necrosis virus (Van Emmlo., et al., 1984) and the
capsid protein of brome mosaic virus (Miglietta and
Kaesberg, 1984).


CHAPTER 2
COMPLEMENTARY DNACLONING AND EXPRESSIONOFTHECAPSID
AND A NUCLEAR INCLUSION-LIKE PROTEIN OF PAPAYA
RINGSPOT VIRUS IN E. COLI
Introduction
The Heidecker and Messing procedure (1983) was selected
for the cloning of PRSV-W because the procedure was reported
to be very efficient and yield a high proportion of full-
length clones. In theory approximately one-sixth of the cDNA
clones produced by this procedure should also express
protein products for the cloned inserts in E. co1i (Helfman
et al. 1983; Heidecker and Messing, 1983). Finally, because
cDNA synthesis is primed by a poly (T) tail that is
covalently attached to the pUC9 plasmid, it was assumed that
the resultant cDNA clones would have originated from the
poly (A) tail on the 3' end of the viral RNA. Such a factor
would have facilitated future mapping studies by providing a
known starting point.
Materials and Methods
Materials
Restriction enzymes and the Klenow fragment of DNA
polymerase I were from Bethesda Research Laboratories, Inc.
(BRL, Gaithersburg, MD 20877). Reverse transcriptase was
from the Seikaguki Company (St. Petersburg, FL 33702). f^5S]
Methionine, ^C- 1 abe 1ed molecular weight standards, and
5


6
I O C
I-labeled protein A were from Amersham (Arlington
Heights, IL 60005). Escherichia co 1 i JM83, pUC8, and pUC9
were from D.C. Loschke. Antisera to three strains of PRSV:
type W, type P, and type T were from D.E. Purcifull, D.
Gonsalves, and L. Quiot-Douine, respectively.
Papaya ringspot virus type W (PRSV-W), formerly
watermelon mosaic virus-1 (Purcifull et al., 1984), was
propagated in pumpkin plants (Cucrbita pepo L. 'Small
Sugar') and purified 2.5 wk after inoculation by the method
of Purcifull et al. (1984). RNA was extracted from purified
virus by treatment for 5 min at 60C with 2.0% sodium dodecyl
sulfate (SDS), 2 mM ethylenediamine tetraacetic acid (EDTA),
and 200 mM ammonium carbonate, pH 9 (Brakke and Van Pelt,
1970a), and isolated on sucrose log-linear gradients (Brakke
and Van Pelt, 1970b). The RNA was then precipitated with
ethanol, centrifuged, and resuspended in a small volume of
sterile, distilled water. The phenol extraction of the RNA
was similar to that described by Maniatis et al. (1982)
except that after the phenol and phenol/chloroform
extractions, the organic phases were removed with a
capillary tube, leaving the aqueous phase and interface.
Only the aqueous phase, containing the RNA, was retained
after the final chloroform extraction. The combined organic
phases were reextracted according to Maniatis et al. (1982).


7
cDNA Synthesis and Molecular Cloning
Complementary (c) DNA clones of PRSV-W were prepared
by the method of Heidecker and Messing (1983). This involved
1) cutting the plasmid, pUC9, with the restriction
endonuclease, Pst 1; 2) tailing the cut plasmid with 40-60
deoxythymidy1 ate residues; 3) adding the viral RNA and using
the poly (T) tail of the plasmid as a primer for first
strand cDNA synthesis; 4) tailing the first strand of cDNA
with dGTP; 5) denaturing and purifying the plasmid with
attached cDNA by centrifugation through alkaline sucrose
gradients; 6) reannealing the size-fractionated plasmid with
attached cDNA to dCTP-tailed plasmid; and 7) filling in the
single-stranded area with the Klenow fragment of DNA
polymerase I (see Appendix A).
Escherichia co1 i JM83 cells were transformed by a
calcium chloride shock treatment and plated onto L plates
containing 50 ug/ml ampicillin and the indicator 5-bromo-4-
chloro-3-indoly1-B-D-ga1 actoside (Xgal) at 40 ug/ml
(Maniatis et al., 1982; see Appendix A). Insertional
inactivation of the B-galactosidase gene of pUC9 (Viera and
Messing, 1982) was used as a color marker to select
bacterial colonies containing pUC9 with a cDNA insert. The
presence of PRSV-W sequences in white colonies was verified
by colony hybridization of cloned DNA with [^P]-dCTP
labeled first-strand cDNA of PRSV-W RNA (Maniatis et al.,
1982). Because Close et al. (1983) reported that not all
insertions into the B-ga1 actosidase fragment of pUC vectors
result in white colonies, a portion of the blue and light


8
blue colonies were also tested for PRSV sequences. Insert
size was determined by restriction endonuclease digestion of
plasmid DNA and agarose gel electrophoresis. Molecular
weight markers were lambda DNA (supplied by D. Pring) and
0X174 RF DNA (BRL), digested with Hind III and Hae III,
respectively.
Detection of Expressed Viral Proteins
Bacterial cells from 12 to 16 hr cultures grown in L
broth (Maniatis et al., 1982) were collected by
centrifugation at 5,000x2, and lysed in 1/10 volume
(original cell suspension) of Laemmli dissociation buffer
(Laemmli, 1970). The lysed cells were centrifuged at
27,000x2 fr 30 min, and the supernatant was retained. Gel
electrophoresis, transfer to nitrocellulose sheets, and
detection of proteins with antibody (Western blots) were
performed as described by de Mejia et al. (1985a). Agar gel
(Ouchter1ony) immunodiffusion tests were conducted as
described by Purcifull and Batchelor (1977). The
immunodiffusion medium contained 0.8% Noble agar, 0.5% SDS,
and 1% sodium azide (Purcifull and Batchelor, 1977).
For immunoprecipitat ions, bacterial cells were labeled
with [^5S]-methionine. In these experiments bacterial
cultures were grown in potassium morpho1inopropane sulfonate
(MOPS) medium (Neidhardt et al., 1974) supplemented with
2% dehydrated methionine assay medium (Difco, Detroit, MI
48232) and 10 uCi/ml []-methionine. The cells were
incubated for 8 to 16 hr at 37 C with shaking, and lysates


9
were prepared as described above.
Immunoprecipitat ions were performed by incubating for 1
hr at room temperature 100 ul of [ ]-methionine-1 abe 1 ed
bacterial lysate with 100 ul of antiserum and 400 ul of 0.5%
Nonidet P-40 in 150 mM NaCl, 5 mM EDTA, 50 mM Tris, and
0.02% sodium azide (NET buffer) containing 1 mg/ml
ovalbumin, and 2 mM methionine (Kessler, 1975). One hundred
microliters of a 10% Staphylococcus aureus protein A
solution was then added, and the incubation was continued
for 20 min. The mixture was washed three times in 0.05% NP-
40 NET buffer and resuspended in 40 ul of Laemmli
dissociation buffer. Before electrophoresis in SDS-
permeated polyacrylamide gels (SDS-PAGE) the samples were
heated in a boiling water bath for 5 min. Products were
visualized by fluorography (Bonner and Laskey, 1974).
Nick translations were performed as described by Rigby
et al. (1977). Southern blots were as described by Maniatis
et al. (1982).
Results
DNA Analysis of Expression Clones
Three of the six resulting cDNA clones expressed viral
proteins and were characterized. Clone Wl-77 contained a
1480 base pair (bp) insert and clone W2-1 contained a 630 bp
insert. Each insert could be excised from the plasmid by
cutting on both sides of the insert, in the polylinker
region of pUC9, with Bam HI and Hind III (Figs. 2-1, 2-2).
Eco RI cut at a site in the polylinker region and also at


10
sites within the inserts of Wl-77 and W2-1, producing
fragments of approximately 580 and 3600 bp for Wl-77, and
550 and 2780 bp for W2-1. Clone Wl-18 contained a 1330 bp
insert with an internal Hind III site (Fig. 2-2). When Hind
III alone was used to restrict Wl-18, fragments of 450 and
3580 bp were observed. Bam HI cut only in the polylinker
region of pUC9; it did not cut the Wl-18 insert.
From Southern blotting analyses, clone Wl-18 was found
to overlap with Wl-77 but not W2-1. When the 880 bp Bam HI
to Hind III fragment of Wl-18 was isolated, nick translated,
and used as a probe, only self homology was observed (Fig.
2-3). When the 450 bp fragment of Wl-18 was used as a
probe, it hybridized to itself and the Wl-77 insert, but not
to the W2-1 insert (Fig. 2-4). The Wl-77 insert, when used
as a probe, hybridized to itself, the W2-1 insert, and only
the 450 bp insert of Wl-18 (Fig. 2-5).
The orientation of Wl-77 in pUC9 is shown in Figure 2-
la. The direction of transcription and translation in
Figure 2-1 is counterclockwise, in pUC9 from the Hind III
site to the Eco RI site. The cDNA insert of clone Wl-18 was
transferred from pUC9 into pUC3 to orient the insert in the
proper direction for transcription and translation (Fig. 2-
lb) .
Identification of Expressed Proteins
Clones which expressed protein were identified by
serological testing. Similar serological results were
observed in Western blots and immunoprecipitat ion tests of


11
protein extracts for each expression clone (Figs. 2-6, 2-7).
Clone Wl-77 produced predominately a polypeptide of Mr
40,000 (40K) that reacted with PRSV capsid antiserum (Fig.
2-6, lane 2; Fig. 2-7, lane 2). When the insert of clone
Wl-18 was in pUC9, no reactions were observed in either
Western blots or in immunoprecipitations. However, when
the Wl-18 insert was in pUC9 a 52K polypeptide that
serologically reacted with an antiserum to TEV 54K nuclear
inclusion protein was formed (Fig. 2-6, lane 8; Fig. 2-7,
lane 11). A number of extra bands were observed with
immunoprecipitation tests with the TEV 54K protein antiserum
(Fig. 2-7, lanes 3, 7, and 11), and with the Western blots
(Fig. 2-6) with either serum. These bands were assumed to
be reactions of the antisera with certain unidentified
bacterial proteins and occurred regardless of whether the
bacteria contained pUC9 or any of the clones. Clone W2-1
produced a 14K polypeptide antigenica1 ly related to PRSV
capsid protein (Fig. 2-6, lane 3).
In Ouchterlony immunodiffusion tests the expressed
protein of Wl-77 reacted homologously with antiserum to


12
Sal I
Hind III Sal I
Fig. 2-1. Locations of cDNA inserts in pUC plasmids:
a. Wl-77 in pUC9. b. Wl-18 in pUC8. Transcription and
translation proceed counterclockwise.


13
Fig. 2-2. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B in an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [32P]-labeled probe consisting of randomly primed
first-strand cDNA of PRSV-W.


14
Fig. 2-3. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [3;i P]-labeled probes consisting of nick translated
880 bp fragment of Wl-18.


15
Fig. 2-4. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [3i P]-labeled probe consisting of nick translated 450
bp fragment of Wl-18.


16
1 2 3 4 1 2 3 4
Fig. 2-5. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [32P]-labeled probe consisting of nick translated
1480 insert of Wl-77.


17
14K-^
Fig. 2-6. Western blot of lysates from E. co 1 i JM83
containing: pUC9 (lanes 1 and 5), clone Wl-77 (lanes 2 and
6)', clone W2-1 (lanes 3 and 7), and clone Wl-18 (lanes 4 and
8). The proteins were analysed by electrophoresis in an
SDS-permeated polyacrylamide gel, electrophoretically
blotted onto nitrocellulose, incubated with an antiserum to
PRSV-P capsid protein (lanes 1-4), or an antiserum to TEV
54K nuclear inclusion protein (lanes 5-8), then incubated
with ,i5I-labeled protein A and autoradiographed. Lane 9
contains protein molecular weight markers. Arrows identify
the estimated sizes of the immunoreactive proteins.


18
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Fig. 2-7. Immunoprecipitat ions of methionine labeled E.
co1i fusion proteins related to PRSV capsid protein and TEV
54K nuclear inclusion protein. The proteins were analysed
on an SDS-permeated 7.5 to 15% gradient polyacrylamide gel
which was processed by f1uorography. Lanes 1, 2, 3, and 4
contain lysate from JM83 containing clone Wl-77; lanes 5, 6,
7, and 8 contain lysate from JM83 containing pUC9, and lanes
9, 10, and 11 contain lysate from JM83 containing cloneWl-
18. Lysates in lanes 2, 6, and 10 were immunoprecipitated
with antiserum to PRSV-P capsid. Lysates in lanes 3, 7, and
11 were immunoprecipitated with antiserum to TEV 54K nuclear
inclusion protein. Lysates in lanes 4 and 8 were
immunoprecipitated with pre-immune serum. Arrows identify
the locations of the major immunoreactive proteins.


