Complementary DNA cloning and expression of the papaya ringspot virus sequences encoding capsid protein and a nuclear in...

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Title:
Complementary DNA cloning and expression of the papaya ringspot virus sequences encoding capsid protein and a nuclear inclusion-like protein
Uncontrolled:
Papaya ringspot virus sequences encoding capsid protein and a nuclear inclusion-like protein, Complementary DNA cloning and expression
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vi, 49 leaves : ill. ; 28 cm.
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English
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Nagel, Julianne, 1957-
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Subjects / Keywords:
Papaya -- Diseases and pests   ( lcsh )
Plant genetic engineering   ( lcsh )
Plant Pathology thesis Ph. D
Dissertations, Academic -- Plant Pathology -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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notis - ACS7345
sobekcm - AA00004881_00001
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Full Text








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.


iii













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










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 coli JM83. In

the Cann et al. procedure (Nucl. Acids Res. 11,1267-1281) an

RNA:cDNA hybrid inserted into pUC19 was used to transform E.

coli JM83. The Cann method was more efficient than the

Heidecker and Messing procedure, both in the number of







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. coli were characterized. The expressed polypeptides

were fusion products with the amino terminus of the oC-donor

fragment of B-galactosidase. Clones W1-77 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-like

proteins of PRSV-W were physically mapped to adjacent

positions through Southern blot analyses of clones Wl-77 and

W1-18 and are presumed to be located at the 3' end of the

viral genome.










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 (10kb) in size with a

genome-linked 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









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

plasmodesmata (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 nonstructural 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 in vitro translations (de Mejia et al., 1985b;

Dougherty and Hiebert,1980). It is probable that these









protein subunits are also formed in infected host cells but

do not aggregate in the nucleus to form structuresapparent

by light microscopy.

Over 25 potyviruses have been translated in 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; Hellman 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, Hellman 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







the transcription and translation of these portions in

Escherichia coli. By serologically identifying the

polypeptides expressed in E. coli 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 nonstructural proteins

and expressed the sequences in E. coli. These nonstructural

proteins are normally extremely difficult to isolate from

infected plants, but by expressing the proteins in E. coli,

sufficient amounts were obtained for characterization and

antibody production. Other examples of plant viral proteins

expressed in E. coli 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. coli (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). [35S]

Methionine, 14C-labeled molecular weight standards, and






125I-labeled protein A were from Amersham (Arlington

Heights, IL 60005). Escherichia coli 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-i (Purcifull et al., 1984), was

propagated in pumpkin plants (Cucurbita 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).







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

deoxythymidylate 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 coli 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-indolyl-B-D-galactoside (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 [32p]-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-galactosidase fragment of pUC vectors

result in white colonies, a portion of the blue and light










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 5,000xj, and lysed in 1/10 volume

(original cell suspension) of Laemmli dissociation buffer

(Laemmli, 1970). The lysed cells were centrifuged at

27,000x2 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

(Ouchterlony) 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 immunoprecipitations, bacterial cells were labeled

with [35S]-methionine. In these experiments bacterial

cultures were grown in potassium morpholinopropane sulfonate

(MOPS) medium (Neidhardt et al., 1974) supplemented with

2% dehydrated methionine assay medium (Difco, Detroit, MI

48232) and 10 uCi/ml [35S]-methionine. The cells were

incubated for 8 to 16 hr at 37 C with shaking, and lysates







were prepared as described above.

Immunoprecipitations were performed by incubating for 1

hr at room temperature 100 ul of [35S]-methionine-labeled

bacterial lysate with 100 ul of antiserum and 400 ul of 0.5%

Nonidet P-40 in 150 mM NaCI, 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 W1-77 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









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 Bar 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 W1-77 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 W1-18 (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 pUC8 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 immunoprecipitation tests of









protein extracts for each expression clone (Figs. 2-6, 2-7).

Clone W1-77 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

W1-18 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 antigenically 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












Sal I
Acc I
Hinc II


NI indII


P






a

Hind III Sal I
Accl
Hind III 4 inser /Hinc II
S Sma l

/ Eco RI
I 0

W1-18




pUC 8
b



Fig. 2-1. Locations of cDNA inserts in pUC plasmids:
a. W1-77 in pUC9. b. W1-18 in pUC8. Transcription and
translation proceed counterclockwise.















ri I
a 3


2700-1


'1480-

880-
630-
450- -






A` B
S2 3 4 1 2 3 4



Fig. 2-2. Restriction analysis and Southern blotting of DNA
from clones W1-77, W2-1, and W1-18. Lanes contain; 1. pUC9,
2. W1-77, 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








C.3








2700-


1480--

880-0'
63 0-
450-"p






A8
1 2 3 4 1 2 3 4


Fig. 2-3. Restriction analysis and Southern blotting of DNA
from clones W1-77, W2-1, and W1-18. Lanes contain; 1. pUC9,
2. W1-77, 3. W2-1, 4. W1-18 DNA cut with Barn 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 [3ZP]-labeled probes consisting of nick translated
880 bp fragment of W1-18.





















