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thesis entitled

Characterization of ChlorOplast DNA
From Wild—type and Mutant Plastids
of Oenothera hookeri str. johansen

presented by

Sara Anne Kaplan

has been accepted towards fulfillment
of the requirements for

M.S. degree in Botany/Plant Pathology

' ,4 L .. ’ I.
(fiQLuaApt £3. Smart

Major professor

Date 4 June 1987

 

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CHARACTERIZATION OF CRLOROPLAST DNA
FROM WILD-TYPE AND MUTANT PLASTIDS

0F OENOZEKRA HOOKERI STE. JORANSEN.

By

Sara Anne Kaplan

A THESIS
Sub-itted to
Michigan State Univeraity
in partial fulfill-ant of the requirements
for the degree of

MASTER OF SCIENCE

Depart-ant of Botany and Plant Pathology
1987

ABSTRACT
CHARACTERIZATION OF CRLOROPLAST DNA

FROM WILD-TYPE AND MUTANT PLASTIDS
OF OENDTFFRZ HOOKER! STR. JORANSEN

By

Sara Anne Kaplan

Chloroplast DNA (chNA) from two plastone nutants and
three wild-type plastone I lines of Oenotbera jobansen were
analyzed by restriction endonuclease napping and Southern
hybridization. Although characterizations indicated that no
major DNA changes could be correlated to the nutation
events, the restriction frag-ant analyses did suggest that
two BaIRI DNA fragnents were variable.

lragnents were localized by Southern hybridization
experinents on the physical nap of the chloroplast genome.
The larger Banal frag-eat is located in the large single
copy region; the other is located within the inverted repeat
at or near the junction of the large single copy region.
Restriction endonuclease napping analysis of the two
variable DaIHI fragnents fron chNA revealed small
differences between the chNAs in the for. of discrete
insertions and deletions. Previously, insertions and
deletions in chNA had been observed only between nore

distantly related groups of plants.

This thesis is dedicated to:
Dr. and Mrs. William D. Kaplan
David H. Kaplan
Professor Frederick I. Kaplan

and the memory of Mr. Kenneth Aberle

ii

ACKNOWLEDGEMENTS

I would like to thank all those who helped me during my
studies at Michigan State University.

I am very thankful to Professor Barb Sears, my major
professor, for her guidance and encouragement, and committee
members Professors Tom Friedman, Dennis Fulbright and Nor-an
Good for their encouragement and stimulating discussions.

Special thanks to the people in the Sears lab: Ellen
Johnson, Dr. Ruth Holfson, Dr. Linda Schnabelrauch, Kin
Blasko, Pung Choo Lee, Han-Ling Chiu, Kelly Higgins,
Mireille Khairallah, and Mike Stine for their friendship and
norale support.

A special thanks also goes to Dr. Mickey Gurevitz for his
helpful criticisms and discussions.

I gratefully acknowledge financial support from a GPOP
fellowship under the supervision of Dr. Cassandra Sim-ens of
Urban Affairs.

Assistance in graphic art in the preparation of the
figures for ny thesis and seminar from Jacqui Soule is also
gratefully acknowledged.

I am also thankful to the faculty, staff and graduate
students in the Botany and Plant Pathology Department for
the encouraging environment, including the Coral Gables Gang
for many stimulating Friday evening discussions.

And a heartfelt thanks to Dr. Bob Creelman for his
assistance particularly in providing ne with word-processing
and graphics software.

Thank-you all, very, very much!

iii

Oenotbere:
”A hopelessly confused and freely hybridizing group, early
introduced into Europe and there cultivated, and, like other
plants of the garden, intermixed; then spreading to waste or
open ground.”

Fernald (1950) Grays Manual of Botany

iv

TABLE OF CONTENTS

page

List of Tables.. ........................................ x
List of Figures.......... ............................... xii
List of Abbreviations.......................... ....... .. xiv
Chapter 1. INTRODUCTION AND LITERATURE REVIEW .......... 1
1.1. Introduction................... ................ 2
1.2. Inheritance of the chloroplast. ................ 3
1.3. Characterization of chloroplast DNA.. .......... 4
1.4. Mapping the chloroplast genome...... ........... 7
1.5. Oenotbers, an important genetic system.. ....... 10
1.6. Mutations of the chloroplast.. ................. 12
1.7. The p1astome.mutator gene ...................... 12
Chapter 2. MATERIALS AND METHODS ....................... 15

2.1. Plant nateria1................................. 16

2.2. Bacterial strains and plasmids ................. 22
2.3. DNA preparation.................. .............. 23
2.3.1. Chloroplast DNA isolation ............... 23

2.3.1.1. Chloroplast isolation fro-
abundant anounts of plant

material ......................... 23

page
2.3.1.2. Isolation of chNA from
limited amounts of plant
material..... .................... 24
2.3.1.2a. Protoplast method .......... 24

2.3.1.2b. Modified whole

cell DNA method ............ 25
2.3.2. Purification of chNA... ................ 26
2.3.3. Precipitation of DNA......... ........... 26
2.3.4. Purification of plasmid DNA.. ........... 27

2.3.4.1. Large scale isolation
of plasnid DNA.... ............... 27
2.3.4.1a. Triton lysis nethod... ..... 27
2.3.4.1b. Physical shearing method... 28
2.3.4.2. Isolation and purification
of plasnid DNA following
ultracentrifugation .............. 29
2.3.4.3. Rapid snall scale isolation

and purification of plasmid

DNA (mini preps)... ..... ... ...... 30

2.4. Restriction endonuclease digestion of DNA ...... 31
2.5. Gel electrophoresis.......... .................. 31
2.5.1. Agarose gel electrophoresis ............. 31
2.5.2. Polyacrylanide gel electrophoresis ...... 32

2.5.3. Calculation of molecular size of

bands separated by electrophoresis ...... 33

vi

page
2.6. Purification of DNA from gels .................. 33
2.6.1. Purification of DNA fragments
from agarose gels ...... . ....... . ........ 33
2.6.1.1. Crush and extract nethod. ........ 33
2.6.1.2. Electroelution onto DEAE
cellulose menbrane ........ . ...... 34
2.6.2. Purification of DNA from polyacrylamide
gels: crush and soak method ............. 35

2.7. Southern transfer of DNA from agarose

gels to nitrocellulose paper........... ........ 36
2.8. Hybridization of southern filters .............. 36
2.9. Nick translation ............................... 37
2.10. Cloning........................ .............. . 38
2.10.1. Preparation of vector DNA. ............. 38
2.10.2. Ligation.... ........................... 39
2.10.3. Transformation............ ............. 39

2.10.3.1. Transformation by the
calcium chloride procedure ...... 39
2.10.3.2. Transformation using a

modification of the Ranahan

procedure............... ........ 40

2.10.4. Screening for recombinant clones ....... 41

Chapter 3. PLASTOME MUTATOR. ........................... 44
3.1. Introduction ................................... 45

vii

3.2. Results...... ...... . ........... . ...............

3.2.1. Clonin‘oeeeooe eeeee es eeeeeeeeeeee e eeeeee

3.2.2. Comparison of restriction endonuclease

Ram 12 between mutant pm}! and its

sibling wild-type line 01.. ...... ......

3.2.3. Pm mutants fall into two
discrete c1asses.. ............... . ......
3.3. Discussion....................... ..............

Chapter 4.

COMPARISON OF CLONED chNA RESTRICTION

FRAGMENTS EamRI 3b AND 12 FROM WILD-TYPE PLASTOME I

LINES OF OINOTWZRA HOOKER! STRAIN JORANSEN. .............

4.1. Introduction...... ................... . ..........

4.2. Results................. ......... . ....... . .....

4.2.1. Cloning.................... .............

4.2.2. Location of the BamRI 3b and 12

variable regions on the

plastome I chNA ........................

Construction of a physical map

for the Dam 12 fragment of the three

wild-type plastome I lines using

restriction endonucleases.. .............
4.2.3.1. Preliminary mapping of

the entire 3.0 kb BamRI

12 fragment .....................

viii

48

51

57

61

63

65

71

71

page
4.2.3.2. Fine mapping of the variable
region of Bam 12. ........... .... 77
4.2.4. Construction of a physical
map of Bam 3b DNAs from
plasmids pOJ118 and pOJoS using
restriction endonucleases....... ........ 97

4.2.5. Comparison of DNA in the variable

regions of Bam 3b and Ben 12 ...... ...... 110

4.3. Discussion.. ............. .... .......... . ...... . 113
Literature Cited ..................................... ... 120
Appendix...................................... .......... 133

Restriction Endonucleases and Their
Recognition Sequences.......... .......... . ........ ... 134

DNA Marker Band Sizes ............ . ................... 135

ix

Table

2.1.
2.2.

2.3.
2.4.

3.1.