19
Fig. 2-8. Serological relationships of capsid protein from
clone Wl-77, PRSV-W, and PRSV-T. Central wells contain: P
= antiserum to PRSV-P capsid, G = antiserum to PRSV-T, N =
pre-immune serum. Peripheral wells contain: c = lysate from
JM83 containing clone Wl-77, g = extract from pumpkin plants
infected with PRSV-T, p = extract from pumpkin plants
infected with PRSV-W, b = lysate from JM83 containing pUC9,
and h = extract from healthy pumpkin plants.


20
either PRSV-W virions or PRSV-P capsid protein (Fig. 2-8),
which is consistent with previous reports that PRSV-P and
PRSV-W are serologically indistinguishable in
immunodiffusion tests (Purcifull et al., 1984, and
references therein). The protein expressed by Wl-77 reacted
heterologously with an antiserum to virions of PRSV-T, a
strain serologically distinct from PRSV-P or PRSV-W (Quiot-
Douine et al., 1985). No reactions were evident with pre-
immune sera or with crude extracts of bacteria containing
only pUC9. The expressed protein from clone W2-1 did not
produce a visible reaction in these tests. Apparently
either the protein was not in a great enough concentration
for a precipitan formation, or perhaps the antisera used
contained antibodies predominately to the amino end of the
capsid protein.
Discussion
Two clones of PRSV-W were identified as expressing
polypeptides that are antigenica1ly related to PRSV capsid
protein, and one clone was demonstrated to produce a
polypeptide antigenica1ly related to a nuclear inclusion
protein of TEV. The expressed proteins are fusion products
because 27 bp exist between the translational start site of
the B-galactosidase fragment and the Pst I site into which
the cDNA was inserted (Viera and Messing, 1982). Therefore,
the first 9 amino acids of each of the expressed proteins
are from the amino terminus of the c(-donor of
B-ga1 actosidase. The plasmid lac promoter of pUC9 is
constitutive for B-galactosidase when the E. co1 i JM83 host


21
is used, and so no inducer was necessary for the expression
of the fusion proteins. Sufficient amounts of the fusion
protein of clone Wl-77 were synthesized that the crude
lysates of bacterial cells could be used in Ouchterlony
immunodiffusion tests.
It was demonstrated with Southern blot analyses that
clones Wl-77 and Wl-18 overlap, and therefore concluded that
the two sequences encoding the capsid and 57K nuclear
inclusion-1 ike proteins are adjacent to one another on the
viral genome. However, their positions on the viral genome
were not established in this study. The location of the
capsid coding region has been identified for two other
potyviruses, pepper mottle virus (Dougherty et al., 1985)
and TEV (Allison et al., 1985) at the 3' end of the viral
RNA. It seems probable, therefore, that the capsid coding
region of PRSV is also located at the 3' end of the viral
RNA. The proposed locations of clones Wl-18 and Wl-77 in
reference to the PRSV-W gene map are illustrated in Figure
2-9.
The cDNA insert in Wl-77 was 1480 bp long and after
subtracting 88 bp for the poly (A) sequence (sequence data
not shown) and a presumed 3' non-coding region of 100 to 300
bp, it could theoretically code for a protein in the range
of 40K to 48K. The predominant antigenic polypeptide found
in either Western blots or in immunoprecipitations of Wl-77
protein was approximately 40K. The amino terminus of
B-ga1 actosidase would account for approximately IK and so


22
the bacterially expressed protein was larger than the capsid
protein from virions by 3K. The additional 3K may represent
the carboxyl end of the 57K nuclear inclusion-1 ike protein.
This would imply that there is no recognized stop codon
between the 57K nuclear inclusion coding region and the
capsid protein coding region in the PRSV-W genome. The 57K
nuclear inclusion coding region has been reported
previously to lie adjacent to and in the same reading frame
as the capsid protein coding region through analysis of in
vitro translation 'read through' or polyprotein products (de
Mejia et al., 1985b; Dougherty and Hiebert, 1980). We were
not able to detect any reaction of the 40K protein with the
antiserum to TEV 54K nuclear inclusion protein in
immunoprecipitations or in Western blots. This is not
surprising considering that such a small part of the nuclear
inclusion protein is presumed to be present and that the
antiserum is not homologous for PRSV.
After subtracting the same 188 to 388 bp to account for
the poly (A) and 3' non-coding regions, clone W2-1 could
theoretically code for a polypeptide of 9K to 16K. The
observed 14K polypeptide serologically related to the capsid
protein is within the predicted size range.
When the Wl-18 insert was in pUC9, no vira1-specif ic
product was made because the insert was oriented 3' to 5'
with respect to the promoter. Transferring the insert into
pUC8 made it 5' to 3' with respect to the promoter and
allowed the transcription and translation of a polypeptide


23
antigenically related to the 54K nuclear inclusion protein
of tobacco etch virus. Apparently the insert was already in
the correct reading frame and no attempts to change the
reading frame were necessary. The polypeptide expressed by
clone Wl-18 was larger than is expected for its coding
capacity. The 1330 bp insert could maximally code for a
polypeptide of 49K; however, a protein of 52K was observed.
One reason for this discrepancy could be that there is no
stop codon in the cDNA insert, as already suggested for Wl-
77, and transcription and translation continue into the 13-
gal actos idase coding area. Indeed, the colony color of Wl-
18 was light blue on indicator plates. The 52K protein is
not large enough to contain both the predicted 49K nuclear
inclusion polypeptide and the 16K B-ga1 actos idase
polypeptide fragment, so some post-translational cleavage
might be occurring. Although the cleavage site is not
known, it would seem likely that since B-ga1 actosidase is a
bacterial protein, it would be more resistant to degradation
in E. coli than the nuclear inclusion protein. Therefore,
the 52K protein may consist of 16K from B-galactos idase and
36K from the nuclear inclusion protein. The remaining 13K
nuclear inclusion cleavage fragment, however, was not
apparent in the Western blots or in the immuno-
precipitations.


24
5VpgJi
60k
9
^nk|40k
Amorphous >
Inclusion
70k
Cylindrical
Inclusion
49k 57k
Nuclear Nuclear
Inclusion Inclusion
1330 bp
W1-18
36k
Capsid
^poly A
W1-77
1480 bp
W2-1
6 30bp
3
Fig. 2-9. Proposed locations of clones Wl-18, W1-77, and
W2-1 on the PRSV-W gene map. For discussion of the PRSV-W
gene map, see Chapter 1.


CHAPTER 3
COMPLEMENTARY DNACLONING OF PAPAYA RINGSPOT VIRUS RNA
BYAN RNA:cDNA HYBRID METHOD
Introduction
The RNA:cDNA hybrid cloning procedure by Cann et al.
(1983) was selected as the second cloning method for PRSV-W.
The authors reported that by using this technique with
poliovirus RNA, which is structurally similar to potyviral
RNA, they produced a set of clones with long, overlapping
inserts which spanned the entire 7 Kb size of the
poliovirus genome. The authors also observed that more of
their clones represented the 5' half of the genome rather
than the 3' half. Since clones of what is presumed to be
the 3' end of the PRSV-W genome had already been produced by
the Heidecker and Messing procedure (1983), this method was
undertaken with the goal of obtaining clones with long cDNA
inserts (>2000 bp) representing an area 5' to those areas
previously cloned. Another reason this procedure was
selected was because of its time efficiency; a minimal
number of steps are involved and the entire procedure can be
completed in two days.
25


26
Materials and Methods
Materials
Restriction enzymes, terminal transferase, deoxy
nucleotides, and {X174 RF Hae III fragments were from
Bethesda Research Laboratories (BRL, Gaithersburg, MD
20877). Reverse transcriptase was from Seikaguki (St.
Petersburg, FL 33702). pUCl9 and E. co1i JM83 were from
D.C. Loschke. Lambda molecular weight markers were from
D.R. Pring.
Papaya ringspot virus type W (PRSV-W) was purified from
inoculated Cucrbita pepo L. 'Small Sugar' by the procedure
of Purcifull et al. (1984). RNA was extracted from purified
virus (Brakke and Van Pelt, 1970a) and isolated on sucrose
log-linear gradients (Brakke and Van Pelt, 1970b). The RNA
was phenol extracted (Maniatis et al., 1982) before being
used in the cloning procedure.
cDNA Synthesis and Molecular Cloning
The method of Cann et al. (1983) was used to produce
cDNA clones of PRSV-W RNA. Complementary DNA synthesis was
primed with oligo dT. After the first strand of cDNA was
made, the RNArcDNA hybrid was tailed on the 3' ends with
dCTP. The pUCl9 vector was cut with Pst I and tailed on the
3' ends with dGTP. The G-tailed vector and the C-tailed
RNA:DNA hybrid were then combined and allowed to circularize
(see Appendix B). Competent E. co 1 i JM83 cells were
transformed and plated onto L plates containing ampicillin
at 50 ug/ul and the indicator 5-bromo-4chloro-3-indoly 1-B-D-
galactoside (Xgal) at 40 ug/ml (Maniatis et al., 1982; see


27
Appendix A). A schematic of the cloning procedure is shown
in Figure 3-1. The resultant 113 white colonies and 19
randomly selected light blue colonies were tested for cDNA
inserts by colony hybridizations (Maniatis et al., 1983;
Close et al., 1983). The hybridization probe was randomly
primed, 22P-labeled, first strand cDNA of PRSV-W.
Cultures of 81 white and 13 light blue colonies that
were positive in the colony hybridizations for PRSV cDNA
inserts were grown overnight in L broth and the plasmid was
purified by the method of Maniatis et al. (1982). Insert
sizes were determined by digesting the plasmid DNAs with Bam
HI and Hind III and electrophoresing the samples on 1.2%
agarose gels along with molecular markers consisting of
lambda DNA Hind III and ¡X174 RF Hae III restriction
fragments.
To determine if the cloned inserts represented the
capsid or 57K nuclear inclusion coding regions of the viral
genome, the plasmid DNAs were analyzed by Southern blot
hybridizations. Duplicate nitrocellulose sheets were
prepared by using sandwich blots to transfer the DNA from
the agarose gels. One of the nitrocellulose sheets was
hybridized with 22P-labeled nick-translated 1480 bp insert
of clone Wl-77, and the other was hybridized with 22P-
labeled, nick-translated 880 bp insert of clone Wl-18.
Results
Eighty-one of the 113 white and 13 of the 19 light blue
colonies were positive in the colony hybridization tests.


28
Of these 94 colonies containing PRSV cDNA inserts, 8 had
inserts between 1000 and 1300 bp, 10 contained inserts
between 800 and 1000 bp, 21 contained inserts between 400
and 800 bp, and the remaining 55 colonies contained inserts
under 400 bp in length.
All of the clones tested in Southern blot analyses
hybridized to either clone Wl-77 or Wl-18. The Southern
blots for 15 of the larger cDNA clones are shown in Figures
3-2 and 3-3. Seven of these clones appeared to be similar
to clone Wl-18. They each contained a Hind III site within
the cDNA insert; and after a double digestion with Hind III
and Bam HI to excise the insert from the vector, the smaller
fragment portion of each clone hybridized with the insert of
Wl-77, and the larger fragment hybridized with the 880 bp
fragment of Wl-18. With two of the clones, W4-27 and W4-56
(Figs. 3-2 and 3-3, lanes 2 and 13), the larger fragment
hybridized to the 1480 bp fragment of Wl-77, and the smaller
fragment hybridized to the 880 bp fragment of Wl-18. Clone
W4-48 hybridized only to the 880 bp fragment of Wl-18 and
did not contain a Hind III site. The remaining five clones
also lacked an internal Hind III site and hybridized only to
Wl-77.