2700s


1480"

880-
630"w








1 2 3 4 1 2 3 4



Fig. 2-4. Restriction analysis and Southern blotting of DNA
from clones W1-77, W2-1, and W1-18. Lanes contain; 1. pUC9,
2. W1-77, 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 [3ZP]-labeled probe consisting of nick translated 450
bp fragment of W1-18.












rm
m- M









2700""


1480" < lp


630- e

450-1-







1 2 3 4 1 2 3 4



Fig. 2-5. Restriction analysis and Southern blotting of DNA
from clones W1-77, W2-1, and W1-18. Lanes contain; 1. pUC9,
2. W1-77, 3. W2-1, 4. W1-18 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 W1-77.













S t.
S*r- 0 1;
S; ; ;
Fft W. a V


M OMM- .- lt-. -*A
"T*


S. -- 29K


1 2 8 4 5 8


7 8 9


Fig. 2-6. Western blot of lysates from E. coli JM83
containing: pUC9 (lanes 1 and 5), clone W1-77 (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 1'25I-labeled protein A and autoradiographed. Lane 9
contains protein molecular weight markers. Arrows identify
the estimated sizes of the immunoreactive proteins.


S
I


40K- "


*- 94K


67K
--m <-52K


A .


- 38K


14K-'-















i S I


















1 2 3 4 5 6 7 8 9 10 11




Fig. 2-7. Immunoprecipitations of 35S methionine labeled E.
coli 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 fluorography. Lanes 1, 2, 3, and 4
contain lysate from JM83 containing clone W1-77; lanes 5, 6,
7, and 8 contain lysate from JM83 containing pUC9, and lanes
9, 10, and 11 contain lysate from JM83 containing clone Wi-
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.










































Fig. 2-8. Serological relationships of capsid protein from
clone W1-77, 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 W1-77, 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.







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 antigenically related to PRSV capsid

protein, and one clone was demonstrated to produce a

polypeptide antigenically 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 <-donor of

B-galactosidase. The plasmid lac promoter of pUC9 is

constitutive for B-galactosidase when the E. coli JM83 host








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-like 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-galactosidase would account for approximately 1K and so







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-like 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 viral-specific

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







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-galactosidase

polypeptide fragment, so some post-translational cleavage

might be occurring. Although the cleavage site is not

known, it would seem likely that since B-galactosidase 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.



































70k 49k 57k 36k


-.. 1 60k


D I I o0n


rU-


ly A 3'


Cylindrical Nuclear Nuclear Capsid
Inclusion Inclusion Inclusion
1480 bp
W1-77
1330 bp
W1-18 630bp
W2-1


Fig. 2-9. Proposed locations of clones W1-18, W1-77, and
W2-1 on the PRSV-W gene map. For discussion of the PRSV-W
gene map, see Chapter 1.


3 V


, 51k 40k


? Amorphous ?
Inclusion













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.






Materials and Methods

Materials

Restriction enzymes, terminal transferase, deoxy

nucleotides, and OX174 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. coli 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 Cucurbita 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 RNA:cDNA hybrid was tailed on the 3' ends with

dCTP. The pUC19 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. coli JM83 cells were

transformed and plated onto L plates containing ampicillin

at 50 ug/ul and the indicator 5-bromo-4chloro-3-indolyl-B-D-

galactoside (Xgal) at 40 ug/ml (Maniatis et al., 1982; see







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, 32P-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 Bar

HI and Hind III and electrophoresing the samples on 1.2%

agarose gels along with molecular markers consisting of

lambda DNA Hind III and OX174 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 32P-labeled nick-translated 1480 bp insert

of clone W1-77, and the other was hybridized with 32p

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.








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 W1-77 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 W1-18. They each contained a Hind III site within

the cDNA insert; and after a double digestion with Hind III

and Barn HI to excise the insert from the vector, the smaller

fragment portion of each clone hybridized with the insert of

W1-77, and the larger fragment hybridized with the 880 bp

fragment of W1-18. 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 W1-77, and the smaller

fragment hybridized to the 880 bp fragment of W1-18. 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

W1-77.























p( UC19





S Pt I




Terminal Transferase


GGGGG --
GGGGG CCCCC-




) Reanneal


------.--.. -AAAAA

Ollgo dT

-- --------AAAAA
TTTTT
Reverse Transcriptase

---- -- --AAAAA
-- TTTTT


Terminal Transferase


- --AAAAACCCCC
TTTTT


Transform


Fig. 3-1. Schematic diagram of Cann et al. (1983) cDNA
cloning procedure.