LIST OF TABLES

Plant lines and their origins............ .........
Modified N.T. Medium for the maintenance of

shoot meristem cultures.. .........................
M.S. Medium.......................................
Marker DNAs used in gel electrophoresis. ..........
Recombinant plasmids of BamEI fragments

3b and 12 and their origins... ..... .. .............
Recombinant plasmids with chNA inserts

from Oenotbera jobansen. ........ ... ...............
Restriction endonucleases tested for

digestion of the Bam 12 insert DNA ................
Single and double digestions of Ram 12

inserts from p0j119.... ...........................
End mapping experiments....... ....................
Single digests of the 1.6 kb insert of

subclone pOJll9a.................... ..............
End mapping for comparison of the 1.6 kb

from all three lines with and without

inserts

attached vector DNA ...............................

9389
17

20

21

33

48

63

72

74

76

85

Table

4.7.

page
Restriction endonucleases tested for
digestion of the Bam 3b insert DNA ................ 98
Digestions of Ram 3b inserts from plasmids
p0j118 and p0jo$......... ...... ........ ........... 99
End-mapping for comparison of the Dam 3b
inserts from lines D (p0j118) and Ci(p0j06) ....... 101

xi

Figure

2.1.
3.1.

3.2.

4.3.

LIST OF FIGURES

page

Pedigree of plant lines. .......................... 19
Comparison of cloned Ben 12 fragments

between mutant p.11 and its sibling wild-type ..... 50
Plastome mutstor induced plastome I

mutants and their sibling wild-types

fall into two distinct c1asses.......... .......... 53
Comparison of chNA between mutant p18

and its sibling wild-type line Ca using

restriction endonuclease digestion and

Southern hybridization......... ................... 56
Physical map of Oenotbera bookeri

strain jobansen plastome 1.. ..... . ..... . .......... 67
Southern Hybridization of Bam 12 insert

DNA to restriction endonuclease digested

Oenotbera plastome I chNA................ ..... ... 70
Restriction endonuclease digestion and agarose

gel electrophoresis of plasmid DNAs from p0j119a,
pOjo4a, pOejla enabling the calculation of the

sizes of the BamHI 12 subclone variable bands ...... 80

xii

Figure page
4.4. Restriction endonuclease double digestions

of the Dam 12 1.6kb subclones... .................. 82
4.5. Restriction endonuclease digestions of

Bam 12 1.6kb subclone inserts to define

the and fragments................... ..... . ........ 87
4.6. Single and double digestions of the

Bam 12 1.6kb insert DNAs.................. ........ 90
4.7. Single and double digestions of the Dam 12

1.6kb insert...................................... 93
4.8. Restriction endonuclease map of the cloned

BamHI 12 fragment....................... ...... .... 96
4.9. Restriction endonuclease digestions of the

Bam 3b 7.5 kb inserts and plasmid DNAs. .......... . 103
4.10. Single and double digestions of the

7.5kb BamHI 3b insert.............. ............... 106
4.11. Restriction endonuclease map of the cloned '

BamHI 3b 7.5kb inserts............. ............... 109
4.12. Polyacrylamide gel electrophoresis of

restriction endonuclease digested Bam 3b

and Dam 12 inserts ........... ..... ........... ..... 112

xiii

LIST OF ABBREVIATIONS

bp base pair(s)

Ci Curie(s)

d day(s)

EMS ethylmethanesulfonate
S gram(s)

h hour(s)

k kilo (1 X 103)

kbp kilo base pair(s)
l liter(s)

m1 mililiter(s)

M Molar

mM mili Molar

min minute(s)

n nano (1 X 10")
rpm revolutions/minute
rt room temperature

s second(s)

F micro (l X 10‘5)
UV. ultra violet

w/v weight/volume

xiv

CRAPTER 1
INTRODUCTION AND

LITERATURE REVIEW

2
1.1. INTRODUCTION

The characterization of the chloroplast as being the part
of the plant responsible for photosynthesis, and for its
other functions, was a long process which took place over
several hundreds of years by some of the great early
scientists including Hales, Priestley, Ingen-Housz, and
deSassure. (Reviewed by Roober 1984, Arnon 1955, and
Rabinowitch 1945). The chloroplast is perhaps the most
conspicuous and, because of its importance to the plant
cell, certainly the most extensively characterized type of
plastid. In addition to photosynthesis, chloroplasts are
responsible for amino acid and fatty acid biosynthesis,
nitrogen assimilation and partial synthesis of chlorophyll,
carotenoids, and plastoquinone (Kirk and Tilney-Bassett
1978).

Many aspects of chloroplast genetics have been
investigated, including the organization, expression, and
the interaction of chloroplast genes with the nuclear genome
of the plant. Because the chloroplast genome, on the whole,
is greatly conserved throughout the plant kingdom (Palmer
1987a, Stein et a1. 1986, Gillham et a1. 1985, and Palmer
1985a, 1985b), it is also the subject of evolutionary
studies. The study of the subgenus Euoenotbera including
the genus Oenotbera has added a wealth of information on the
study of chloroplast inheritance. Species within this plant

group have an unusual arrangement of chromosomes, three well

3
characterized nuclear genome types and five genetically
distinct plastid types (reviewed by Kutzelnigg and Stubbe
1974, Kirk and Tilney-Bassett 1978, Cleland 1972, and
Burnham 1962). Genetic studies of the chloroplast also
include the induction and characterization of mutants.
Plastid genome (plastome) mutations have been characterized
in many plants including Oenotbera (reviewed by Sears and
Boerner 1986). In one case, a variety of chloroplast
mutations in Oenotbera chloroplast DNA have been induced by

a nuclear gene (Plastome mutator) (Epp 1972, 1973).
1.2. INHERITANCE OF THE CHLOROPLAST.

In 1900, the Mendelian laws of inheritance were
rediscovered. A few years later, investigators found
exceptions to these laws which showed the manner in which
chloroplast traits were inherited. Work by Correns in 1909
and Baur in 1909 (reviewed by Gillham 1978, Kirk and Tilney-
Bassett 1978, and Cleland 1972) revealed two types of
chloroplast inheritance: uniparental and biparental.
Correns’ work on .Mfirabjljs jalapa showed that only the
female parent contributed plastid traits to the offspring.
This mode of inheritance has been designated uniparental—
maternal. Baur performed reciprocal crosses on Pelargonium
between green and variegated plants, and found green, white

and variegated progeny in the F1 generation. This mode of

4
inheritance has been designated as biparental. Oenotbera
displays biparental inheritance of plastids. Even in cases
of biparental inheritance, plastids from the maternal parent
are generally favored (Sears 1983, Gillham 1978, and Cleland
1972). Since the work of Baur and Correns, chloroplast
inheritance in many plants has been examined. In most of
the algae and ferns examined to date, the predominant type
of inheritance is uniparental. Conifers display biparental
inheritance although it is skewed towards paternal
transmission of plastids (Ohba 1971, reviewed by Sears 1980
and Kirk and Tilney-Bassett 1978). Among the angiosperms,
uniparental-maternal inheritance appears to be the most
common pattern, as it has been observed in BOX of
angiosperms studied so far. However, biparental inheritance
may be more widespread than was previously thought. Through
the use of mutant plastids, Schmitz and Kowallik (1986) have
shown that Epilobium, a species previously believed to have
uniparental-maternal chloroplast inheritance, does display

some degree of biparental chloroplast inheritance.
1.3. CHARACTERIZATION OF CHLOROPLAST DNA.

Evidence indicating the presence of nucleic acids in the
chloroplast first came from a report by Chiba (1951), using
Feulgen staining techniques. In the cytological studies

which followed, including autoradiography, electron

5

microscopy, high speed density centrifugation, . and
reassociation kinetics, the presence of nucleic acids (DNA
and RNA) in the chloroplast was confirmed. (Kowallik and
Herrmann 1972, Gunning 1965, and Ris and Plant 1962).
Chloroplast DNA in broad bean was demonstrated as being
distinct from nuclear DNA due to a higher ratio of adenine
to guanine (Kirk 1963). Wells and Birnsteil (1969)
distinguished chloroplast DNA from mitochondrial DNA by the
chloroplast DNA’s lack of 5-methy1cytosine. The circular
nature and location of the chloroplast DNA molecule within
the chloroplast was established through the work of Manning
et a1. (1971). Subsequent work by investigators
demonstrated that chloroplast DNA ocurred naturally in
supercoiled form (Gunning 1965, and His and Plant 1962), was
contained within discrete areas called nucleoids and that
there were 20 - 50 copies of the DNA per chloroplast with an
average density of around 1.697 g / cm3, and a d(G + C)
content of 383 for most higher plants (Herrmann et a1. 1975,
Kolodner and Tewari 1972, Herrmann and Possingham 1980, and
Kowallik and Herrmann 1972).

Chloroplast DNA has been characterized from lower plants
including the algae, bryophytes, and ferns. Some algae
which have been investigated include: Iantbopbyceae
(Yellow-green: Faucberis sessilis (Herrmann and Possingham
1980, Hahn and Herrmann 1977), Chrysopbyceae (golden algae):

011stbidiscus Ieteus (Aldrich and Catallico 1981),

6
Bacillarjopbyceae (diatoms): (Palmer 1985b), Phaeopbyceae
(brown algae): Dictyota dice-a (Kushel and Kowallik 1985)
and Fwydaiella littoral}: and Spbacelaria sp. (Dalmon and
Loiseaux 1983), Chloropbycopbyta (green algae):

Chlamydomonas (Rochaix 1978), Acetabularia (Green and Burton
1970), and from Englena gracjlis (Gray and Hallick 1978).