AAAAA
I
Pst I
Terminal Transferase
Oligo dT
v
AAAAA
TTTTT
Reverse Transcriptase

AAAAA
TTTTT
Terminal Transferase
ccccc
AAAAACCCCC
TTTTT
Reanneal
Transform
Fig. 3-1. Schematic diagram of Cann et al.
cloning procedure.
(1983) cDNA


30
b
Fig. 3-2. Restriction analysis and Southern blotting of
cloned DNA. Lane 1 contains 0X174 RF Hae III fragments. The
other lanes contain: 2. W4-27, 3. W4-42, 4. W4-43, 5. W4-
48, 6. W4-35, 7. W4-37, 8. W4-38, 9. W4-47, 10. W4-50, 11.
W4-51, 12. W4-55, 13. W4-56, 14. W4-60, 15. W4-66, 16. W4-
72, 17. Wl-18, 18. Wl-77 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A is the
gel stained with ethidium bromide. Panel B is an
autoradiograph of the the DNA transferred onto
nitrocellulose and hybridized with a [ 5P]-labe led probe
consisting of nick translated 1480 bp insert of Wl-77.


31
1353-
1078
872^
603-
310-
b
Fig. 3-3. Restriction analysis and Southern blotting of
cloned DNA. Lane 1 contains 0X174 RF Hae III fragments. The
other lanes contain: 2. W4-27, 3. W4-42, 4. W4-43, 5. W4-
48, 6. W4-35, 7. W4-37, 8. W4-38, 9. W4-47, 10. W4-50, 11.
W4-51, 12. W 4 5 5, 13. W4-56, 14. W4-60, 15. W4-66, 16. W4-
72, 17. Wl-18, 18. Wl-77 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A is the
gel stained with ethidium bromide. Panel B is an
autoradiograph of the the DNA transferred onto
nitrocellulose and hybridized with a [^P] labeled probe
consisting of nick translated 880 bp fragment of Wl-18.


32
Clones W4-42 and W4-38 (Figs. 3-2 and 3-3, lanes 3 and
8) appeared to extend about 100 bp further towards the 5'
end of the viral genome than did Wl-18. Clones W4-27, W4-
42, W4-43, and W4-48 were light-blue in color on indicator
plates which may indicate that nuclear inclus ion-1 ike
proteins were being expressed.
Discussion
Many more clones were produced by the Cann et al.
method (1983) than by the Heidecker and Messing method
(1983) (94 vs 6 clones, respectively). The Cann method was
also much less time consuming than the Heidecker and Messing
procedure. Unfortunately, the goal of obtaining large
clones from an area of the viral genome 5' to clone Wl-18
was not met. Clones W4-42 and W4-38 did extend about 100 bp
further than Wl-18 toward the 5' end, but the remaining
clones all represented an area of the genome previously
cloned by the Heidecker and Messing procedure. Apparently
the reaction conditions were not optimal and cDNA synthesis
did not proceed past the 57K nuclear inclusion coding
region.
It would seem advantageous in future cloning
experiments to prime the cDNA reactions either with random
primers or with a small segment from the 5' end of clone W4-
42. Another approach would be to sequence the 5' end of W4-
42 to find a unique restriction site. A synthetic
oligonucleotide could then be prepared to the area
immediately 3' to the restriction site and used to prime


33
the synthesis of new clones. The
later be joined to W4-42 through
to form longer clones.
new clones could <
the unique restric
Iso
ion site


CHAPTER 4
CONCLUSIONS
Two cloning procedures were used sucessfully to prepare
cDNA clones of PRSV-W. The Cann et al. method (1983),
however, involved fewer manipulations and more clones were
produced. In the Heidecker and Messing procedure (1983),
the addition of T-tails to the cloning vector can cause the
appearance of long poly (A) sequences on cDNA clones which
may have been primed by short, internal A-rich regions.
Clones containing these long poly (A) regions may possibly
be misidentified as representing the 3' end of the viral RNA.
Clone Wl-77 has a poly (A) sequence on one end
approximately 88 bp in size. Wl-77 is assumed to represent
the 3' end of the viral RNA, not because it contains poly
(A), but because it causes the expression of a polypeptide
serologically related to the capsid protein. The capsid
protein coding regions of two other potyviruses have been
located on the 3' end of the genome through direct
comparisons of the amino acid sequences of the capsid
proteins with the viral RNA sequences (Allison et al., 1985;
Dougherty et al., 1985). The capsid protein coding regions
of 23 potyviruses, including PRSV, have also been mapped to
the 3' ends of the viral RNAs from analyses of products
formed during i_n vitro translations (Hiebert, unpublished).
Clone Wl-18 was mapped to an internal location on the
RNA, adjacent and 5' to clone Wl-77. It is unknown whether
34


this clone originated from a short internal A-rich region in
the viral RNA or whether a recombinationa1 event occurred
during the cloning procedure to yield this internal clone.
Sequencing studies should resolve the issue.
The expression of plant viral genes in bacteria has
many potential uses. Bacteria can produce viral proteins
that are not easily extracted from infected plants, and
antisera produced to these proteins would not be
contaminated with antibodies to plant antigens. For the
production of such large amounts of proteins, however,
vectors other than pUC8, 9, and 19 are recommended. Under
the best conditions in this study, the expressed viral-
specific proteins represented about 5% of the total soluble
proteins (data not shown). Other expression vectors are
available that express polypeptides for inserted sequences
at levels of up to 30 to 50% of the total E. £0_1 _i cel lular
protein (Amann et al., 1983; Masui et al., 1984; Schoner et
al., 1985). The expression of the cloned inserts is tightly
regulated with these vectors because the expressed products
can be toxic to the bacterial host. In the pUC plasmids
that were used in this study, the lac promoter is
constitutive when the E. co1i JM83 host is used, which may
cause a selection pressure towards those clones that do not
express the insert at a high level.


APPENDIX A
RECIPES FOR HEIDECKER AND MESSING CLONING PROCEDURE
Digestion of pUC9 DNA wi th PST I_
20 ul pUC9 DNA (1 ug/ul)
110 ul H2O
15 ul BRL 10X Core buffer (500 mM Tris, pH 8.0,100 mM
MgCl2' 500 NaCl)
5.0 ul PST I (9 U/ul)
150 ul total
incubate 37 C, 90 min or longer
phenol extract, ethanol precipitate,
resuspend in 20 ul H2O
C-tailing of pUC9
10 ul pUC9, cut with PST I
28.67 ul H20
10.5 ul 5X tailing buffer (500 mM K-cacodylate pH 7.2,10 mM
CoCl2, 1 mM DTT)
1 ul 1.0 mM dCTP
0.33 ul 32P dCTP
2 ul terminal transferase (20 U)
52.5 total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate 3 times
resuspend in 10 ul 10 mM Tris, pH 7.6, 10 mM NaCl,
1 mM EDTA
36


37
T-tailing of pUC9
10 ul pUC9, cut with PST I
10 ul 1 M K-cacodylate, pH 7.0
21 ul 20
0.5 ul 0.1 M DTT
2 ul 1 mM dTTP
2 ul 32P dTTP (25 uCi)
1.5 ul terminal transferase (15 U)
5 ul 10 mM C0CI2
52 ul total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate 3 times, warm to room
temperature before centrifuging.
resuspend in 10 ul 10 mM Tris pH 7.6, 10 mM NaCl, 1 mM EDTA
cDNA Synthesis
6.4 ul H20
1 ul 12 mM dNTP's
1 ul 1.05 M KC1
1 ul 0.75 M Tris pH 8.2
0.5 ul 300 mM MgCl2
1 ul 30 mM DTT
0.6 ul RNasin 25 U/ul
0.5 ul.750 ug/ml actinomycin D
1 ul T-tailed pUC9 (1 ug/ul)
1 ul PRSV-W RNA (5 ug/ul)
1 ul reverse transcriptase (10 U/ul)
final cone.
8 00 uM
7 0 mM
50 mM
10 mM
2 mM
1 U/ul
25 ug/ml
40 nM
120 nM
100 U/ml
15 ul total


38
incubate 37 C, 90 min.
phenol extract, ethanol precipitate 3 times, warm to
room temp, before centrifuging, resuspend in 10 ul H20
G-tailing reaction
10 ul cDNA
4 ul 1 M K-cacodylate
1 ul 1 mM dGTP
1 ul 0.05 M DTT
2 ul 20 mM MnCl2
1 ul terminal transferase (15 (J/ul)
19 ul total
incubate 37 C, 15 min.
phenol extract, ethanol precipitate
resuspend in 50 ul 10 mM Tris, pH 7.6, 10 mM NaCl, 1 mM EDTA
Alkaline Sucrose Gradient
Centrifugation
Recipe
for
10 5-20% linear
alkaline sucrose
gradients
with a
60%
cushion
Layer
g. sucrose
ml buffer*
amt. p*
1. 5%
.5625
11.25
1.12
2. 10%
1.125
11.25
1.12
3. 15%
1.687
11.25
1.12
4. 20%
2.250
11.25
1.12
5. 60%
3.000
5.00
0.50
buffer: 0.2 M NaOH, 0.8 M NaCl, 1 mM EDTA
Add 50 ul of 5% sucrose layer to G-tailed cDNA. Layer
on gradient. Centrifuge in 50.1 rotor at 36K, 4 C, for 17
hr. Fractionate gradient by puncturing bottom of tube and
collecting 0.3 ml fractions. Measure amt. of radioactivity
by Cerenkov radiation. Pool samples from bottom of tube
that contain radioactivity, but do not use any fraction
after the peak fraction.
Add 3 ul of C-tailed pUC9. Dialyse over night at 4 C
against 3 changes of 10 mM Tris, pH 7.6, 10 mM NaCl, 1 mM
EDTA. Add 25 ug/ml carrier tRNA and ethanol precipitate.


39
Resuspend in 50 ul 10 mM Tris, pH 7.6, 10 mM NaCl, ImM
Reannealing Reaction
50 ul DNA
320 ul 100% formamide (deionized)
20 ul 2.5 M NaCl
10 ul 1 M Tris pH 8
600 ul H2<0
1000 ul total
final cone.
1-5 ul/ml
32%
50 mM
10 mM
incubate 37 C for 24 hr. Dialyse overnight at 4 C
against 100 mM NaCl,10 mMTris, pH 8.0, 1 mM EDTA.
Ethano lprecipitate, resuspend in 43.5 ul H20.
Fill-in Reaction final cone.
43.5 ul DNA
1 ul 2.5 M NaCl
50
mM
1 ul 1 M Tris pH 7.6
20
mM
1.67 ul 300 mM MgCl2
10
mM
1 ul 0.05 M DTT
1
mM
1 ul 5 mM dNTP1s
100
uM
0.8 ul Klenow Frag. DNA Pol. I
3
U
50 ul total
EDTA.
incubate 15 C for 60 min., then room temperature for 60 min.
phenol extract, ethanol precipitate, resuspend in 50 ul
10 mM Tris pH 7.6, 10 mM NaCl, 1 mM EDTA.