1353-. -4
1078-
872- -880
603-
-450






310-
1353-,
1078-
872- e I
603-

310- g i m a


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. W1-18, 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 [32P]-labeled probe
consisting of nick translated 1480 bp insert of W1-77.






























1353 -14
1078--
872- -880
603-
-450













310-
1353-.

872-


310-


b






Fig. 3-3. Restriction analysis and Southern blotting of
cloned DNA. Lane 1 contains XX174 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. W1-18, 18. W1-77 DNA cut with Barn 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 [3ZP]-labeled probe
consisting of nick translated 880 bp fragment of W1-18.






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 inclusion-like

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 W1-18

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 new clones could also

later be joined to W4-42 through the unique restriction site

to form longer clones.










CHAPTER 4
CONCLUSIONS

Two cloning procedures were used successfully 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 W1-77 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 in vitro translations (Hiebert, unpublished).

Clone W1-18 was mapped to an internal location on the

RNA, adjacent and 5' to clone W1-77. It is unknown whether







this clone originated from a short internal A-rich region in

the viral RNA or whether a recombinational 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. coli cellular

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. coli 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 with PST I

20 ul pUC9 DNA (1 ug/ul)

110 ul H20

15 ul BRL 10X Core buffer (500 mM Tris, pH 8.0,100 mM
MgC12, 500 mM NaC1)

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 H20



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
CoC12, 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 NaC1,
1 mM EDTA








T-tailing of pUC9

10 ul pUC9, cut with PST I

10 ul 1 M K-cacodylate, pH 7.0

21 ul H20

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 CoCI2

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 NaC1, 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 MgC12

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)

15 ul total


final conc.



800 uM

70 mM

50 mM

10 mM

2 mM

1 U/ul

25 ug/ml

40 nM

120 nM

100 U/ml







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 U/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 NaC1, 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. per tube

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 NaC1, 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 NaC1, 1 mM
EDTA. Add 25 ug/ml carrier tRNA and ethanol precipitate.








Resuspend in 50 ul 10 mM Tris, pH 7.6, 10 mM NaCI, ImM EDTA.



Reannealing Reaction final cone.

50 ul DNA 1-5 ul/ml

320 ul 100% formamide deionizedd) 32%

20 ul 2.5 M NaCI 50 mM

10 ul 1 M Tris pH 8 10 mM

600 ul H20

1000 ul total

incubate 37 C for 24 hr. Dialyse overnight at 4 C
against 100 mM NaC1,10 mMTris, pH 8.0, 1 mM EDTA.
Ethanolprecipitate, resuspend in 43.5 ul H20.




Fill-in Reaction final cone.

43.5 ul DNA

1 ul 2.5 M NaC1 50 mM

1 ul 1 M Tris pH 7.6 20 mM

1.67 ul 300 mM MgC12 10 mM

1 ul 0.05 M DTT 1 mM

1 ul 5 mM dNTP's 100 uM

0.8 ul Klenow Frag. DNA Pol. I 3 U

50 ul total

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 NaC1, 1 mM EDTA.






Transformation Procedure

1. Inoculate 20 ml of L broth in a 100 ml flask with 1 ml of
an overnight culture of E. coli JM83. Incubate culture at
37 C with vigorous shaking until reaching an OD550 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 CaC12, 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 NaC1, 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 NaC1
bring volume up to 1 liter, autoclave

L plates: Add 15 g/l Bacto Agar to L broth before
autoclaving. After autoclaving, cool media to 55 C
before adding 50 mg/l ampicillin and 2 ml of a 20
mg/ml stock solution of Xgal in dimethyl formamide
(40 mg/l final concentration)












APPENDIX B
RECIPES FOR CANN ET AL. RNA:cDNA HYBRID CLONING PROCEDURE

G-tailing of pUC19

181 ul H20

50 ul 5X BRL tailing buffer (500 mM K-cacodylate, 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 H20


cDNA Synthesis

12.4 ul H20

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/U1)

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 NaC1, 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 NaC1, 20 mM Tris, pH 7.2, 1 mM EDTA).