Chloroplast DNAs have also been characterized in the
bryophyte, ”archantja polymorpba (Ohyama et a1. 1986) and
some ferns from the genus almonds (Stein et a1. 1986).
Chloroplast DNA from many higher plants has been
examined. Research on chNA of gymnosperms is not as
extensive as the research on angiosperms chNA with only one
gymnosperm, Ginkgo biloba, (Palmer and Stein 1986)
characterized thus far. Restriction mapping and sequencing
of chNA has occurred in some 200 species representing 33
families of angiosperms including both monocotyledonous and
dicotyledonous plants (Palmer 1987a, Palmer and Stein 1986,
and Palmer 1985). Some representative angiosperm families
which have been extensively studied are the Leguminosae
(Palmer 1987a), Solanaceae (Shinozaki et a1. 1986, Sugiura
et a1. 1986) and the Omagraceae (Gordon et a1. 1982, Gordon

et a1. 1981).

7

1.4. MAPPING TEE CHLOROPLAST GENOME.

During the past ten years, the circularity of chloroplast
DNAs has been observed as a general feature of many plant
groups from the primitive to the advanced. Their sizes have
been calculated either by contour length measurement of
electron micrographs or by summing up the molecular weights
of restriction endonuclease cut DNA fragments.

By far the most powerful technique used in the
characterization of chNA is the use of restriction fragment
length polymorphisms (RFLPs). The first use of RFLPs in the
comparison of chNAs was by Atchison, Nhitfeld and Bottomley
(1976). This group used the restriction endonuclease EcoRI
in digests of chNA from different plant species as well as
on plants within the same genus. They found that although
the chNAs are similar in gross base composition, they show
distinctly different restriction digestion patterns.
Subsequently, RFLPs have been used as the primary method in
determining the gene order in chNA (Nhitfeld and Bottomley,
1983). Restriction mapping has generated the preliminary
data necessary for the sequencing of the entire sequences of
chNAs from tobacco (Shinozaki et a1. 1986) and a liverwort
(Ohyama et a1. 1986).

Studies involving restriction fragment cloning in
conjunction with cell free translation and immunological

techniques allowed the location of many genes on chNA

8

including those for thylakoid proteins (Herrmann et a1.
1983). This study also reported a 50 kilobase (kb)
inversion between spinach and Oenotbera in the large single
copy region of the chNA. This inversion is located between
the ATPase and the cytochrome f genes and between the large
sub-unit of RuBISCO and psbA genes (figure 4.1). Inversions
have also been found in the large single copy region of
wheat and mung bean chNA when compared with spinach (Palmer
et a1. 1982).

All chloroplast DNAs isolated are circular molecules
ranging from 120 kbp in pea to 217 kbp in geranium (Palmer
et al. 1987b). Most chloroplast DNAs are represented by
only one size class with the exceptions of Acetabularia
(Green and Burton 1970), .Euglena gracjljs (Jenni et a1.
1981), Phylsiells littoralis and Spbacelaria sp. (Dalmon at
al. 1983), green and brown algae. In higher plants, rice
was reported by Moon et a1. (1987) to consist of
heterogeneous chNA molecules. With respect to plastome
size, the green algae are the most divergent group, with
chNA molecules ranging from 85 kbp to 292 kbp (Palmer
1985a, 1985b).

In general, the chloroplast DNA molecule is divided into
four parts: two areas of single copy DNA designated as the
large and small single copy regions, respectively, separated
by two large inverted repeats ranging from 10 - 11 kbp to as

large as 76 kbp (Palmer et al. 1987a, Stein and Palmer 1986

8

including those for thylakoid proteins (Herrmann et a1.
1983). This study also reported a 50 kilobase (kb)
inversion between spinach and Oenotbera in the large single
copy region of the chNA. This inversion is located between
the ATPase and the cytochrome f genes and between the large
sub-unit of RuBISCO and psbA genes (figure 4.1). Inversions
have also been found in the large single copy region of
wheat and mung bean chNA when compared with spinach (Palmer
et a1. 1982).

All chloroplast DNAs isolated are circular molecules
ranging from 120 kbp in pee to 217 kbp in geranium (Palmer
et al. 1987b). Most chloroplast DNAs are represented by
only one size class with the exceptions of Acetabulerie
(Green and Burton 1970), .tuglena gracjljs (Jenni et a1.
1981), Phylaiella littoralis and Spbecelarie sp. (Dalmon et
a1. 1983), green and brown algae. In higher plants, rice
was reported by Moon et al. (1987) to consist of
heterogeneous chNA molecules. Nith respect to plastome
size, the green algae are the most divergent group, with
chNA molecules ranging from 85 kbp to 292 kbp (Palmer
1985a, 1985b).

In general, the chloroplast DNA molecule is divided into
four parts: two areas of single copy DNA designated as the
large and small single copy regions, respectively, separated
by two large inverted repeats ranging from 10 - 11 kbp to as

large as 76 kbp (Palmer et al. 1987a, Stein and Palmer 1986

9

and Palmer 1985a, 1985b). The single copy regions are
relatively d(A + T) rich, the repeated regions are
relatively d(G + C) rich in their base compositions (Gillham
et a1. 1985). The unique regions of the chloroplast DNA
encode the majority of chloroplast proteins including the
large subunit of ribulose-l,5-bisphosphate carboxylase-
oxygenase (RuBISCO), proteins associated with of
photosystems I and II and proteins of the ATPase complex.
The inverted repeats contain genes for ribosomal proteins
and some tRNAs. The ribosomal genes are similar to
prokaryotic ribosomal genes and are arranged in operons
(Shinozaki 1986, reviewed by Gillham et a1. 1985, Palmer
1985a). The plastome contains spacer regions between genes.
Compared to plant nuclear and mitochondrial genomes these
are much smaller and fewer in quantity (Shinozaki et a1.
1986, Gillham 1985, Palmer 1985b, Gordon et a1. 1981, 1982).

The inverted repeat regions in the chloroplast genome
allow reciprocal recombinations to occur. This results in
chloroplast DNA molecules in isomeric form differing only in
the relative orientation of their single copy sequences
(Palmer 1983). The inverted repeats are considered to be
primitive (Palmer et al. 1987a) and are thought to confer
stability to the molecule since a strong correlation has
been shown between the presence of the inverted repeat and

the rarity of gross rearrangements in the chloroplast DNA

10
molecules which contain them (Palmer 1985a, Mubumbila et
a1. 1984, Palmer and Thompson 1981, 1982).

Small rearrangements occur within chNA (Palmer et al.
1987a) and are less common within the chloroplast genome
containing inverted repeats, but they do occur (Palmer
1985a, Gordon et a1. 1982, Palmer and Thompson 1982, Palmer
et al 1987a). In the case of Oenotbera, its five
genetically- classified plastome types can be distinguished
by a series of small insertions and deletions within the
spacer regions of the chloroplast DNA, however, these
differences do not alter the overall restriction
endonuclease DNA fragment order (Gordon et a1. 1982).

With the exception of the geranium chloroplast genome
(Palmer et al. 1987b), most of the chloroplast genome does
not contain large areas of repeated sequences outside the
inverted repeats (Palmer et al. 1987a). Small repeated
sequences have been reported in the chloroplast DNA of
subclover (Palmer et al. 1987a), wheat (Bowman and Dyer

1986) and Chlamydomonas (Palmer 1985b).

1.5. OINOTHIRJ, AN IMPORTANT GENETIC SYSTEM.

The genus Oenotbera (evening primrose), sub-genus
.Fuoenotbera, is an important genetic system but it is also a
complicated one. The subgenus luoenotbera has three basic

haploid genome types designated A, B, and C, and may give

11
six combinations. The group also contains five genetically
distinct plastid types. The plastid types vary in their
restriction endonuclease digestion patterns as well as their
ability to green in certain nuclear backgrounds. The five
plastome types also differ in their DNA replication rates.
Plastome type I is the fastest, and plastome type IV is the
slowest. Oenotbere has been the focus of many experiments
in genetics, cytogenetics and cytoplasmic inheritance ever
since deVries’ first publication in 1901. deVries’ work was
continued by Renner and his students and is extensively
reviewed by Burnham (1962), Tilney-Bassett (1978), and
Cleland (1972). The work performed by Banner, his students
and Cleland established Oenotbera as having a unique
characteristic in its organization of chromosomes. Most
Cenotbera species contain 7 pairs of chromosomes. The
chromosomes may be linked together in a chain or ring at the
first meiotic division (Cleland 1972). These rings are the
result of a series 'of equal subterminal reciprocal
translocations between chromosome ends. Such translocations
are deleterious in most plants because exchanges are
ordinarily unequally spaced. Unequal translocations, in
*most cases, result in a high level of non—disjunction and
consequent sterility. In Oenotbera, however, the
translocations that have survived are the result of equal
exchanges so that the centromeres are evenly spaced in the

circles and little sterility ensues. Crossing-over between

12
these homologous regions serves to stabilize the chromosomes
in the ring or chain configuration. In the ring
arrangements, independent assortment and crossing over of
ring chromosomes are effectively suppressed during meiosis.
Oenotbera has other characteristics which make it the plant
of choice to study cytoplasmic inheritance including
biparental inheritance of plastids, and the ability to

perform crosses between species.