40
Transformation Procedure
1. Inoculate 20 ml of L broth in a 100 ml flask with 1 ml of
an overnight culture of E. co1 i JM83. Incubate culture at
37 C with vigorous shaking until reaching an ODcrg of 0.5 (2
to 4 hr).
2. Pour the culture into a sterile capped centrifuge tube
and cool on ice 10 min. Centrifuge the cell suspension at
6,000 rpm in an SW 34 rotor for 5 min at 4 C.
3. Decant the supernatant. Gently resuspend the pellet in
10 ml sterile, ice-cold CaCl2, 10 mM Tris, pH 8.0. Incubate
suspension on ice 15 min.
4. Centrifuge the cell suspension at 6,000 rpm for 5 min at
4 C.
5. Decant the supernatant. Resuspend pellet in 1.33 ml
sterile, ice-cold 50 mM CaCl2 10 mM Tris, pH 8.0.
6. Aliquot 0.2 ml volumes into sterile prechilled tubes.
Incubate on ice for 1 hr.
7. Add up to 100 ul of plasmid DNA in 10 mM Tris, pH 7.6, 10
mM NaCl, 1 mM EDTA. Incubate on ice 30 min.
8. Heat shock the cells by transferring tubes to a 42 C
water bath for 2 min.
9. Add 1 ml of L broth to each tube and incubate at 37 C
without shaking for 30 to 60 min.
10. Spread the transformed cells on to 10 or more L plates
containing 50 ug/ul ampicillin and 40 ug/ml Xgal. Let
plates dry, invert, and incubate at 37 C 12 to 16 hr.
L broth: 10 g Tryptone
5 g Yeast Extract
5 g NaCl
bring volume up to 1 liter, autoclave
L plates: Add 15 g/1 Bacto Agar to L broth before
autoclaving. After autoclaving, cool media to 55 C
before adding 50 mg/1 ampicillin and 2 ml of a 20
mg/ml stock solution of Xgal in dimethyl formamide
(40 mg/1 final concentration)


41
APPENDIX B
RECIPES FOR CANN ET AL. RNA:cDNA HYBRID CLONING PROCEDURE
G-tailing of pUC!9
181 u1 H20
50 ul 5X BRL tailing buffer (500 mM K-cacody1 ate, pH 7.2,
10 mM CoCl2' 1 mM DTT)
15 ul pUCl9 DNA cut with PST I (1 ug/ul)
1 ul 500 uM dGTP
3 ul terminal transferase (60 U)
250 ul total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate
resuspend in 15 ul H2O
cDNA Synthesis
12.4 ul H2O
5 ul 1.0 M Tris pH 8.3
5 ul 2.0 M NaCl
1 ul 0.8 M MgCl2
1 ul 0.5 M DTT
4 ul RNasin (25 U/ul)
0.5 ul oligo dT 12-18 (1 ug/ul)
2 ul PRSV RNA (1 ug/ul)
5 ul 10 mM dATP
5 ul 10 mM dTTP
5 ul 10 mM dGTP
1 ul 100 uM dCTP


0.5 ul 32P dCTP (5 uCi)
2.6 ul Reverse transcriptase (10-16 U/Ul)
50 ul total
incubate 42 C, 15 min; add 2 ul 10 mM dCTP
incubate 42 C, 45 min; add 10 ul 0.2 M EDTA
incubate 65 C, 5 min
dilute 10 X with BRL NACS buffer C (0.5 M NaCl, 20 mM Tris,
pH 7.2, 1 mM EDTA) and run column according to
manufacturer's directions (BRL) elute sample with NACS
buffer D (2.0 M NaCl, 20 mM Tris, pH 7.2, 1 mM EDTA).
C-tailing of cDNA
10 ul RNA:cDNA hybrid
28 ul H2o
10 ul 5X tailing buffer
1 ul 100 ul uM dCTP
1 ul terminal transferase (40 U)
50 ul total
incubate 37 C, 15 min.
phenol extract, ethanol precipitate
resuspend in 15 ul H2o
Reannealing Reaction
15 ul RNAtcDNA hybrid
170.5 ul H20
4 ul 0.5 M Tris pH 7.5, 10 mM EDTA
10 ul 2 M NaCl
0.5 ul G-tailed pUC19
200 ul total
incubate 65 C, 5 min, then incubate 45 C, 2 h
slow cool to room temp.
Transform competent E. co1i JM83 cells as in Appendix A.


APPENDIX C
VARIOUS OTHER RECIPES
cDNA Synthesis for Hybridization Probes
25.5 ul H20
5 ul 10X cDNA buffer (500 mM Tris, pH 8.3, 80 uiM MgCl2,
250 mM NaCl)
2.5 ul 100 mM DTT
1.8 ul RNasin (25 U/ul)
1.3 ul actinomycin D (700 ug/ml)
3 ul 10 mM dATP
3 ul 10 mM dTTP
3 ul 10 mM dGTP
0.5 ul random primers (5 ug/ul)
2.0 ul PRSV-W RNA (1 ug/ul)
1.8 ul reverse transcriptase (10-16 U/ul)
0.6 u132P-dCTP (5 uCi)
50.0 ul total
incubate at 42 C, 30 min
add 0.5 ul 10 mM dCTP
incubate at 42 C, 60 min
add 3.0 ul 6 N NaOH
incubate at 56 C, 60 min.
Plasmid Purification
Grow cultures overnight in a 250 ml flask containing 40 ml L
broth at 37 C with shaking. Cool culture in ice 30 min.
Centrifuge 10K for 10 min. Decant supernatant.
43


Resuspend pellet in:
0.48 ml 25% sucrose, 50 mM Tris pH 8.0 (ST)
vortex
0.16 ml 10 mg/ml lysozyme in ST (make fresh)
incubate on ice 5 min
0.40 ml 0.2 M EDTA pH 8.0
incubate on ice 5 min
1.02 ml Triton lysis buffer
(1 ml 10% Triton X100, 5 ml 1.0 M Tris pH 8.0, 31.25
ml 0.2 M EDTA pH 8.0, 62.75 ml H20)
incubate on ice 15 min, swirl occasionally
Centrifuge 15K, 30 min; collect supernatant
Phenol extract 2 times; ethanol precipitate; resuspend in
250 u1 H2-
Nick Translation
0.5 ul DNA (1 ug/ul)
5.0 ul 10X NTB (500 uM Tris pH 7.9, 50 uM MgCl2, 100 uM 2-
mercaptoethanol)
34 ul H20
5.0 ul 10X NTP (40 uM each dATP, dTTP, dGTP)
5.0 ul DNase I (1 ul of 50 ug/ml stock in 500 ul H20)
0.5 ul 32P dCTP (5 uCi)
50 ul total
incubate 15 C, 4 h
add 2.5 ul 10% SDS, and 2 ul of 0.35% bromophenol blue
Run through a 1 ml Sephadex G-50 column with NTB buffer,
collect first radioactive peak (when the bromophenol blue is
about at the 80 cc mark on a 1 ml Tuberculin syringe)


LITERATURE CITED
Adlerz, W. C. 1972. Momordica charantia as a source
watermelon mosaic virus 1 for cucurbit crops in
Beach County, Florida. Plant Dis. Reptr. 56,
of
Pa lm
463-567.
All
ison, R. F., Sorenson, J. C., Kelly, M. E., Armstrong, F.
B., and Dougherty, W. G. (1985). Sequence
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BIOGRAPHICAL SKETCH
Julianne Nagel was born in Colver, Pennsylvania, on
December 12, 1957, and grew up in Johnstown, PA. She
graduated from Ferndale Area High School in 1975. She
completed her undergraduate degree in horticulture at
Pennsylvania State University, graduating in 1978 with the
title of Student Marshall of the College of Agriculture. In
1979 she enrolled at the University of Florida, Plant
Pathology Department, and obtained a Master of Science
degree under the direction of Dr. F. W. Zettler in 1981.
From 1981 until 1982 she was employed as a laboratory
technician II for Dr. F.W. Zettler. She began her Ph.D.
research in 1982 in the same department under the direction
of Dr. E. Hiebert and completed her degree in 1985. She is
currently a postdoctoral research associate with Dr. R.
Shepherd in the Plant Pathology Department at the University
of Kentucky.
49


I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of
Philosophy.
.j'
Ernest Hiebert, Chairman
Professor of Plant Pathology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of
Philosophy.
L. Curtis Hannah
Professor of Horticultural Science
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation, foj; £he degree^ of,Poctor of
Philosophy. ) // / J
David C. Loschke
Assistant Professor
of Plant Pathology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for th^o^gree of Doctor of
Philosophy.
Daryl R. Pri*ig
Professor of Plant Pathology


i n
I certify that I have read this study and that
my opinion it conforms to accepta-ble standards of
scholarly presentation and is fuJyly adequate, in scope
and quality, as a dissertati>ri fpr the degree of Doctor of
Philosophy. r\i
Francis W. Zettler
Professor of Plant Pathology
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School,
and was accepted as partial fulfillment of the requirements


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COMPLEMENTARY DNA CLONING AND EXPRESSION OF THE PAPAYA
RINGSPOT VIRUS SEQUENCES ENCODING CAPSID PROTEIN
AND A NUCLEAR INCLUSION-LIKE PROTEIN
By
Julianne Nagel
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA FOR
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1985

In memory of my father, Robert C. Nagel.

ACKNOWLEDGEMENTS
I would like to thank my family for their faith
encouragement. I am grateful to my major professor, Ernest
Hiebert, for allowing me great freedom in my research and
for always being available when I needed help. I would also
like to thank the members of my committee, D.R. Pring, D.C.
Loschke, F.W. Zettler, and L.C. Hannah, for sharing their
expertise and generously allowing me access to their
laboratories and supplies.
I am indebted to Maria de Mejia for convincing me to
work with PRSV instead of DMV. I appreciated John Payne's
help with the computers and his decency in not graduating
before me. Margarita Licha, Feiko Ferwerda, Kris Figura,
and Gene Crawford are thanked for their friendship and for
preventing my starvation by supplying me with countless
lunches and dinners. Finally, I thank Randy Ploetz for his
love and encouragement.
in

TABLE OF CONTENTS
Page
DEDICATION ii
ACKNOWLEDGEMENTS iii
ABSTRACT V
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: COMPLEMENTARY DNA CLONING AND EXPRESSION
OF THE CAPSID AND A NUCLEAR INCLUSION-LIKE
PROTEIN OF PAPAYA RINGSPOT VIRUS IN E. COLI
Introduction 5
Materials and Methods 5
Results 9
Discussion 20
CHAPTER 3: COMPLEMENTARY DNA CLONING OF PAPAYA RINGSPOT
VIRUS BY AN RNA:CDNA HYBRID METHOD
Introduction 25
Materials and Methods 26
Results 27
Discussion 32
CHAPTER 4: CONCLUSIONS 34
APPENDIX A: RECIPES FOR HEIDECKER AND MESSING CLONING
PROCEDURE 36
B: RECIPES FOR CANN ET AL. CLONING PROCEDURE... 41
C: VARIOUS OTHER RECIPES 43
LITERATURE CITED 45
BIOGRAPHICAL SKETCH 49
IV

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of
the Requirements for the Degree of Doctor of Philosophy
COMPLEMENTARY DNA CLONING AND EXPRESSION OF THE PAPAYA
RINGSPOT VIRUS SEQUENCES ENCODING CAPSID PROTEIN
AND A NUCLEAR INCLUSION-LIKE PROTEIN
By
Julianne Nagel
May, 1985
Chairman: Ernest Hiebert
Major Department: Plant Pathology
Papaya ringspot virus type W (PRSV-W), formerly
watermelon mosaic virus 1, is a member of the potyvirus
group of plant viruses. The potyviral genome consists of a
single plus-stranded RNA, about 10,000 nucleotide residues
in molecular size, with a small protein attached to the 5'
end and a poly (A) tail on the 3' end. In this research,
two complementary DNA (cDNA) cloning methods were used to
clone portions of the PRSV-W genome. Poly (T) was used to
prime cDNA synthesis in both procedures. In the Heidecker
and Messing procedure (Nucl. Acids Res. 11,4891-4906) the
vector was pUC9 and the viral RNA was copied into double-
stranded DNA before transforming Escherichia co 1 i JM83. In
the Cann et al. procedure (Nucl. Acids Res. 11,1267-1281) an
RNA:cDNA hybrid inserted into pUCl9 was used to transform EL
co1 i JM83. The Cann method was more efficient than the
Heidecker and Messing procedure, both in the number of
v

clones produced, and in the length of time required to
complete the procedure. The clones produced by either
procedure represented approximately the same area of the
genome.
Three cDNA clones that expressed viral gene products in
E. co1i were characterized. The expressed polypeptides
were fusion products with the amino terminus of the oC-donor
fragment of B-galactosidase. Clones Wl-77 and W2-1 were
1480 and 630 base pairs (bp) in size and expressed fusion
products with apparent molecular weights of 40,000 and
14,000, respectively, which were serologically related to
PRSV capsid protein. Clone Wl-18 was 1330 bp in size and
produced a 52K product that was serologically related to a
54K nuclear inclusion protein of tobacco etch virus. The
sequences encoding the capsid and 57K nuclear inclusion-1 ike
proteins of PRSV-W were physically mapped to adjacent
positions through Southern blot analyses of clones Wl-77 and
Wl-18 and are presumed to be located at the 3' end of the
viral genome.
vi