C-tailing of cDNA

10 ul RNA:cDNA hybrid

28 ul H20

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
resuspend in 15 ul H20


Reannealing Reaction

15 ul RNA:cDNA hybrid

170.5 ul H20

4 ul 0.5 M Tris pH 7.5,

10 ul 2 M NaCI

0.5 ul G-tailed pUC19


precipitate


10 mM EDTA


200 ul total

incubate 65 C, 5 min, then incubate 45 C, 2 h
slow cool to room temp.

Transform competent E. coli 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 mM MgC12,
250 mM NaCI)

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 ul32P-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.








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 ul H20.


Nick Translation

0.5 ul DNA (1 ug/ul)

5.0 ul 10X NTB (500 uM Tris pH 7.9, 50 uM MgC12, 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 of
watermelon mosaic virus 1 for cucurbit crops in Palm
Beach County, Florida. Plant Dis. Reptr. 56, 463-567.

Allison, R. F., Sorenson, J. C., Kelly, M. E., Armstrong, F.
B., and Dougherty, W. G. (1985). Sequence
determination of the capsid protein gene and flanking
regions of tobacco etch virus: Evidence for the
synthesis and processing of a polyprotein in potyvirus"
genome expression. Proc. Natl. Acad. Sci., in press.

Amann, E., Brosius, J., Ptashne, M., (1983). Vectors
bearing a hybrid trp-lac promoter useful for regulated
expression of cloned genes in Escherichia coli. Gene
25, 167-178.

Bethesda Research Laboratories, Inc. (1980). BRL
recommendations for gel electrophoresis of low M.W.
range proteins. Focus 2, No. 6, p. 6.

Bonner, W. M., and Laskey, R. A. (1974). A film detection
method for tritium labeled protein and nucleic acids in
polyacrylamide gels. Eur. J. Biochem. 46, 83-88.

Brakke, M. K., and Van Pelt, N. (1970a). Properties of
infectious ribonucleic acid from wheat streak mosaic
virus. Virology 42, 699-706.

Brakke, M. K., and Van Pelt, N. (1970b). Linear-log
gradient for estimating sedimentation coefficients of
plant viruses and nucleic acids. Anal. Biochem. 38,
56-64.

Cann, A. J., Stanway, G., Hauptmann, R., Minor, P. D.,
Schild, G. C., Clarke, L. D., Mountford, R. C., Almond,
J. W. (1983). Poliovirus type 3: molecular cloning of
the genome and nucleotide sequence of the region
encoding the protease and polymerase proteins. Nucl.
Acids Res. 11, 1267-1281.

Christie, R. G., and Edwardson, J. R. (1977). Light and
electron microscopy of plant virus inclusions. Fla.
Agric. Exp. Stn. Monogr. Ser. No. 9. 150p.








Close, T. J., Christmann, J. L., and Rodriguez, R. L.
(1983). M13 bacteriophage and pUC9 plasmids containing
DNA inserts but still capable of B-galactosidase
-complementation. Gene 23, 131-136.

Daubert, S., Richins, R., Shepherd, R. J., and Gardner,
R. C. (1982). Mapping of the coat protein gene of
cauliflower mosaic virus by its expression in a
prokaryotic system. Virology 122, 444-449.

de Mejia, M. V. G., Hiebert, E., and Purcifull, D. E.
(1985a). Isolation and partial characterization of
the amorphous, cytoplasmic inclusions associated with
infections caused by two potyviruses. Virology 142,
24-33.

de Mejia, M. V. G., Hiebert, E., Purcifull, D. E.,
Thornbury, D. W., and Pirone, T. P. (1985b).
Identification of potyviral amorphous inclusion protein
as a nonstructural, virus-specific protein related to
helper component. Virology 142, 34-43.

Dougherty, W. G., Allison, R., Parks, T. D., Johnston, R.
E., Feild, M. J., and Armstrong, F. B. (1985).
Nucleotide sequence at the 3' terminus of pepper mottle
virus genomic RNA: Evidence for an alternative mode of
potyvirus capsid protein organization. Virology, in
press.

Dougherty, W. G., and Hiebert, E. (1980). Translation of
potyvirus RNA in a rabbit reticulocyte lysate: Cell-
free translation strategy and a genetic map of the
potyviral genome. Virology 104, 183-194.

Hari, V. (1981). The RNA of tobacco etch virus: Further
characterization and detection of protein linked to
RNA. Virology 112, 392-399.

Hari, V., Siegel, A., Rozek, C., and Timberlake, W. E.
(1979). The RNA of tobacco etch virus contains poly
(A). Virology 92, 568-571.

Heidecker, G., and Messing, J. (1983). Sequence analysis of
zein cDNAs obtained by an efficient mRNA cloning
method. Nucl. Acids Res. 11, 4891-4906.