1.6. MUTATIONS OF THE CHLOROPLAST.

Studies of the chloroplast genome including its role in
‘the physiology and genetics of the plant have been greatly
advanced through the investigation of mutations in the
plastome (Boerner and Sears 1986, and Gordon et a1. 1980).

Plastome mutants affecting many physiological and genetical
processes have been isolated and studied in a large number

of plants (reviewed by Boerner and Sears 1986).

1 . 7. THE PLASTONI ”UTA TOR GENE.

A mutator gene has been defined as a gene which controls
the frequency of occurrence of mutation events in a
particular system (Cox 1976). The mutator genes found in
prokaryotic systems may serve as useful models for those
found in eukaryotic organelles, since mitochondria and

chloroplasts are considered to be prokaryotic in nature

13

(Palmer 1985b and Bohnert et a1. 1982). Mutator genes have
been shown to affect many cellular processes in bacteria
including the DNA polymerases and DNA repair (Cox 1976).
They may also affect removal and replacement of specific
nitrogenous bases (Coulandre et al. 1978), nucleotide
removal, replacement, photomonomerization of uv-induced
thymine dimers, and recombination (Kimball 1979), or the
induction of a transposable element (Kleckner 1981).
Mutator genes which cause a variety of non-Mendelian
mutations in mitochondria of yeast (Becker and Floury 1985),
and chloroplasts of Arabidopsis (Redei 1973, Redei and
Plurad 1973) and of Oenotbere (Epp 1972, 1973) have been
identified. The mutator genes of higher plants have been
called "plastome mutator” (Epp 1972, 1973) or "chloroplast
mutator” (Redei 1973). Prior to the isolation of a plastome
mutator strain in Oenotbera, the number and type of plastome
mutants was limited primarily to spontaneous mutations most
of which were described by Kutzelnigg and Stubbe (1974).
These spontaneous mutations arise at a frequency of 0.01-
1.33 in a population (Michaelis 1969). The frequency of
mutations induced by plastome mutator is 303. Since then,
investigators have had some success in creating plastome
mutants in higher plants by the use of chemical mutagens
(reviewed by Boerner and Sears 1986).

Two types of plastome mutator systems in plants have been
demonstrated to data. One type always results in the

expression of the same mutant phenotype as seen in

14

albostrjans of barley (Boerner et al. 1976), and iojap of
maize (Shumway and Meier 1967, Thompson et al. 1983). These
mutators are possibly the cause of‘a loss of chloroplast
ribosomes during a critical stage in the development of the
plant. The second type of plastome mutator is found in
Arabidopsis (Redei 1973, Redei and Plurad 1973), Petunia
(Potrykus 1970), and Oenotbera (Epp 1972, 1973). These
mutators induce a variety of chloroplast mutations.

It is the plastome mutator (pm) gene of Oenotbera which
is the focus of chapter 3 of this thesis. Chapter 4 deals
with the characterization of small discrete insertions or
deletions found of the chloroplast DNA during comparisons of

wild-type plants of Oenotbera hooker! str. johansen.

CHAPTER 2

MATERIALS AND METHODS

15

16

2.1. PLANT MATERIAL.

Three wild-type cultivars and two plastome mutator
induced mutants of Oenotbers jobsnsen were used in the
studies. Table 2.1 lists the plant lines their genotypes
and their phenotypes. The pedigree of these lines is shown
in Figure 2.1.

Wild-type lines D and Cr were grown in the greenhouse in
sandy soil. Wild-type line C: was maintained as shoot
meristem cultures since it was available only in an A/C
nuclear background (plastome I plants are not as vigorous in
the A/C background as they are in their natural A/A nuclear
background (Kutzelnigg and Stubbe 1974), and therefore need
to be grown on nutrient medium). The mutant plants pm8’and
p.11 along with wild-type line C: were maintained as shoot
meristem cultures, in accordance with Gamborg (1975) and
Stubbe and Herrmann (1982) on a modified N.T. medium (Nakata
and Takabe 1971) containing a 1:6 ratio of BAP (benzylamino
purine) to NAA (napthylacetic acid). Mutant p.11 grew
better and had more leaf expansion when grown on M/S medium
(Murashige and Skoog 1962) with a 1:2 ratio of auxin to
cytokinin (Table 2.3). The plants were maintained as shoot
meristem cultures-and were transferred to fresh plates every

three to four weeks as needed.

17

Table 2.1 Plant lines and their origins. Mutant lines
arose as sectors on plants homozygous for the pm gene.
Capital letters denote the nuclear genome type. Roman
numerals denote the plastome type. The genetic composition
of wild-type 0. jobsnsen is AAI. Five digit numbers
represent the year on the field and field designation.

 

Female Male

Line parent parent Genotype Phegotxpe
D Job AAI Job AAI AA wild-type
c; 81/124 AAI Job AAIV nu wild-type
C2 80/960 AAI Job ACIV P'AC wild-type
p.11 80/960 p.11 Job ACIV P'AC pm]!

AAI white
pm8 80/959 me Job ACIV P'AC pm8

AAI pale green

18

Figure 2.1. Pedigree of Plant Lines. Pedigree of plant
lines from Epp’s Cornell Oenotbera johansen pm pm plastome I
line. The five digit numbers following the arrows represent
the year in which the seed was planted (first two digits)
and the field plot number (last three digits). Roman
numerals refer to plastome type and capital letters refer to
the genome type. Line D is a different 0. jobsnsen isolate
from the Duesseldorf collection and is not included in this

pedigree.

19

 

 

 

 

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nuwmvfl AHUIV cacao .m. xvoum QEM oohm 3
H; ”\ xuoam Mflfld xuoum Wad x N
N—Eu >~UI x “Tam >HU¢ x mEm :0 ......d >~U¢ x MANN
@ ...-e ace-m as: .cs.a ems use... not .a if. ace-a
» 9
>Hkmo? X rmu\um _ col? 9
» awmx \aw mmmx
ow
om-\w

 

H groans—a

ecu. Em Eu macaw

 

0C.—

comCOLOW .O

_—OCLOU

mwzHJ Hzmli no mmmoHowd

Figure 2.1

Table 2.2. Modified N.T. Medium for the maintenance of
shoot meristem cultures.

N.T. Medium

 

Component Amount per liter
N.T.-major elements stock 125 ml
N.T.-minor elements stock 1 ml
N.T. vitamin solution 10 ml
NAA solution 5 m1
BAP solution 10 ml
Sucrose 60 g
Agar 8 g

The pH was adjusted to 5.8 with KOH prior to addition of
agar and then autoclaved.

N.T. stock solutions:

N.T.Major elements

 

 

Component Amount per liter, pH5.8
NHeNOe 6.6 g

KNOa 7.6 g

CaClaoZHzO 1.76 g

MgSOa'7HaO 9.86 g

KHzPOa 5.44 g

NazEDTA 0.30 g

Fe-citrate 0.22 g

HaBOa 0.05 g

MnSOe'HzO 0.18 g

ZnSOs'7H20 0.07 g

N.T.Minor Elements Amount per 100 pl
KI 83.0 mg

Nae M00402H20 25.0 mg
CuSanSHzo 2.5 mg
CoSan7H20 3.0 mg
N.T.VitamingSolptiop Amount per 100 ml
Meso (Myo-)-Inositol 1.000 g
Thiamine-H01 0.010 g

NAA Stock

Napthylacetic acid 30 mg/100 ml

BAP Stock Solution
Benzylamino purine 10 mg/ 100 ml

Table 2.3. M.S. Medium

Component

M/S stock solution
M/S stock solution
M/S stock solution
M/S stock solution
NazFeEDTA

thiamine
Pyridoxine
nicotinic acid

BAP

NAA

myo-inositol
sucrose
(2(N-Morpholinol-

ethanesulfonic Acid)(MES)

agar

The pH was adjusted
and than autoclaved.

addition of agar,

ewoaahn

M18 stock solutions

Component

M18 1
NHsNOs
KNOs

MlS 2 (Sulfate)
MgSOaO7H20

MnSOQOHzo
ZnSOe‘7HzO
CuSOeOSHzO

MZS 3 (Halide)
CaCleZHzo

KI
CoClaOSHzO

M18 4 (PBMO)
KHzPOe

HaBOa
NazMoOe‘ZHzO

MZS medium

Amount

20
10
10
10
10

FHOF‘HWO

100
60

m1
ml
ml
ml
ml
ml
ml
ml
ml
ml
IS
S

0.5 g

8.0 g

per liter

(2.5 mg/ml)
(0.5 mg/ml)
(0.5 mg/ml)
(1 Isl-1)
(1 Is/Il)

to 5.8 with l N KOH prior to the

Amount per liter

82.5

95

K

22
2.2. BACTERIAL STRAINS AND PLASMIDS.