CHAPTER 1
INTRODUCTION
Papaya ringspot virus is a potyvirus which causes
destructive diseases of papaya and cucurbits. The virus
limits the production of papaya in Caribbean countries,
Florida, Hawaii, India, South America, and Taiwan (Yeh et
al., 1984; and references therein) and causes serious losses
in cucurbit crops worldwide (Purcifull et al., 1984). The
strain of PRSV used in this study was type W, which until
1984, was known as watermelon mosaic virus 1 (Purcifull et
al., 1984). The type W strain differs from the prototype,
PRSV-P, only in one host range characteristic; type W does
not infect papaya (Purcifull et al., 1984). In Florida
PRSV-W overwinters in the wild cucurbit hosts, Melothria
pendula and Momordica charantia, and is transmitted
nonpersistently by many species of aphids (Adlerz, 1972;
Purcifull et al., 1984).
The PRSV particle is a long flexuous rod approximately
780 nm in length and 12 nm in width. The coat is
constructed of identical, repeating subunits each with a
molecular weight of 36,000 (36K) (Purcifull et al., 1984).
The potyviral genome consists of a single, plus-stranded
RNA, ca. 10,000 nucleotide residues (lOkb) in size with a
genome-1 inked protein on the 5' end (Hari, 1981) and a poly
(A) tail on the 3' end (Hari et al., 1979). At least seven
proteins are thought to be encoded by the genome (de Mejia
1

et al., 1985b). Two of the proteins of PRSV aggregate
separately in the cytoplasm of host plant cells to form
cylindrical inclusions (Christie and Edwardson, 1977) and
amorphous inclusions (de Mejia et al., 1985a). The
cylindrical inclusions are composed of 70K protein subunits
which array themselves to form 'pinwheel' structures
attached to the plasma membrane, frequently over
p1asmodesmata (Christie, personal communication). Later in
infection, the cylindrical inclusions usually move away from
the plasmalemma and aggregate near the nucleus (Christie and
Edwardson, 1977). The amorphous inclusions consist of
aggregations of a 51K protein subunit along with associated,
uncharacterized RNA (de Mejia et al., 1985a). The 51K
protein is the first and only nonstructura1 protein of
potyviruses to which a function has been ascribed; it
appears to be a helper factor which enables potyviruses to
be transmitted by aphids (de Mejia et al., 1985b).
Certain potyviruses, such as tobacco etch virus (TEV),
form a third type of inclusion in the nucleus of host plant
cells. These nuclear inclusions are composed of an
equimolar mixture of two viral proteins, a 49K protein
subunit and a 54K protein subunit (Knutsen et al., 1974).
Other potyviruses, such as PRSV, that are not known to form
nuclear inclusions _in vivo have been demonstrated to form
products serologically related to the 49K and 54K proteins
of TEV during i_n vitro translations (de Mejia et al., 1985b;
Dougherty and Hiebert,1980 ) . It is probable that these

3
protein subunits are also formed in infected host cells but
do not aggregate in the nucleus to form structures apparent
by light microscopy.
Over 25 potyviruses have been translated _i_n vitro
(Hiebert, unpublished) and their products have been
immunoprecipitated with various antisera including those to
the capsid, cylindrical inclusions, amorphous inclusions,
and the 2 nuclear inclusion proteins. Through analysis of
the immunoprecipitated products, gene maps have been
proposed to locate the protein coding regions on the viral
genome (Dougherty and Hiebert, 1980; Heilman et al., 1980;
Vance and Beachy, 1984; Xiong, 1985; de Mejia et al.,
1985b). Each of these gene maps differs to some degree; for
example, Heilman et al. (1980) and Vance and Beachy (1984)
report that the capsid protein coding region is located near
the 5' end of the genome while others define its position at
the 3' end (Dougherty and Hiebert, 1980; Xiong, 1985; de
Mejia et al., 1985b). At the present time the potyviral
gene map is controversial. The map which was used as a
reference in this study is by Xiong (1985), and when adapted
for the molecular weights of the PRSV proteins is 5' end;
60K 'unknown' protein; 51K amorphous inclusion protein; 40K
'unknown' protein; 70K cylindrical inclusion protein; 49K
nuclear inclusion protein; 57K nuclear inclusion protein;
36K capsid protein; 3' end.
The primary objective of this research was to develop a
system to clone portions of a potyviral genome and obtain

4
the transcription and translation of these portions in
Escherichia co1i. By serologically identifying the
polypeptides expressed in E. co1i and correlating them with
specific areas of the viral genome, the gene map may then be
verified. This approach was taken by Daubert et al. (1982)
to map the location of the capsid protein gene of
cauliflower mosaic virus on the viral genome.
Also through such an expression system, polypeptides
could potentially be obtained for the two 'unknown' coding
regions of the potyviral genome. The products of these
coding regions are not known to aggregate in host cells and
have not been purified or used to produce antisera; their
existence has been demonstrated only in _i_n vitro translation
products. Saito et al. (1984) cloned cDNA fragments coding
for parts of two tobacco mosaic virus nonstructura1 proteins
and expressed the sequences in E. co1 i. These nonstructura1
proteins are normally extremely difficult to isolate from
infected plants, but by expressing the proteins in E. co1i,
sufficient amounts were obtained for characterization and
antibody production. Other examples of plant viral proteins
expressed in E. co1i include the capsid protein of satellite
tobacco necrosis virus (Van Emmlo., et al., 1984) and the
capsid protein of brome mosaic virus (Miglietta and
Kaesberg, 1984).

CHAPTER 2
COMPLEMENTARY DNACLONING AND EXPRESSIONOFTHECAPSID
AND A NUCLEAR INCLUSION-LIKE PROTEIN OF PAPAYA
RINGSPOT VIRUS IN E. COLI
Introduction
The Heidecker and Messing procedure (1983) was selected
for the cloning of PRSV-W because the procedure was reported
to be very efficient and yield a high proportion of full-
length clones. In theory approximately one-sixth of the cDNA
clones produced by this procedure should also express
protein products for the cloned inserts in E. co1i (Helfman
et al. 1933; Heidecker and Messing, 1983). Finally, because
cDNA synthesis is primed by a poly (T) tail that is
covalently attached to the pUC9 plasmid, it was assumed that
the resultant cDNA clones would have originated from the
poly (A) tail on the 3' end of the viral RNA. Such a factor
would have facilitated future mapping studies by providing a
known starting point.
Materials and Methods
Materials
Restriction enzymes and the Klenow fragment of DNA
polymerase I were from Bethesda Research Laboratories, Inc.
(BRL, Gaithersburg, MD 20877). Reverse transcriptase was
from the Seikaguki Company (St. Petersburg, FL 33702). f^5S]
Methionine, ^C- 1 abe 1ed molecular weight standards, and
5

6
I O C
I-labeled protein A were from Amersham (Arlington
Heights, IL 60005). Escherichia co 1 i JM83, pUC8, and pUC9
were from D.C. Loschke. Antisera to three strains of PRSV:
type W, type P, and type T were from D.E. Purcifull, D.
Gonsalves, and L. Quiot-Douine, respectively.
Papaya ringspot virus type W (PRSV-W), formerly
watermelon mosaic virus-1 (Purcifull et al., 1984), was
propagated in pumpkin plants (Cucúrbita pepo L. 'Small
Sugar') and purified 2.5 wk after inoculation by the method
of Purcifull et al. (1984). RNA was extracted from purified
virus by treatment for 5 min at 60C with 2.0% sodium dodecyl
sulfate (SDS), 2 mM ethylenediamine tetraacetic acid (EDTA),
and 200 mM ammonium carbonate, pH 9 (Brakke and Van Pelt,
1970a), and isolated on sucrose log-linear gradients (Brakke
and Van Pelt, 1970b). The RNA was then precipitated with
ethanol, centrifuged, and resuspended in a small volume of
sterile, distilled water. The phenol extraction of the RNA
was similar to that described by Maniatis et al. (1982)
except that after the phenol and phenol/chloroform
extractions, the organic phases were removed with a
capillary tube, leaving the aqueous phase and interface.
Only the aqueous phase, containing the RNA, was retained
after the final chloroform extraction. The combined organic
phases were reextracted according to Maniatis et al. (1982).

7
cDNA Synthesis and Molecular Cloning
Complementary (c) DNA clones of PRSV-W were prepared
by the method of Heidecker and Messing (1983). This involved
1) cutting the plasmid, pUC9, with the restriction
endonuclease, Pst 1; 2) tailing the cut plasmid with 40-60
deoxythymidy1 ate residues; 3) adding the viral RNA and using
the poly (T) tail of the plasmid as a primer for first
strand cDNA synthesis; 4) tailing the first strand of cDNA
with dGTP; 5) denaturing and purifying the plasmid with
attached cDNA by centrifugation through alkaline sucrose
gradients; 6) reannealing the size-fractionated plasmid with
attached cDNA to dCTP-tailed plasmid; and 7) filling in the
single-stranded area with the Klenow fragment of DNA
polymerase I (see Appendix A).
Escherichia co1 i JM83 cells were transformed by a
calcium chloride shock treatment and plated onto L plates
containing 50 ug/ml ampicillin and the indicator 5-bromo-4-
chloro-3-indoly1-B-D-ga1 actoside (Xgal) at 40 ug/ml
(Maniatis et al., 1982; see Appendix A). Insertional
inactivation of the B-galactosidase gene of pUC9 (Viera and
Messing, 1982) was used as a color marker to select
bacterial colonies containing pUC9 with a cDNA insert. The
presence of PRSV-W sequences in white colonies was verified
by colony hybridization of cloned DNA with [^^P]-dCTP
labeled first-strand cDNA of PRSV-W RNA (Maniatis et al.,
1982). Because Close et al. (1983) reported that not all
insertions into the B-ga1 actosidase fragment of pUC vectors
result in white colonies, a portion of the blue and light

8
blue colonies were also tested for PRSV sequences. Insert
size was determined by restriction endonuclease digestion of
plasmid DNA and agarose gel electrophoresis. Molecular
weight markers were lambda DNA (supplied by D. Pring) and
0X174 RF DNA (BRL), digested with Hind III and Hae III,
respectively.
Detection of Expressed Viral Proteins
Bacterial cells from 12 to 16 hr cultures grown in L
broth (Maniatis et al., 1982) were collected by
centrifugation at 5,000x£, and lysed in 1/10 volume
(original cell suspension) of Laemmli dissociation buffer
(Laemmli, 1970). The lysed cells were centrifuged at
27,000xc[ for 30 min, and the supernatant was retained. Gel
electrophoresis, transfer to nitrocellulose sheets, and
detection of proteins with antibody (Western blots) were
performed as described by de Mejia et al. (1985a). Agar gel
(Ouchter1ony) immunodiffusion tests were conducted as
described by Purcifull and Batchelor (1977). The
immunodiffusion medium contained 0.8% Noble agar, 0.5% SDS,
and 1% sodium azide (Purcifull and Batchelor, 1977).
For immunoprecipitat ions, bacterial cells were labeled
with [^5S]-methionine. In these experiments bacterial
cultures were grown in potassium morpho1inopropane sulfonate
(MOPS) medium (Neidhardt et al., 1974) supplemented with
2% dehydrated methionine assay medium (Difco, Detroit, MI
48232) and 10 uCi/ml []-methionine. The cells were
incubated for 8 to 16 hr at 37 C with shaking, and lysates

9
were prepared as described above.
Immunoprecipitat ions were performed by incubating for 1
hr at room temperature 100 ul of [ ]-methionine-1 abe 1 ed
bacterial lysate with 100 ul of antiserum and 400 ul of 0.5%
Nonidet P-40 in 150 mM NaCl, 5 mM EDTA, 50 mM Tris, and
0.02% sodium azide (NET buffer) containing 1 mg/ml
ovalbumin, and 2 mM methionine (Kessler, 1975). One hundred
microliters of a 10% Staphylococcus aureus protein A
solution was then added, and the incubation was continued
for 20 min. The mixture was washed three times in 0.05% NP-
40 NET buffer and resuspended in 40 ul of Laemmli
dissociation buffer. Before electrophoresis in SDS-
permeated polyacrylamide gels (SDS-PAGE) the samples were
heated in a boiling water bath for 5 min. Products were
visualized by fluorography (Bonner and Laskey, 1974).
Nick translations were performed as described by Rigby
et al. (1977). Southern blots were as described by Maniatis
et al. (1982).
Results
DNA Analysis of Expression Clones
Three of the six resulting cDNA clones expressed viral
proteins and were characterized. Clone Wl-77 contained a
1480 base pair (bp) insert and clone W2-1 contained a 630 bp
insert. Each insert could be excised from the plasmid by
cutting on both sides of the insert, in the polylinker
region of pUC9, with Bam HI and Hind III (Figs. 2-1, 2-2).
Eco RI cut at a site in the polylinker region and also at