Helfman, D. M., Feramisco, J. R., Fiddes, J. C., Thomas, G.
P., and Hughes, S. H. (1983). Identification of
clones that encode chicken tropomysin by direct
immunological screening of a cDNA expression library.
Proc. Natl. Acad. Sci. 80, 31-35.







Hellman, G. M., Shaw, J. G., Lesnaw, J. A., Pirone, T. P.,
and Rhoads, R. E. (1980). Cell-free translation of
tobacco vein mottling RNA. Virology 106, 207-216.

Kessler, S. (1975). Rapid isolation of antigens from cells
with a staphylococcal protein A-antibody absorbent:
Parameters of the interaction of antibody-antigen
complexes with protein A. J. Immunol. 115, 1617-1624.

Knuhtsen, H., Hiebert, E., and Purcifull, D. E. (1974).
Partial purification and some properties of tobacco
etch virus intranuclear inclusions. Virology 61, 200-
209.

Laemmli, U. K. (1970). Cleavage of the structural proteins
during the assembly of the head of bacteriophage
T-4. Nature (London) 227, 680-685.

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982).
Molecular cloning, a laboratory manual. Cold Spring
Harbor Laboratory, New York. 545p.

Masui, Y., Mizuno, T., Inouye, M. (1984). Novel high-level
expression cloning vehicles: 104-fold amplification of
Escherichia coli minor protein. Bio/Technology 2,
81-85.

Miglietta, J. J., and Kaesberg, P. (1984). Synthesis of
brome mosaic viral proteins in E. coli. Presented at
the American Society for Virology 1984 Meetings. July
22-26. Madison, WI.

Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1974).
Culture medium for enterobacteria. J. Bacteriol. 119,
736-747.

Purcifull, D. E., and Batchelor, D. L. (1977).
Immunodiffusion tests with sodium dodecyl sulfate (SDS)
treated plant viruses and plant viral inclusions. Fla.
Agric. Expt. Sta. Tech. Bull. No. 788.

Purcifull, D. E., Edwardson, J. R., Hiebert, E., and
Gonsalves, D. (1984). Papaya ringspot virus. CMI/AAB
Descr. Plant Viruses No. 292.

Quiot-Douine, L., Purcifull, D. E., Hiebert, E., and de
Mejia, M. V. G. (1985). Serological relationships and
in vitro translation of an antigenically distinct
strain of papaya ringspot virus type W (watermelon
mosaic virus 1). Phytopath., in press.








Rigby, P. W. J., Dieckman, M., Rhodes, C., and Berg, P.
(1977). Labeling deoxy-ribonucleic acid to high
specific activity in vitro by nick translation with DNA
polymerase I. J. Mol. Biol. 113, 237-251.

Saito, T., Watanabe, Y., Ooshika, I., Meshi, T., and Okada,
Y. (1984). Studies using antibodies against non-
structural proteins of TMV. Sixth International
Congress of Virology. Sendai, Japan. Abstract W42-1.

Schoner, R. G., Ellis, L. F., and Schoner, B. E. (1985).
Isolation and purification of protein granules from
Escherichia coli cells overproducing bovine growth
hormone. Bio/Technology 3, 151-154.

Van Emmelo, J., Ameloot, P., Plaetinck, G., and Fiers, W.
(1984). Controlled synthesis of the coat protein of
satellite tobacco necrosis virus in Escherichia coli.
Virology 136, 32-40.

Vance, V. B., and Beachy, R. N. (1984). Translation of
soybean mosaic virus RNA in vitro: Evidence of protein
processing. Virology 132, 271-281.

Viera, J., and Messing, J. (1982). The pUC9 plasmids, an
Ml3mp7- derived system for insertion mutagenesis and
sequencing with synthetic universal primers. Gene 19,
259-268.

Xiong, Z. (1985). Purification and partial characterization
of peanut mottle virus and detection of peanut stripe
virus in peanut seeds. M.S. Thesis, Univ. of Florida,
Gainesville. 113p.

Yeh, S. D., Gonsalves, D., Provvidenti, R. (1984).
Comparative studies on host range and serology of
papaya ringspot virus and watermelon mosaic virus 1.
Phytopath. 74, 1081-1085.














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.









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.
J / /

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 dis tat f e degree ofoctor of
Philosophy.


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 agree of Doctor of
Philosophy.


Daryl R. Pri g
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 fu ly adequate, in scope
and quality, as a dissertatib6 fir the degree of Doctor of
Philosophy.


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
for the degree of Doctor of Philosophy.


May, 1985 ,
Dean, C6llege of Agric lture


Dean, Graduate School
































































UNIVERSITY OF FLORIDA


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