The bacteria Escherichia coli (E. calf) strains E08654:
[gal I} gal T, trp.R,.met B, bed.R, sup E, Inc 77 (Bork et
al 1976) and HBlOl: [bed S (re-,me-), rec A, are, pro A,
Inc 7, gal I} rps L, xy1,.mt1, sup.5,] (Boyer and Roulland-
Dussoix 1969) were used in this study. They were maintained
on Luria Broth (L.B.) plates for working cultures and as
frozen cultures for long term storage. Luria Broth consists
of : 5 g NaCl, 10 g Bacto tryptone (Difco), 5 g yeast
extract (Difco), l g glucose [(15 g Agar(Difco or Sigma)]
per liter (Maniatis et a1. 1982). 3. col! storage medium
consists of: 0.7 g KaHP04, 0.3 g KHzPOe, 0.05 g Na-Citrate,
and 0.01 g Mg804, 50 X Glycerine per liter. Frozen cultures
were maintained at -20°C.

Bacterial strains containing the plasmid pBR322 were
maintained on L.B. plates containing either 80 pg/ml
ampicillin (amp) or 12.5 pg/ml tetracycline (tet).
Recombinant plasmids containing inserts cloned into the tet
resistance gene were maintained on L.B. + amp plates, or as
frozen cultures lacking antibiotics in the storage medium

described above.

23

2.3. DNA PREPARATION.

2.3.1. Chloroplast DNA Isolation.
2.3.1.1. Chloroplast isolation from abundant amounts of
plant material.

For plants which were grown in the greenhouse, leaf
material was abundant. Chloroplasts were first purified as
described below. DNA was then extracted from the isolated
organelles.

The leaves of Oenotbera contain compounds, such as
phenolics, which make the isolation of chloroplasts
difficult. Because young leaves of Oenotbera plants contain
lower concentrations of phenolic compounds and other
secondary metabolites than do older ones, most of the leaves
were trimmed from the plants about three weeks before the
scheduled harvest in order to promote new growth. These
plants were then placed in the dark one to four days before
harvest in order to deplete their starch reserves. 200 to
250 g of leaves were then harvested and washed in distilled
H20, damp dried and weighed. The leaves were homogenized in
a buffer containing 68 sorbitol, 6 mM EDTA, 1 mM ascorbic
acid, 3 mM cysteine, 0.153 (w/v) polyvinyl pyrrolidone
(P7P), 0.1x bovine serum albumin (BSA), 50 mM Tris pH 7.5.
This buffer is based on the one described by Herrmann (1982)
with modifications adapted from Somerville et al. (1981),

Loomis (1974), and Galliard (1974). The homogenate was

24

filtered through one layer of 100 micron mesh gauze followed
by filtration through two layers of miracloth (Calbiochem).
The chloroplasts were pelleted at 6,000 rpm at 4°C and were
washed in the same buffer described above, but lacking PVP.
The chloroplasts were then purified over a 108 - 80% sucrose
step gradient buffered with lOmM Tris pH8.0, lmM EDTA. The
band containing the chloroplasts was separated and diluted
with 50 mM Tris, 20 mM EDTA pH 7.5 and pelleted at 10,000
rpm 4°C for 10 min. The chloroplast pellet was resuspended
in an equal volume of 50 mM Tris, 100 mM EDTA, 15 mM NaCl pH
8.5 for subsequent lysis.

2.3.1.2. Isolation of chNA from limited amounts of plant
material.

Plant material from tissue culture was generally in a
much more limited supply than material from greenhouse grown
plants.

2.3.1.2a. Protoplast method.

Protoplasts were isolated from 10 to 50 g of tissue. The
tissue was first chopped with a razor blade and placed in
glass petri plates (5 g per plate) in a solution of 22
Cellulysin, 1% Macerase, lx Driselase, 138 Mannitol in a
buffer containing 27.2 mg KHzPOa, 101.0 mg KNOa, 246.0 mg
MgSOeO7HzO, 0.16 mg KI, 0.025 mg Cu80405H20, 1.48 g CaClz-
2H20 per liter at pH 5.8. The plant material was incubated
overnight at room temperature in the dark. Undigested plant

material was separated from the protoplasts by filtration

25

through an 80 micron stainless steel mesh screen. The
filtered protoplasts were pelleted by centrifugation at
2,000 rpm at room temperature for 15 min. The pellet was
resuspended in a buffer containing 400 mM Sorbitol, 5 mM
EDTA, 100 mM Tris pH 8.0, 0.13 BSA, and 0.32
Mercaptoethanol and allowed to equilibrate on ice for 20
min.

The protoplasts were lysed by forcing the suspension
through an 18 gauge needle three times. The lysate was
pelleted by centrifugation in a swinging bucket (Sorvall
HB4) rotor at 4°C at 4,500 rpm for 5 min. The pellet was
resuspended in an equal volume of 50 mM Tris pH 8.0, 20 mM
EDTA for organelle lysis and chNA isolation.
2.3.1.2b. Modified whole cell DNA method.

The whole cell DNA method was modified from DeBonte and
Matthews (1984). 10 to 50 g of plant material was
homogenized in the chloroplast isolation buffer described
above and filtered through one layer of 100 micron mesh and
one layer of miracloth. The homogenate was then pelleted by
centrifugation at 9,000 rpm in a Sorvall GSA rotor at 4°C
for 20 min. The pellet was resuspended in chloroplast wash
buffer and pelleted as before. The pellet was then

resuspended in 5 ml 50mM Tris, 20mM EDTA pH 8.5 for lysis.

26

2.3.2. PURIFICATION OF chNA.

The chloroplasts were lysed and the chNA was liberated
from the membranes to which they associate by the addition
of 1x Sarkosyl and 1 mg/ml Pronase, followed by gentle
mixing at 4°C for 4 h. Chloroplast DNA was separated from
nuclear DNA by CsCl buoyant density equilibrium
centrifugation in the presence of bisbenzamide (Hoechst
33258) (Mueller and Gautier 1975, and Preisler 1978). 20
p‘ bisbenzimide and 1.1 g/ml CsCl were added per ml of
lysate and the refractive index was adjusted to 1.3960.
CsCl gradients were run in a Sorvall OTD-B6 ultracentrifuge
with vertical rotors T7865 for small volume samples or a
T7850 rotor for samples greater than 30 ml. Gradients were
run at 40,000 rpm to 42,000 rpm at 1900 for 12 to 15 h.

The upper band which contained chNA was removed from the
gradient by fractionation. Bisbenzimide was removed by at
least three extractions using CsCl- or NaCl-saturated
isopropanol. The salt was removed by dialysis at 4°C with

three changes of 10 mM Tris pH 8.0, lmM EDTA (TE).
2.3.3. PRECIPITATION OF DNA.
DNA was precipitated by the addition of either 2/3 volume

5M NHaOAC or 1/20 volume 3M NaOAC, 1 pl 20 mg/ml glycogen

and 2.5 volumes absolute ethanol at -20°C for at least 4 h.

 

at
re
tr;

Dre

27
Large volumes greater than 3 ml were centrifuged at 8,000
rpm at 4°C for l h in a Sorvall HB4 rotor. Small volumes
were centrifuged in a microfuge at 4°C for 15 min. The
purified chNA was then resuspended in the appropriate

amount of 10 mM Tris pH 8.0, 0.1 mM EDTA (T 1/10 E).

2.3.4. PURIFICATION OF PLASMID DNA.

2.3.4.1. Large Scale isolation of Plasmid DNA.

For purification of large amounts of plasmid DNA, two
methods were used depending on the size of the plasmid.
2.3.4.1a. Triton Lysis method.

This method has been modified from Maniatis et al.
(1982). It was used for plasmids larger than 15,000 bp. A 1
liter culture of E. coli containing the plasmid was grown in
L.B. + amp in a shaker incubator at 37°C until the cell
culture reached an optical density (O.D.) 050 of 0.4. A
final concentration of 170 pg/ml Chloramphenicol was added
for amplification of the plasmid. Incubation of the
cultures continued at 37°C for at least 12 h or overnight.