10
sites within the inserts of Wl-77 and W2-1, producing
fragments of approximately 580 and 3600 bp for Wl-77, and
550 and 2780 bp for W2-1. Clone Wl-18 contained a 1330 bp
insert with an internal Hind III site (Fig. 2-2). When Hind
III alone was used to restrict Wl-18, fragments of 450 and
3580 bp were observed. Bam HI cut only in the polylinker
region of pUC9; it did not cut the Wl-18 insert.
From Southern blotting analyses, clone Wl-18 was found
to overlap with Wl-77 but not W2-1. When the 880 bp Bam HI
to Hind III fragment of Wl-18 was isolated, nick translated,
and used as a probe, only self homology was observed (Fig.
2-3). When the 450 bp fragment of Wl-18 was used as a
probe, it hybridized to itself and the Wl-77 insert, but not
to the W2-1 insert (Fig. 2-4). The Wl-77 insert, when used
as a probe, hybridized to itself, the W2-1 insert, and only
the 450 bp insert of Wl-18 (Fig. 2-5).
The orientation of Wl-77 in pUC9 is shown in Figure 2-
la. The direction of transcription and translation in
Figure 2-1 is counterclockwise, in pUC9 from the Hind III
site to the Eco RI site. The cDNA insert of clone Wl-18 was
transferred from pUC9 into pUC3 to orient the insert in the
proper direction for transcription and translation (Fig. 2-
lb) .
Identification of Expressed Proteins
Clones which expressed protein were identified by
serological testing. Similar serological results were
observed in Western blots and immunoprecipitat ion tests of

11
protein extracts for each expression clone (Figs. 2-6, 2-7).
Clone Wl-77 produced predominately a polypeptide of Mr
40,000 (40K) that reacted with PRSV capsid antiserum (Fig.
2-6, lane 2; Fig. 2-7, lane 2). When the insert of clone
Wl-18 was in pUC9, no reactions were observed in either
Western blots or in immunoprecipitations. However, when
the Wl-18 insert was in pUC9 a 52K polypeptide that
serologically reacted with an antiserum to TEV 54K nuclear
inclusion protein was formed (Fig. 2-6, lane 8; Fig. 2-7,
lane 11). A number of extra bands were observed with
immunoprecipitation tests with the TEV 54K protein antiserum
(Fig. 2-7, lanes 3, 7, and 11), and with the Western blots
(Fig. 2-6) with either serum. These bands were assumed to
be reactions of the antisera with certain unidentified
bacterial proteins and occurred regardless of whether the
bacteria contained pUC9 or any of the clones. Clone W2-1
produced a 14K polypeptide antigenica1 ly related to PRSV
capsid protein (Fig. 2-6, lane 3).
In Ouchterlony immunodiffusion tests the expressed
protein of Wl-77 reacted homologously with antiserum to

12
Sal I
Hind III Sal I
Fig. 2-1. Locations of cDNA inserts in pUC plasmids:
a. Wl-77 in pUC9. b. Wl-18 in pUC8. Transcription and
translation proceed counterclockwise.

13
Fig. 2-2. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9r
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B in an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [32P]-labeled probe consisting of randomly primed
first-strand cDNA of PRSV-W.

14
Fig. 2-3. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [3;i P]-labeled probes consisting of nick translated
880 bp fragment of Wl-18.

15
Fig. 2-4. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [3¿ P]-labeled probe consisting of nick translated 450
bp fragment of Wl-18.

16
1 2 3 4 1 2 3 4
Fig. 2-5. Restriction analysis and Southern blotting of DNA
from clones Wl-77, W2-1, and Wl-18. Lanes contain; 1. pUC9,
2. Wl-77, 3. W2-1, 4. Wl-18 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A, gel
stained with ethidium bromide. Panel B is an autoradiograph
of the DNA transferred onto nitrocellulose and hybridized
with a [32P]-1 abe 1ed probe consisting of nick translated
1480 insert of Wl-77.

17
14K-^
Fig. 2-6. Western blot of lysates from E. co 1 i JM83
containing: pUC9 (lanes 1 and 5), clone Wl-77 (lanes 2 and
6)', clone W2-1 (lanes 3 and 7), and clone Wl-18 (lanes 4 and
8). The proteins were analysed by electrophoresis in an
SDS-permeated polyacrylamide gel, electrophoretically
blotted onto nitrocellulose, incubated with an antiserum to
PRSV-P capsid protein (lanes 1-4), or an antiserum to TEV
54K nuclear inclusion protein (lanes 5-8), then incubated
with ,i5I-labeled protein A and autoradiographed. Lane 9
contains protein molecular weight markers. Arrows identify
the estimated sizes of the immunoreactive proteins.

18
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a
<
<*-52K
1 2 3 4 56789 10 11
â– 3 e
Fig. 2-7. Immunoprecipitations of methionine labeled E.
co1i fusion proteins related to PRSV capsid protein and TEV
54K nuclear inclusion protein. The proteins were analysed
on an SDS-permeated 7.5 to 15% gradient polyacrylamide gel
which was processed by f1uorography. Lanes 1, 2, 3, and 4
contain lysate from JM83 containing clone Wl-77; lanes 5, 6,
7, and 8 contain lysate from JM83 containing pUC9, and lanes
9, 10, and 11 contain lysate from JM83 containing clone Wl-
18. Lysates in lanes 2, 6, and 10 were immunoprecipitated
with antiserum to PRSV-P capsid. Lysates in lanes 3, 7, and
11 were immunoprecipitated with antiserum to TEV 54K nuclear
inclusion protein. Lysates in lanes 4 and 8 were
immunoprecipitated with pre-immune serum. Arrows identify
the locations of the major immunoreactive proteins.

19
Fig. 2-8. Serological relationships of capsid protein from
clone Wl-77, PRSV-W, and PRSV-T. Central wells contain: P
= antiserum to PRSV-P capsid, G = antiserum to PRSV-T, N =
pre-immune serum. Peripheral wells contain: c = lysate from
JM83 containing clone Wl-77, g = extract from pumpkin plants
infected with PRSV-T, p = extract from pumpkin plants
infected with PRSV-W, b = lysate from JM83 containing pUC9,
and h = extract from healthy pumpkin plants.

20
either PRSV-W virions or PRSV-P capsid protein (Fig. 2-8),
which is consistent with previous reports that PRSV-P and
PRSV-W are serologically indistinguishable in
immunodiffusion tests (Purcifull et al., 1984, and
references therein). The protein expressed by Wl-77 reacted
heterologously with an antiserum to virions of PRSV-T, a
strain serologically distinct from PRSV-P or PRSV-W (Quiot-
Douine et al., 1985). No reactions were evident with pre-
immune sera or with crude extracts of bacteria containing
only pUC9. The expressed protein from clone W2-1 did not
produce a visible reaction in these tests. Apparently
either the protein was not in a great enough concentration
for a precipitan formation, or perhaps the antisera used
contained antibodies predominately to the amino end of the
capsid protein.
Discussion
Two clones of PRSV-W were identified as expressing
polypeptides that are antigenica1ly related to PRSV capsid
protein, and one clone was demonstrated to produce a
polypeptide antigenica1ly related to a nuclear inclusion
protein of TEV. The expressed proteins are fusion products
because 27 bp exist between the translational start site of
the B-galactosidase fragment and the Pst I site into which
the cDNA was inserted (Viera and Messing, 1982). Therefore,
the first 9 amino acids of each of the expressed proteins
are from the amino terminus of the oC-donor of
B-ga1 actosidase. The plasmid lac promoter of pUC9 is
constitutive for B-galactosidase when the E. co1 i JM83 host

21
is used, and so no inducer was necessary for the expression
of the fusion proteins. Sufficient amounts of the fusion
protein of clone Wl-77 were synthesized that the crude
lysates of bacterial cells could be used in Ouchterlony
immunodiffusion tests.
It was demonstrated with Southern blot analyses that
clones Wl-77 and Wl-18 overlap, and therefore concluded that
the two sequences encoding the capsid and 57K nuclear
inclusion-1 ike proteins are adjacent to one another on the
viral genome. However, their positions on the viral genome
were not established in this study. The location of the
capsid coding region has been identified for two other
potyviruses, pepper mottle virus (Dougherty et al., 1985)
and TEV (Allison et al., 1985) at the 3' end of the viral
RNA. It seems probable, therefore, that the capsid coding
region of PRSV is also located at the 3' end of the viral
RNA. The proposed locations of clones Wl-18 and Wl-77 in
reference to the PRSV-W gene map are illustrated in Figure
2-9.
The cDNA insert in Wl-77 was 1480 bp long and after
subtracting 88 bp for the poly (A) sequence (sequence data
not shown) and a presumed 3' non-coding region of 100 to 300
bp, it could theoretically code for a protein in the range
of 40K to 48K. The predominant antigenic polypeptide found
in either Western blots or in immunoprecipitations of Wl-77
protein was approximately 40K. The amino terminus of
B-ga1 actosidase would account for approximately IK and so

22
the bacterially expressed protein was larger than the capsid
protein from virions by 3K. The additional 3K may represent
the carboxyl end of the 57K nuclear inclusion-1 ike protein.
This would imply that there is no recognized stop codon
between the 57K nuclear inclusion coding region and the
capsid protein coding region in the PRSV-W genome. The 57K
nuclear inclusion coding region has been reported
previously to lie adjacent to and in the same reading frame
as the capsid protein coding region through analysis of in
vitro translation 'read through' or polyprotein products (de
Mejia et al.f 1985b; Dougherty and Hiebert, 1980). We were
not able to detect any reaction of the 40K protein with the
antiserum to TEV 54K nuclear inclusion protein in
immunoprecipitations or in Western blots. This is not
surprising considering that such a small part of the nuclear
inclusion protein is presumed to be present and that the
antiserum is not homologous for PRSV.
After subtracting the same 188 to 388 bp to account for
the poly (A) and 3' non-coding regions, clone W2-1 could
theoretically code for a polypeptide of 9K to 16K. The
observed 14K polypeptide serologically related to the capsid
protein is within the predicted size range.
When the Wl-18 insert was in pUC9, no vira1-specif ic
product was made because the insert was oriented 3' to 5'
with respect to the promoter. Transferring the insert into
pUC8 made it 5' to 3' with respect to the promoter and
allowed the transcription and translation of a polypeptide

23
antigenically related to the 54K nuclear inclusion protein
of tobacco etch virus. Apparently the insert was already in
the correct reading frame and no attempts to change the
reading frame were necessary. The polypeptide expressed by
clone Wl-18 was larger than is expected for its coding
capacity. The 1330 bp insert could maximally code for a
polypeptide of 49K; however, a protein of 52K was observed.
One reason for this discrepancy could be that there is no
stop codon in the cDNA insert, as already suggested for Wl-
77, and transcription and translation continue into the B-
galactosidase coding area. Indeed, the colony color of Wl-
18 was light blue on indicator plates. The 52K protein is
not large enough to contain both the predicted 49K nuclear
inclusion polypeptide and the 16K B-ga1 actosidase
polypeptide fragment, so some post-trans1 ationa1 cleavage
might be occurring. Although the cleavage site is not
known, it would seem likely that since B-ga1 actosidase is a
bacterial protein, it would be more resistant to degradation
in E. coli than the nuclear inclusion protein. Therefore,
the 52K protein may consist of 16K from B-galactosidase and
36K from the nuclear inclusion protein. The remaining 13K
nuclear inclusion cleavage fragment, however, was not
apparent in the Western blots or in the immuno-
precipitations.

24
5’VpgJi
60k
9
^tk40k
Amorphous â– >
Inclusion
70k
Cylindrical
Inclusion
49k , 57k
Nuclear Nuclear
Inclusion Inclusion
1330 bp
W1-18
36k
Capsid
^â– poly A
W1-77
â– 1480 bp
W2-1
6 30bp
3’
Fig. 2-9. Proposed locations of clones Wl-18, W1-77, and
W2-1 on the PRSV-W gene map. For discussion of the PRSV-W
gene map, see Chapter 1.