The cells were pelleted at 3,000 rpm at 4°C for 30 min.,
washed with 20 ml 25 mM Tris pH 8.0, 10 mM EDTA,and pelleted
at 6,000 rpm at 4°C for 10 min. The pellet was carefully
resuspended in 15 ml 50 mM Tris pH 8.0, 253 sucrose and
‘transferred to a 40 ml centrifuge tube. 3 ml of a freshly

Prepared solution of 5 mg/ml lysozyme (Sigma) was added and

28

the cells were incubated on ice for 15 min. 15 m1 of Triton
1ytic solution (3 m1 103 w/v Triton X-100, 75 m1 0.2M EDTA,
15 ml 1.0M Tris pH 8.0, 7 ml H20 per 100ml) was added and
the cells were incubated on ice for another 30 min. The
lysate was then centrifuged in a Ti 60 rotor (Beckman) at
35,000 rpm at 4°C for 30 min in a Sorvall OTD-B6
ultracentrifuge. The top of the supernatant containing the
plasmid DNA was carefully removed with a siliconized large
bore Pipette. 100 pl (10 mg/ml)of a RNase A stock solution
was added to the plasmid DNA, followed by incubation at 37°C
for 45 min. 5M NaCl was added to a final concentration of
0.5M and the nucleic acids were precipitated with an equal
volume of isopropanol at -20°C for at least 2 h. The
nucleic acids were then pelleted in a Sorvall HB4 rotor at
6,000 rpm at 4°C for 1 h. The pellet was dried and
reconstituted in 6 ml TE. For each ml, 180 pl of a 10
mg/ml Ethidium bromide (EtBr) stock solution and 1.1 g CsCl
was added. The refractive index was adjusted to 1.3915
followed by ultracentrifugation at 42,000 rpm at 19°C for
12 to 15 h.

2.3.4.1b. Physical Shearing Method.

The physical shearing method was obtained as a personal
communication from M. Gurevitz (developed by John Williams,
DuPont), and was used to purify plasmid DNA from small
plasmids of less than 15,000 bp. A 200 ml culture of cells

was grown, amplified and pelleted as described previously.

29

The cells were resuspended in 5 ml 103 sucrose in TES buffer
(50 mM Tris pH 8.5, 50 mM NaCl, 5 mM EDTA). 100 pl lysozyme
(35 mg/ml) and 10 pl RNase A (10 mg/ml) were added followed
by incubation at 37°C for 10 min. 5 ml 23 sarkosyl in TES
buffer was added, the lysate resumed incubation at 37°C for
another 10 min. The DNA was then sheared through a 10 ml
glass pipette until the solution was less viscous and would
fall dropwise from the pipette. The lysate was then
transferred to a tared 125 ml erlenmeyer flask, 1.5 m1 EtBr
(10 mg/ml) was added and the weight was adjusted to 23 g
with TES buffer. To this solution, 21 g solid CsCl was
added and the refractive index adjusted to 1.3915, followed
by ultracentrifugation in a Sorvall T7850 rotor at 40,000
rpm at 19°C for at least 18 h.

2.3.4.2. Isolation and purification of Plasmid DNA
following ultracentrifugation.

Following ultracentrifugation, the gradient was
fractionated to recover the lower band containing
supercoiled plasmid DNA. The EtBr was removed by extraction
with sec-butanol. CsCl was removed by dialysis in ddeO at
room temperature for 1.5 h. The volume was reduced to 1 ml
by several extractions of sec-butanol, followed by phenol
and CHCla (CHCle : isoamylalcohol 24 : 1) extractions. The

DNA was precipitated as previously described.

30
2.3.4.3. Rapid small scale isolation and purification of
plasmid DNA (mini preps).

The mini prep method is a modification of a procedure
described by Maniatis et a1. (1982). Mini preps were used
to isolate small quantities (1 to 5 pg) of plasmid DNA in
order to screen for recombinants in cloning experiments or
any time small quantities of DNA were sufficient.

Bacterial cultures were grown in 5 ml L.B. + amp in a
shaker incubator at 37°C for 8 h or overnight. Cells were
pelleted at 3,000 rpm at 4°C for 5 min. if from liquid
culture, transferred to 1.5 ml microfuge tubes and pelleted
by a brief spin in a microfuge. Occasionally, cells were
taken directly from an L.B. + amp plate. The pellet was
resuspended in 350 p1 STET buffer (83 sucrose, 0.53 Triton
X-100, 50 mM EDTA, 10 mM Tris pH 8.0). 25 pl Lysozyme (1
mg/ml) was added, the samples then placed in a boiling water
bath for 40 s. followed by centrifugation in a microfuge for
10 min. The top 200 p1 of the supernatant was removed and
to it 3 p1 of RNase A (1 mg/ml) was added and the samples
incubated at 37°C for 30 min. The samples were then
extracted with phenol/CHCla, and precipitated. Plasmid DNA
was then pelleted as described above, resuspended in T 1/10E

and checked for purity on a small agarose gel.

31

2.4. RESTRICTION ENDONUCLEASE DIGESTION OF DNA.

Restriction endonucleases were purchased from the following
companies: International Biotechnologies, Inc., Boehringer
Mannheim Biochemicals, Bethesda Research Laboratories, and
New England Biolabs. Reactions were carried out according
to company specifications using 1 to 3 units of enzyme per
pg of DNA in a reaction volume of 20 pl per 1 pg DNA for 2
to 4 h. Enzyme digestion reactions were stopped by the
addition of EDTA to a final concentration of 10 mM. If the
restricted DNA needed to be reacted with another enzyme
under different conditions, or needed to be concentrated to

a smaller volume, the DNA was precipitated.
2.5. GEL ELECTROPHORESIS.

2.5.1. Agarose gel electrophoresis.

Agarose gel electrophoresis was performed as described by
Maniatis et a1. (1982). The concentration of the agarose
gels depended upon the sizes of the DNA fragments which
needed to be resolved from restriction endonuclease
digestions. Electrophoresis buffer TAE (.004M Tris, 0.001M
EDTA pH 8.0 with acetic acid) was used for resolution of
large molecular weight fragments of 10 kbp or more, and TBE
(0.089M Tris, 0.089 boric acid, 0.002M EDTA pH 8.0 with HCl)

for the resolution of DNA fragments less than 10 kbp. In

32

order to be able to monitor the progress of the
electrophoresis, a running dye of 0.13 bromophenol blue
(BPB) in 303 glycerol was added to the samples. Ethidium
bromide (EtBr) was added to the gel and the running buffers
at a final concentration of 0.5 pg/ml. It was then possible
to monitor the progress of the electrophoresis by watching
the dye and by looking directly at the DNA using a hand held
UV light. Gels were run at room temperature at a current of
25 to 50 mAmps with constant voltage.

2.5.2. Polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) was set up
according to Maniatis et a1. (1982). The concentration of
the polyacrylamide gels varied from 53 to 203 depending on
the sizes of the DNA fragments. The running-buffer for PAGE
was exclusively TBE lacking EtBr. PAGE was run at room
temperature at 10 to 15 mAmps (50 volts).

In order to obtain the best resolution, sanples were
loaded in volumes not exceeding 5 p1 and run with 1/10
volume of 10x loading dye (0.253 bromophenol blue, 0.253
xylene cyanol, 253 Ficoll (type 400) in H20). Following
electrophoresis, the gels were stained in a buffer of
1 pg/liter EtBr in TBE for 15 to 30 min, examined on a UV

transilluminator, and photographed.

 

Pro

33
2.5.3. Calculation of molecular size of bands separated by
electrophoresis.

The approximate sizes of DNA bands on electrophoresis
gels were calculated as described by Sealey and Southern
(1982) by extrapolation from a plot of log molecular weight
versus distance traveled of marker DNAs listed in table 2.3.
The entire ranges of fragment sizes are listed in the

appendix.

Table 2.4. Marker DNAs used in gel electrophoresis.

' Fragment
Markgr ‘DNA Size Range

1 phage hdigested with 25kb - 0.5kb
HindIII + EcoRI

2 BRL 123 bp ladder 4000bp - 123bp

3 pBR322 DNA digested 1600bp - 50bp
with Hian

4 pBR322 DNA digested 620bp - 50bp
with MspI

2.6. PURIFICATION OF DNA FROM GELS.

2.6.1. Purification of DNA fragments from agarose gels.
2.6.1.1. Crush and extract method.

This method was used for the purification of DNA
fragments from agarose gels containing many bands in close

proximity. DNA digestions were run on the lowest possible

34

concentration of agarose which would provide resolution of
the band(s) of interest. The band(s) were then located
under UV light and removed from the gel with a sterile
scalpel. The gel piece was then placed in a microfuge tube
and frozen at -70°C for 10 min. The blunt and of a small
spatula was then used to crush the frozen piece of gel while
still in the microfuge tube. An equal volume of phenol was
added, mixed gently and frozen at -70°C for 10 min, followed
by centrifugation in a microfuge for 5 min. The aqueous
layer containing the DNA was removed and saved on ice while
the organic layer was back extracted with an equal volume of
TE as before. The aqueous layers were pooled, extracted two
more times with phenol, and twice with CHCla: isoamylalcohol
(24:1). The DNA was then precipitated.
2.6.1.2. Electroelution onto DEAE Cellulose Membrane.

Schleicher and Schuell (SSS) NA-45 DEAE cellulose
membrane was used when isolating DNA fragments which were
easily separated by electrophoresis. Restricted DNA was run
on a 0.63 agarose gel until a good separation was achieved.
A block was cut from the gel just below the band of
interest. A piece of SSS membrane was cut to fit and was
inserted into the gap. The gel block was replaced behind
the membrane and the current set at 75 mAmps until the band
of DNA moved onto the membrane (about 20 min). .The progress
was monitored by a hand held UV lamp. The membrane was then

removed from the gel, washed briefly in sterile ddeO and

 

NE

wE

35

placed into a microfuge tube containing 400 pl 1.5M NaCl, 10
mM Tris pH 7.5, 1 mM EDTA. The membrane holding the DNA was
then incubated at 65°C for 4 h. Following incubation, the
membrane was removed and discarded, the DNA was then
precipitated.

2.6.2. Purification of DNA from polyacrylamide gels: Crush
and Soak method.

The EtBr stained gel was placed on a UV transilluminator;
the band(s) of interest were located and carefully removed
with a sterile scalpel. Each gel fragment was then placed
into a 5 ml disposable syringe fitted with an 18 gauge
needle which had been cut to 1/4 of its original length.
The syringe was placed above a microfuge tube, the whole
system was then placed in a 30 ml corex centrifuge tube and
was spun at 5,000 rpm at room temperature in a desk top
centrifuge for 10 min. Elution buffer (0.5M NHaOAC, lmM
EDTA pH 8.0) was added to the crushed acrylamide, and the
slurry was incubated with shaking at 37°C for at least 8 h
or for overnight. The acrylamide was separated from the DNA
by centrifugation through a quick-sap (Isolab) tube. 5M
NHeOAC was added to a final concentration of 2M, and the DNA

was precipitated.

36
2.7. SOUTHERN TRANSFER OF DNA FROM AGAROSE GELS TO

NITROCELLULOSE PAPER.

Southern transfer of DNA to nitrocellulose paper was done

as described by Maniatis et a1. (1982).

2.8. HYBRIDIZATION OF SOUTHERN FILTERS.

A slight modification of the technique described by
Southern (1975) and Maniatis et a1. (1982) was used.
Nitrocellulose filters were prehybridized at 68°C for 8 h or
overnight in 6! SSC (1! SSC is 0.15M NaCl, 0.015M NaCitrate,
pH 7.0), 0.53 SDS, 5: Denhardts solution (0.5 g Ficoll, 0.5
g polyvinylpyrrolidone, 0.5 g), 100 pg/ml denatured salmon
sperm DNA, Hybridizations with radioactive DNA probes were
performed in 6X SSC, 0.01M EDTA, 53 SDS, 100 pg/ml denatured
salmon sperm DNA. Hybridizations were done at 68°C when
homologous probes were used. The filters were washed in 2X
SSC, 0.53 SDS at room temperature for 5 min., 2X SSC, 0.13
SDS at room temperature for 15 min. and 2 washes in 0.1x
SSC, 0.53 SDS at 68°C for 30 min. each. For heterologous
probes, the final washes were at less stringent
temperatures. The filters were allowed to air dry followed

by autoradiography.

37

2.9. NICK TRANSLATION.

Radioactive probes were prepared by nick translation with
32P labeled nucleotides (NEN-DuPont). The procedure used was.

a modification of that of Rigby et al. (1977). 0.5 to 1 pg

DNA was labeled in a reaction volume of 50 pl containing
50mM Tris pH 7.8, 7.5mM MgClz, 10mM mercaptoethanol, 0.05
mg/ml BSA, and one or more 32P labeled dNTPs (20 to 40 pCi
specific activity greater than 600 pCi). Cold dNTPs were
added at a concentration of 12.5 nM, omitting the ones used
to label the DNA. The DNA substrate was then digested by
adding 4 p1 fresh DNase I (BMB)(100 ng/ml) for l min. at
room temperature immediately followed by the addition of l
to 5 units of DNA polymerase I (BRL, BMB, or NE Biolabs) and
incubation at 14°C for 1 to 2 h. The unincorporated a3P
nucleotides were separated from the labeled DNA over a
Sephacryl S-200 (Pharmacia) column. The incorporation of
33P into the DNA was quantified with 08-81 (Whatman) ion-
exchange paper as described by Maniatis et a1. (1982) and by
measurements in a Beckman LS-l33 scintillation counter.

The nick-translated DNA (probe) was denatured by heating

at 100°C for 3 min. before adding to the hybridization mix.

38

2.10. CLONING.

Comparison of the chNAs required large amounts of
material. In order to obtain sufficient quantities of these
chNAs, the chNA fragments of interest were isolated and
cloned into plasmid pBR322 using 5. con HB101.

2.10.1. Preparation of Vector DNA.

For cloning of BamHI chNA fragments, plasmid pBR322 was
cut with the restriction endonuclease BamHI. In order to
prevent recircularization of non-recombinant plasmids, BamHI
digested pBR322 was treated with alkaline phosphatase
(Calbiochem). For this reaction, 5 units of phosphatase for
each pg pBR322 DNA was added to the restriction endonuclease
digestion. Following incubation at 37°C for 30 min, the
phosphatase was inactivated by incubation at 70°C for 30 min
followed by 3 extractions with phenol and 3 extractions with
CHCla. The phosphatased pBR322 DNA was then precipitated.

Cloning DNA fragments which had been digested with BamHI
+ EcoRI did not require phosphatase treatment of the vector.
A BamHI/EcoRI digestion of the pBR322 vector yields two DNA
fragments of 3986 bp and 376 bp which are easily separable
by gel .electrophoresis. The larger band was isolated from
the gel with SSS membrane as described above, and ligated to

the chNA to be cloned.

39
2.10.2. Ligation.

Ligation of DNA was carried out with an excess of insert
DNA at a final concentration of 0.1 pg/pl in a reaction
containing 66 mM Tris pH 7.5, 1 mM EDTA, 10 mM MgClz, 0.1
mg/ml BSA, 10 mM DTT, 0.1 mM ATP and ligase. The amount of
ligase added ranged from 1 to 5 units, depending on the age
of the enzyme. The ligation reaction was incubated at 4°C
for 12 to 18 h. The reaction was monitored by gel
electrophoresis.

2.10.3. Transformation.
2.10.3.1. Transformation by the Calcium Chloride Procedure.

Transformation using the calcium chloride procedure was
done as described by Maniatis et al. (1982) with the
following modifications:

I. Transformation competent cells: 100 m1 L.B. was
inoculated with 1 m1 of an overnight culture of E. colj
strain HB101. The cells were grown with shaking at 37°C
until the 0.0.050 reached 0.4, or approximately 3 x 108
cells/ml. The cells were transferred to a sterile
centrifuge jar and chilled on ice for l h. The suspension
was centrifuged at 8,000 rpm at 4°C for 5 min. and gently
resuspended in ice cold sterile 50 mM CaClz. The cells were
incubated on ice for 20 min. and pelleted again. The cells
were resuspended in 8 ml ice-cold 50 mM CaClz and 153
glycerol and allowed to sit on ice overnight or were frozen

immediately in liquid nitrogen in 100 pl alliquots in pre-

40
chilled microfuge tubes. The competent cells were stored at
-80°C. The efficiency of transformation ranged from 5 x 105
to l x 107 per pg intact pBR322 DNA; the cells remain
competent for up to 3 months.

II. Transformation: Approximately 50 to 200 ng of DNA
in ligation buffer was added directly to 100 pl of E. colj
HB101 transformation competent cells. The cells were
incubated on ice for l h, followed by a heat pulse at 42°C
for 2 1/2 min., placed on ice, and transferred into 1 m1
L.B., and incubated at 37°C without shaking for 2 h. The
cells were then plated on L.B. + amp plates and incubated at
37°C for no more than 12 h.
2.10.3.2. Transformation using a modification of the
Hanahan procedure.

The protocol for this modification of the Hanahan
procedure was obtained through Ellen Johnson (personal
communication).

I. Preparation of transformation competent cells: A
modification of the procedure developed by Hanahan (1983)
was used when a high transformation efficiency was required.
This procedure was used to clone the BamHI 3b fragments.
100 m1 SOB medium (20 g Bacto tryptone, 5 g yeast extract, 5
g NaCl, 5 m1 (0.5M) KCl, 10 ml (10 mM) MgSO; per liter) was
inoculated with 1 ml cells from a 2.5 m1 overnight culture

in SOB medium. The cells were grown to an O.D.eso of 0.5 to

0.6 with shaking at 37°C. Cells were collected in a

 

41
centrifuge tube which had been prerinsed with SOB and
chilled. The cells were then incubated on ice for 5 min.
The cells were pelleted in an HB4 rotor at 2,500 rpm at 4°C
for 15 min. The pellet was gently resuspended in 33.3 ml of
ice cold FSB (10 mM KAc, 100 mM KCl, 45 mM MnClz 4 H20, 10
mM CaClz), and incubated on ice for 5 min, and pelleted once
again. The cells were resuspended in 8 ml ice-cold FSB and
280 pl DMSO was added, with gentle mixing. The cells were
incubated on ice for 5 min, followed by the addition of
another 280 pl DMSO. The cells were then aliquotted into
chilled microfuge tubes (200 pl /tube) and immediately
frozen in liquid nitrogen. The transformation competent
cells were stored at -80°C.