CHAPTER 3
COMPLEMENTARY DNACLONING OF PAPAYA RINGSPOT VIRUS RNA
BYAN RNA:cDNA HYBRID METHOD
Introduction
The RNA:cDNA hybrid cloning procedure by Cann et al.
(1983) was selected as the second cloning method for PRSV-W.
The authors reported that by using this technique with
poliovirus RNA, which is structurally similar to potyviral
RNA, they produced a set of clones with long, overlapping
inserts which spanned the entire 7 Kb size of the
poliovirus genome. The authors also observed that more of
their clones represented the 5' half of the genome rather
than the 3' half. Since clones of what is presumed to be
the 3' end of the PRSV-W genome had already been produced by
the Heidecker and Messing procedure (1983), this method was
undertaken with the goal of obtaining clones with long cDNA
inserts (>2000 bp) representing an area 5' to those areas
previously cloned. Another reason this procedure was
selected was because of its time efficiency; a minimal
number of steps are involved and the entire procedure can be
completed in two days.
25

26
Materials and Methods
Materials
Restriction enzymes, terminal transferase, deoxy
nucleotides, and $X174 RF Hae III fragments were from
Bethesda Research Laboratories (BRL, Gaithersburg, MD
20877). Reverse transcriptase was from Seikaguki (St.
Petersburg, FL 33702). pUCl9 and E. co1i JM83 were from
D.C. Loschke. Lambda molecular weight markers were from
D.R. Pring.
Papaya ringspot virus type W (PRSV-W) was purified from
inoculated Cucúrbita pepo L. 'Small Sugar' by the procedure
of Purcifull et al. (1984). RNA was extracted from purified
virus (Brakke and Van Pelt, 1970a) and isolated on sucrose
log-linear gradients (Brakke and Van Pelt, 1970b). The RNA
was phenol extracted (Maniatis et al., 1982) before being
used in the cloning procedure.
cDNA Synthesis and Molecular Cloning
The method of Cann et al. (1983) was used to produce
cDNA clones of PRSV-W RNA. Complementary DNA synthesis was
primed with oligo dT. After the first strand of cDNA was
made, the RNArcDNA hybrid was tailed on the 3' ends with
dCTP. The pUCl9 vector was cut with Pst I and tailed on the
3' ends with dGTP. The G-tailed vector and the C-tailed
RNA:DNA hybrid were then combined and allowed to circularize
(see Appendix B). Competent E. co 1 i JM83 cells were
transformed and plated onto L plates containing ampicillin
at 50 ug/ul and the indicator 5-bromo-4chloro-3-indoly 1-B-D-
galactoside (Xgal) at 40 ug/ml (Maniatis et al., 1982; see

27
Appendix A). A schematic of the cloning procedure is shown
in Figure 3-1. The resultant 113 white colonies and 19
randomly selected light blue colonies were tested for cDNA
inserts by colony hybridizations (Maniatis et al., 1983;
Close et al., 1983). The hybridization probe was randomly
primed, 22P-labeled, first strand cDNA of PRSV-W.
Cultures of 81 white and 13 light blue colonies that
were positive in the colony hybridizations for PRSV cDNA
inserts were grown overnight in L broth and the plasmid was
purified by the method of Maniatis et al. (1982). Insert
sizes were determined by digesting the plasmid DNAs with Bam
HI and Hind III and electrophoresing the samples on 1.2%
agarose gels along with molecular markers consisting of
lambda DNA Hind III and ¡ÍX174 RF Hae III restriction
fragments.
To determine if the cloned inserts represented the
capsid or 57K nuclear inclusion coding regions of the viral
genome, the plasmid DNAs were analyzed by Southern blot
hybridizations. Duplicate nitrocellulose sheets were
prepared by using sandwich blots to transfer the DNA from
the agarose gels. One of the nitrocellulose sheets was
hybridized with 22P-labeled nick-translated 1480 bp insert
of clone Wl-77, and the other was hybridized with 22P-
labeled, nick-translated 880 bp insert of clone Wl-18.
Results
Eighty-one of the 113 white and 13 of the 19 light blue
colonies were positive in the colony hybridization tests.

28
Of these 94 colonies containing PRSV cDNA inserts, 8 had
inserts between 1000 and 1300 bp, 10 contained inserts
between 800 and 1000 bp, 21 contained inserts between 400
and 800 bp, and the remaining 55 colonies contained inserts
under 400 bp in length.
All of the clones tested in Southern blot analyses
hybridized to either clone Wl-77 or Wl-18. The Southern
blots for 15 of the larger cDNA clones are shown in Figures
3-2 and 3-3. Seven of these clones appeared to be similar
to clone Wl-18. They each contained a Hind III site within
the cDNA insert; and after a double digestion with Hind III
and Bam HI to excise the insert from the vector, the smaller
fragment portion of each clone hybridized with the insert of
Wl-77, and the larger fragment hybridized with the 880 bp
fragment of Wl-18. With two of the clones, W4-27 and W4-56
(Figs. 3-2 and 3-3, lanes 2 and 13), the larger fragment
hybridized to the 1480 bp fragment of Wl-77, and the smaller
fragment hybridized to the 880 bp fragment of Wl-18. Clone
W4-48 hybridized only to the 880 bp fragment of Wl-18 and
did not contain a Hind III site. The remaining five clones
also lacked an internal Hind III site and hybridized only to
Wl-77.

AAAAA
Terminal Transferase
Oligo dT
v
AAAAA
TTTTT
Reverse Transcriptase
â–¼
AAAAA
TTTTT
Terminal Transferase
ccccc
AAAAACCCCC
TTTTT
j Reanneal
Transform
Fig. 3-1. Schematic diagram of Cann et al.
cloning procedure.
(1983) cDNA

30
b
Fig. 3-2. Restriction analysis and Southern blotting of
cloned DNA. Lane 1 contains 0X174 RF Hae III fragments. The
other lanes contain: 2. W4-27, 3. W4-42, 4. W4-43, 5. W4-
48, 6. W4-35, 7. W4-37, 8. W4-38, 9. W4-47, 10. W4-50, 11.
W4-51, 12. W4-55, 13. W4-56, 14. W4-60, 15. W4-66, 16. W4-
72, 17. Wl-18, 18. Wl-77 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A is the
gel stained with ethidium bromide. Panel B is an
autoradiograph of the the DNA transferred onto
nitrocellulose and hybridized with a [ 5¿P]-labe led probe
consisting of nick translated 1480 bp insert of Wl-77.

31
1353-—
1078—
872^—
603-
310-
b
Fig. 3-3. Restriction analysis and Southern blotting of
cloned DNA. Lane 1 contains 0X174 RF Hae III fragments. The
other lanes contain: 2. W4-27, 3. W4-42, 4. W4-43, 5. W4-
48, 6. W4-35, 7. W4-37, 8. W4-38, 9. W4-47, 10. W4-50, 11.
W4-51, 12. W 4 - 5 5, 13. W4-56, 14. W4-60, 15. W4-66, 16. W4-
72, 17. Wl-18, 18. Wl-77 DNA cut with Bam HI and Hind III
and electrophoresed on a 1.2% agarose gel. Panel A is the
gel stained with ethidium bromide. Panel B is an
autoradiograph of the the DNA transferred onto
nitrocellulose and hybridized with a [3,2 P ] - labeled probe
consisting of nick translated 880 bp fragment of Wl-18.

32
Clones W4-42 and W4-38 (Figs. 3-2 and 3-3, lanes 3 and
8) appeared to extend about 100 bp further towards the 5'
end of the viral genome than did Wl-18. Clones W4-27, W4-
42, W4-43, and W4-48 were light-blue in color on indicator
plates which may indicate that nuclear inclus ion-1 ike
proteins were being expressed.
Discussion
Many more clones were produced by the Cann et al.
method (1983) than by the Heidecker and Messing method
(1983) (94 vs 6 clones, respectively). The Cann method was
also much less time consuming than the Heidecker and Messing
procedure. Unfortunately, the goal of obtaining large
clones from an area of the viral genome 5' to clone Wl-18
was not met. Clones W4-42 and W4-38 did extend about 100 bp
further than Wl-18 toward the 5' end, but the remaining
clones all represented an area of the genome previously
cloned by the Heidecker and Messing procedure. Apparently
the reaction conditions were not optimal and cDNA synthesis
did not proceed past the 57K nuclear inclusion coding
region.
It would seem advantageous in future cloning
experiments to prime the cDNA reactions either with random
primers or with a small segment from the 5' end of clone W4-
42. Another approach would be to sequence the 5' end of W4-
42 to find a unique restriction site. A synthetic
oligonucleotide could then be prepared to the area
immediately 3' to the restriction site and used to prime

33
the synthesis of new clones. The
later be joined to W4-42 through
to form longer clones.
new clones could <
the unique restric
Iso
ion site

CHAPTER 4
CONCLUSIONS
Two cloning procedures were used sucessfully to prepare
cDNA clones of PRSV-W. The Cann et al. method (1983),
however, involved fewer manipulations and more clones were
produced. In the Heidecker and Messing procedure (1983),
the addition of T-tails to the cloning vector can cause the
appearance of long poly (A) sequences on cDNA clones which
may have been primed by short, internal A-rich regions.
Clones containing these long poly (A) regions may possibly
be misidentified as representing the 3' end of the viral RNA.
Clone Wl-77 has a poly (A) sequence on one end
approximately 88 bp in size. Wl-77 is assumed to represent
the 3' end of the viral RNA, not because it contains poly
(A), but because it causes the expression of a polypeptide
serologically related to the capsid protein. The capsid
protein coding regions of two other potyviruses have been
located on the 3' end of the genome through direct
comparisons of the amino acid sequences of the capsid
proteins with the viral RNA sequences (Allison et al., 1985;
Dougherty et al., 1985). The capsid protein coding regions
of 23 potyviruses, including PRSV, have also been mapped to
the 3' ends of the viral RNAs from analyses of products
formed during i_n vitro translations (Hiebert, unpublished).
Clone Wl-18 was mapped to an internal location on the
RNA, adjacent and 5' to clone Wl-77. It is unknown whether
34

this clone originated from a short internal A-rich region in
the viral RNA or whether a recombinationa1 event occurred
during the cloning procedure to yield this internal clone.
Sequencing studies should resolve the issue.
The expression of plant viral genes in bacteria has
many potential uses. Bacteria can produce viral proteins
that are not easily extracted from infected plants, and
antisera produced to these proteins would not be
contaminated with antibodies to plant antigens. For the
production of such large amounts of proteins, however,
vectors other than pUC8, 9, and 19 are recommended. Under
the best conditions in this study, the expressed viral-
specific proteins represented about 5% of the total soluble
proteins (data not shown). Other expression vectors are
available that express polypeptides for inserted sequences
at levels of up to 30 to 50% of the total E. c_o_l_i cel lular
protein (Amann et al., 1983; Masui et al., 1984; Schoner et
al., 1985). The expression of the cloned inserts is tightly
regulated with these vectors because the expressed products
can be toxic to the bacterial host. In the pUC plasmids
that were used in this study, the lac promoter is
constitutive when the E. co1i JM83 host is used, which may
cause a selection pressure towards those clones that do not
express the insert at a high level.

APPENDIX A
RECIPES FOR HEIDECKER AND MESSING CLONING PROCEDURE
Digestion of pUC9 DNA wi th PST I_
20 ul pUC9 DNA (1 ug/ul)
110 ul H2O
15 ul BRL 10X Core buffer (500 mM Tris, pH 8.0,100 mM
MgCl2' 500 NaCl)
5.0 ul PST I (9 U/ul)
150 ul total
incubate 37 C, 90 min or longer
phenol extract, ethanol precipitate,
resuspend in 20 ul H2O
C-tailing of pUC9
10 ul pUC9, cut with PST I
28.67 ul H20
10.5 ul 5X tailing buffer (500 mM K-cacodylate pH 7.2,10 mM
CoCl2, 1 mM DTT)
1 ul 1.0 mM dCTP
0.33 ul 32P dCTP
2 ul terminal transferase (20 U)
52.5 total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate 3 times
resuspend in 10 ul 10 mM Tris, pH 7.6, 10 mM NaCl,
1 mM EDTA
36

37
T-tailing of pUC9
10 ul pUC9, cut with PST I
10 ul 1 M K-cacodylate, pH 7.0
21 ul Ü2®
0.5 ul 0.1 M DTT
2 ul 1 mM dTTP
2 ul 32P dTTP (25 uCi)
1.5 ul terminal transferase (15 U)
5 ul 10 mM C0CI2
52 ul total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate 3 times, warm to room
temperature before centrifuging.
resuspend in 10 ul 10 mM Tris pH 7.6, 10 mM NaCl, 1 mM EDTA
cDNA Synthesis
6.4 ul H20
1 ul 12 mM dNTP's
1 ul 1.05 M KC1
1 ul 0.75 M Tris pH 8.2
0.5 ul 300 mM MgCl2
1 ul 30 mM DTT
0.6 ul RNasin 25 U/ul
0.5 ul.750 ug/ml actinomycin D
1 ul T-tailed pUC9 (1 ug/ul)
1 ul PRSV-W RNA (5 ug/ul)
1 ul reverse transcriptase (10 U/ul)
final cone.
8 00 uM
7 0 mM
50 mM
10 mM
2 mM
1 U/ul
25 ug/ml
40 nM
120 nM
100 U/ml
15 ul total

38
incubate 37 C, 90 min.
phenol extract, ethanol precipitate 3 times, warm to
room temp, before centrifuging, resuspend in 10 ul H20
G-tailing reaction
10 ul cDNA
4 ul 1 M K-cacodylate
1 ul 1 mM dGTP
1 ul 0.05 M DTT
2 ul 20 mM MnCl2
1 ul terminal transferase (15 (J/ul)
19 ul total
incubate 37 C, 15 min.
phenol extract, ethanol precipitate
resuspend in 50 ul 10 mM Tris, pH 7.6, 10 mM NaCl, 1 mM EDTA
Alkaline Sucrose Gradient Centrifugation
Recipe for 10 5-20% linear alkaline sucrose gradients
with a 60% cushion
Layer
g. sucrose
ml buffer*
amt ♦ £1
1. 5%
.5625
11.25
1.12
2. 10%
1.125
11.25
1.12
3. 15%
1.687
11.25
1.12
4. 20%
2.250
11.25
1.12
5. 60%
3.000
5.00
0.50
♦buffer: 0.2 M NaOH, 0.8 M NaCl, 1 mM EDTA
Add 50 ul of 5% sucrose layer to G-tailed cDNA. Layer
on gradient. Centrifuge in 50.1 rotor at 36K, 4 C, for 17
hr. Fractionate gradient by puncturing bottom of tube and
collecting 0.3 ml fractions. Measure amt. of radioactivity
by Cerenkov radiation. Pool samples from bottom of tube
that contain radioactivity, but do not use any fraction
after the peak fraction.
Add 3 ul of C-tailed pUC9. Dialyse over night at 4 C
against 3 changes of 10 mM Tris, pH 7.6, 10 mM NaCl, 1 mM
EDTA. Add 25 ug/ml carrier tRNA and ethanol precipitate.

39
Resuspend in 50 ul 10 mM Tris, pH 7.6, 10 mM NaCl, ImM
Reannealing Reaction
50 ul DNA
320 ul 100% formamide (deionized)
20 ul 2.5 M NaCl
10 ul 1 M Tris pH 8
600 ul H20
1000 ul total
final cone.
1-5 ul/ml
32%
50 mM
10 mM
incubate 37 C for 24 hr. Dialyse overnight at 4 C
against 100 mM NaCl,10 mMTris, pH 8.0, 1 mM EDTA.
Ethano lprecipitate, resuspend in 43.5 ul H20.
Fill-in Reaction final cone.
43.5 ul DNA
1 ul 2.5 M NaCl
50
mM
1 ul 1 M Tris pH 7.6
20
mM
1.67 ul 300 mM MgCl2
10
mM
1 ul 0.05 M DTT
1
mM
1 ul 5 mM dNTP1s
100
uM
0.8 ul Klenow Frag. DNA Pol. I
3
U
50 ul total
EDTA.
incubate 15 C for 60 min., then room temperature for 60 min.
phenol extract, ethanol precipitate, resuspend in 50 ul
10 mM Tris pH 7.6, 10 mM NaCl, 1 mM EDTA.

40
Transformation Procedure
1. Inoculate 20 ml of L broth in a 100 ml flask with 1 ml of
an overnight culture of E. co1 i JM83. Incubate culture at
37 C with vigorous shaking until reaching an ODerg of 0.5 (2
to 4 hr).
2. Pour the culture into a sterile capped centrifuge tube
and cool on ice 10 min. Centrifuge the cell suspension at
6,000 rpm in an SW 34 rotor for 5 min at 4 C.
3. Decant the supernatant. Gently resuspend the pellet in
10 ml sterile, ice-cold CaCl2, 10 mM Tris, pH 8.0. Incubate
suspension on ice 15 min.
4. Centrifuge the cell suspension at 6,000 rpm for 5 min at
4 C.
5. Decant the supernatant. Resuspend pellet in 1.33 ml
sterile, ice-cold 50 mM CaCl2» 10 mM Tris, pH 8.0.
6. Aliquot 0.2 ml volumes into sterile prechilled tubes.
Incubate on ice for 1 hr.
7. Add up to 100 ul of plasmid DNA in 10 mM Tris, pH 7.6, 10
mM NaCl, 1 mM EDTA. Incubate on ice 30 min.
8. Heat shock the cells by transferring tubes to a 42 C
water bath for 2 min.
9. Add 1 ml of L broth to each tube and incubate at 37 C
without shaking for 30 to 60 min.
10. Spread the transformed cells on to 10 or more L plates
containing 50 ug/ul ampicillin and 40 ug/ml Xgal. Let
plates dry, invert, and incubate at 37 C 12 to 16 hr.
L broth: 10 g Tryptone
5 g Yeast Extract
5 g NaCl
bring volume up to 1 liter, autoclave
L plates: Add 15 g/1 Bacto Agar to L broth before
autoclaving. After autoclaving, cool media to 55 C
before adding 50 mg/1 ampicillin and 2 ml of a 20
mg/ml stock solution of Xgal in dimethyl formamide
(40 mg/1 final concentration)

41
APPENDIX B
RECIPES FOR CANN ET AL. RNA:cDNA HYBRID CLONING PROCEDURE
G-tailing of pUC!9
181 u1 H20
50 ul 5X BRL tailing buffer (500 mM K-cacody1 ate, pH 7.2,
10 mM CoCl2' 1 mM DTT)
15 ul pUCl9 DNA cut with PST I (1 ug/ul)
1 ul 500 uM dGTP
3 ul terminal transferase (60 U)
250 ul total
incubate 37 C, 30 min.
phenol extract, ethanol precipitate
resuspend in 15 ul H2O
cDNA Synthesis
12.4 ul H2O
5 ul 1.0 M Tris pH 8.3
5 ul 2.0 M NaCl
1 ul 0.8 M MgCl2
1 ul 0.5 M DTT
4 ul RNasin (25 U/ul)
0.5 ul oligo dT 12-18 (1 ug/ul)
2 ul PRSV RNA (1 ug/ul)
5 ul 10 mM dATP
5 ul 10 mM dTTP
5 ul 10 mM dGTP
1 ul 100 uM dCTP

0.5 ul 32P dCTP (5 uCi)
2.6 ul Reverse transcriptase (10-16 U/Ul)
50 ul total
incubate 42 C, 15 min; add 2 ul 10 mM dCTP
incubate 42 C, 45 min; add 10 ul 0.2 M EDTA
incubate 65 C, 5 min
dilute 10 X with BRL MACS buffer C (0.5 M NaCl, 20 mM Tris,
pH 7.2, 1 mM EDTA) and run column according to
manufacturer's directions (BRL) elute sample with NACS
buffer D (2.0 M NaCl, 20 mM Tris, pH 7.2, 1 mM EDTA).
C-tailing of cDNA
10 ul RNA:cDNA hybrid
28 ul H2o
10 ul 5X tailing buffer
1 ul 100 ul uM dCTP
1 ul terminal transferase (40 U)
50 ul total
incubate 37 C, 15 min.
phenol extract, ethanol precipitate
resuspend in 15 ul H2o
Reannealing Reaction
15 ul RNA:cDNA hybrid
170.5 ul H20
4 ul 0.5 M Tris pH 7.5, 10 mM EDTA
10 ul 2 M NaCl
0.5 ul G-tailed pUC19
200 ul total
incubate 65 C, 5 min, then incubate 45 C, 2 h
slow cool to room temp.
Transform competent E. co1i JM83 cells as in Appendix A.

APPENDIX C
VARIOUS OTHER RECIPES
cDNA Synthesis for Hybridization Probes
25.5 ul H20
5 ul 10X cDNA buffer (500 mM Tris, pH 8.3, 80 inM MgCl2,
250 mM NaCl)
2.5 ul 100 mM DTT
1.8 ul RNasin (25 U/ul)
1.3 ul actinomycin D (700 ug/ml)
3 ul 10 mM dATP
3 ul 10 mM dTTP
3 ul 10 mM dGTP
0.5 ul random primers (5 ug/ul)
2.0 ul PRSV-W RNA (1 ug/ul)
1.8 ul reverse transcriptase (10-16 U/ul)
0.6 u132P-dCTP (5 uCi)
50.0 ul total
incubate at 42 C, 30 min
add 0.5 ul 10 mM dCTP
incubate at 42 C, 60 min
add 3.0 ul 6 N NaOH
incubate at 56 C, 60 min.
Plasmid Purification
Grow cultures overnight in a 250 ml flask containing 40 ml L
broth at 37 C with shaking. Cool culture in ice 30 min.
Centrifuge 10K for 10 min. Decant supernatant.
43

Resuspend pellet in:
0.48 ml 25% sucrose, 50 mM Tris pH 8.0 (ST)
vortex
0.16 ml 10 mg/ml lysozyme in ST (make fresh)
incubate on ice 5 min
0.40 ml 0.2 M EDTA pH 8.0
incubate on ice 5 min
1.02 ml Triton lysis buffer
(1 ml 10% Triton X100, 5 ml 1.0 M Tris pH 8.0, 31.25
ml 0.2 M EDTA pH 8.0, 62.75 ml H20)
incubate on ice 15 min, swirl occasionally
Centrifuge 15K, 30 min; collect supernatant
Phenol extract 2 times; ethanol precipitate; resuspend in
250 u1 H2°-
Nick Translation
0.5 ul DNA (1 ug/ul)
5.0 ul 10X NTB (500 uM Tris pH 7.9, 50 uM MgCl2, 100 uM 2-
mercaptoethanol)
34 ul H20
5.0 ul 10X NTP (40 uM each dATP, dTTP, dGTP)
5.0 ul DNase I (1 ul of 50 ug/ml stock in 500 ul H20)
0.5 ul 32P dCTP (5 uCi)
50 ul total
incubate 15 C, 4 h
add 2.5 ul 10% SDS, and 2 ul of 0.35% bromophenol blue
Run through a 1 ml Sephadex G-50 column with NTB buffer,
collect first radioactive peak (when the bromophenol blue is
about at the 80 cc mark on a 1 ml Tuberculin syringe)

LITERATURE CITED
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BIOGRAPHICAL SKETCH
Julianne Nagel was born in Colver, Pennsylvania, on
December 12, 1957, and grew up in Johnstown, PA. She
graduated from Ferndale Area High School in 1975. She
completed her undergraduate degree in horticulture at
Pennsylvania State University, graduating in 1978 with the
title of Student Marshall of the College of Agriculture. In
1979 she enrolled at the University of Florida, Plant
Pathology Department, and obtained a Master of Science
degree under the direction of Dr. F. W. Zettler in 1981.
From 1981 until 1982 she was employed as a laboratory
technician II for Dr. F.W. Zettler. She began her Ph.D.
research in 1982 in the same department under the direction
of Dr. E. Hiebert and completed her degree in 1985. She is
currently a postdoctoral research associate with Dr. R.
Shepherd in the Plant Pathology Department at the University
of Kentucky.
49

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of
Philosophy.
<_/
Ernest Hiebert, Chairman
Professor of Plant Pathology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of
Philosophy.
“2 &.
L. Curtis
Professor
â– l.
-zu—
Hannah
of Horticultural Science
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation, foj; £he degree^ of,Poctor of
Philosophy. ) // / J
CU4
David C. Loschke
Assistant Professor
of Plant Pathology
I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for th^o^gree of Doctor of
Philosophy.
Daryl R. PrUng
Professor of Plant Pathology

I certify that I have read this study and that in
my opinion it conforms to accepta-ble standards of
scholarly presentation and is fuJ/ly adequate, in scope
and quality, as a dissertati¿>ri fpr the degree of Doctor of
Philosophy. Ai
Francis W. Zettler
Professor of Plant Pathology
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School,
and was accepted as partial fulfillment of the requirements

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