Transformation efficiencies with this method yielded 1 x
10° colonies per pg intact pBR322 DNA for HB101 cells and 1
x 107 colonies per pg DNA for E08654 cells. Cells remained
competent for up to 3 months.

II. Transformation: 10 ng of DNA was added to thawed
competent cells, and incubated on ice for 30 min. The cells
were heat-pulsed at 40°C for 2 min followed by the addition
of L.B. broth. The cells were incubated at 37°C with
shaking for l h. and spread on L.B. + amp selection plates
and incubated at 37°C for no more than 12 h.

2.10.4. Screening for recombinant clones.
Screening for recombinant colonies of bacteria was done

as described by Gergen et al. (1979) and Grunstein and

 

fr

p3

I01

 

 

Ill

I81

the

Ir:

581:

et.‘

 

42

Hogness (1975) with the following modifications. After
transformation, ampr colonies were transferred to L.B. + amp
and L.B. + tet grid plates and grown overnight at 37°C.
Colonies displaying an ampr tet' phenotype were transferred
to L.B. + amp grid plates. This second grid contained both
positive and negative controls. The positive controls were
0. johansen chNA clones carrying Ban 3a, Bam 3b, or Ban 12
fragments. The negative control contained the plasmid
pBR322.

The colonies were grown on the screening grids for no
more than 12 h at 37°C. A sterile Nhatman 541 circle was
placed on top of the bacterial colonies. After a few
minutes, the filters holding the bacterial colonies were
removed from the plates.

In order to lyse the cells in the colonies and denature
the DNA, the filters were washed twice with agitation for 5
min. in each of the following solutions:, 0.5M NaOH, 0.5M
Tris pH 7.5, and 2X SSC (1X SSC is 0.15M NaCl, 0.015M
NaCitrate, pH 7.0). The filters were rinsed briefly in 953
ethanol and air dried.

The filters were prehybridized in sealed plastic bags for
at least 5 h. at 55°C to 68°C in 0.53 N P 40 (Sigma), 100
pg/ml denatured salmon sperm DNA, 6X SET ( 0.9M NaCl, 180nM
Tris pH 8.0, 6 mM EDTA). Hybridization was done at 0° - 3°C
below the Ta (temperature of denaturation) which, under the

most stringent conditions in the study was 68°C. The

43

hybridization buffer was the same as the prehybridization
buffer but included 1 pg/filter denatured pBR322 DNA and 33F
nick translated probe. The hybridization was done overnight
(at least 8 h).

Following hybridization, the filters were removed from
the plastic bags and washed three times for 5 min. each in
6! SSC at just below the Ta (for the most stringent

condition). The filters were allowed to air dry, followed

by autoradiography.

CHAPTER 3

PLASTOME MUTATOR

44

45

3.1 INTRODUCTION

Plastome mutants are a useful resource for the study of
the genetic content and the contribution of plastid genes to
chloroplast development (reviewed by Boerner and Sears
1986). Initially, such mutations were obtained mainly
through the isolation of spontaneous mutants (Kutzelnigg and
Stubbe 1974, Stubbe and Herrmann 1982). To obtain
additional plastid mutants, Epp (1972, 1973) tried to induce
chloroplast mutations in Oenotbera bookeri strain johansen
using irradiation and chemical mutagens including
ethylmethanesulfonate (EMS). Although, in immediate terms,
Epp was unsuccessful at obtaining chloroplast mutations, his
chemical mutagenesis experiments with EMS resulted in an M2
plant line with a high frequency of chloroplast mutations.
In this line, Epp was able to identify a recessive nuclear
gene which, when homozygous, induces a variety of non-
Mendelian mutations differing in phenotypic expression (Epp
1972, 1973). He called the nuclear gene plastome mutator
(pm). Once induced, the plastid mutations are not dependent
upon the presence of the nuclear pm gene. They are
inherited in a non-Mendelian fashion.

Although Epp discontinued his work with the plastome
mutator for about ten years, he sent seeds to Professor N.

Stubbe at the University of Duesseldorf who continued to

46
propagate the line. The pm plant lines used in Sears’ lab
were derived from Stubbe’s stocks.
Flowers on shoots which carried newly-arisen mutant

sectors were emasculated and used as the female parent in

crosses with wild-type plants. Such a cross places the
mutant plastids into a nuclear background which is
heterozygous for the pm gene. Heterozygous pm/+ progeny

lack the plastome mutator activity and thus provide a stable
nuclear background for the maintenance of the mutant
plastids. These seeds were then surface sterilized and
germinated on MS medium. From the resulting seedlings,
plant meristem cultures were initiated and mutant sectors
were selected and maintained on N.T.-agar medium. Initial
characterizations by Sears demonstrated that two mutants,
,pmfi’and p.11 appeared to have an altered BamHI restriction
endonuclease digestion pattern when compared to a wild-type
sibling (line Ca). Two chloroplast DNA fragments, Bam 3b
and 12 were identified as being different in size from the
wild-type fragments by approximately 200 bp. (Sears 1983,
Sears and Kaplan 1984). These observations led to the
proposal that the pm gene could have caused a major
rearrangement or transposition within chloroplast DNA. To
test this possibility, my initial project was to isolate the
chloroplast DNA fragments which differed between wild-type
and mutant plastids and to characterize them using

restriction endonuclease mapping.

47

3.2. RESULTS

3.2.1. CLONING.

The twelfth largest BamHI fragment (Bam 12) was one of
the fragments which was thought to have a different mobility
in mutant pal]. In order to amplify this DNA fragment it
was cloned. chNAs from the plant lines designated p.11 and
Cr, shown in Figure 2.1, using the protoplast method.

The Bam 12 fragment was purified from gels using the
crush and extract method described in chapter 2 and was.
inserted into pBR322. .E. coli strain HB101 host cells were
transformed using the calcium chloride procedure. Following
selection for the amp’, tet' phenotype, recombinant plasmids
containing the Bam 12 fragment were identified through
Southern hybridization as described in section 2.2.7.3. The
clone designations are shown in Table 3.1 and include a
clone from the chNA clone library which had been
constructed in the Sears laboratory from a wild-type 0.
johansen strain from the Duesseldorf collection. This line
shall be referred to as "Line 0” in this thesis. The insert
from plasmid pOjll9 from line D was nick-translated and was

used as the hybridization probe.

Table 3.1. Recombinant plasmids of BamHI fragments 3b and
12 and their origins.

Plasmid Bam 12 Plasmid Bam 3b
Plant Line Clone Name Clone Name
0 903119 pOjllB
Cl pOjo4 pOjo6
pmJI mell

Recombinant plasmid DNAs containing Bam 12 were isolated

using the physical shearing method.

3.2.2. COMPARISON OF RESTRICTION ENDONUCLEASE DAM 12

BETWEEN MUTANT p.11 AND ITS SIBLING MILD-TYPE LINE Cl.

Plasmids containing the Bam 12 fragments from mutant p11]
and line C1 were compared using digestions of BamHI combined
with one of the following enzymes: EcoRI, HindIII, Hinfl,
Tan and HaeIII (Figure 3.1). The double digestion with
BamHI was necessary in order to release the insert from the
plasmid and eliminate any differences in the restriction
endonuclease patterns which may have been due to the
orientation of the insert during ligation. Each one of the
bands representing the chNA insert appear to be identical

between the two clones (Figure 3.1).

49

Figure 3.1. Comparison of cloned Bam 12 fragments between
mutant p.11 and its sibling wild-type. Cloned Bam 12 insert
fragments of wild-type line Cl (pOjo4), mutant p.11 (me11)
and vector (V) plasmid pBR322 control were digested with
BamHI and one of the following enzymes: Hian, Tan, or
HaeIII. DNA was electrophoresed on 83 PAGE.

50

Hian Tan HaeIZZ'

 

I DWI pm]l pml
V CI H V Cl II V CI H

 

 

’ riga’re 3.1 I if"

51
3.2.3. Pm'MUTANTS FALL INTO TNO DISCRETE CLASSES.

In a collaborative project with Ellen Johnson, Linda
Schnabelrauch and Ruth Nolfson, chNAs from a number of pm
mutant lines were digested with restriction endonucleases
BamHI and HaeIII, electrophoretically separated on a 23
agarose gel, Southern blotted and probed with 32P nick
translated total chNA from wild-type line C1. One of these
autoradiograms is shown in Figure 3.2. Examination of the
bands indicated by arrows in the figure reveals that the
mutant chNAs fit into two discrete classes: Mutants pm?
and pmfl’do not resemble either wild-type line D or line Cl
particularly in the mobilities of bands 3b and 12. As
suggested by the restriction digestion of fragment Bam 12
described in section 3.2.2, mutant p.11 seems to be
identical to line Cr.

These results indicated that the chNA differences
observed previously were not correlated with the occurrence
of particular mutations. Rather, the existence of two
classes of chNAs suggests that chNA polymorphisms were
pre-existing among the descendants of Epp’s original pm
line. To compare chNA from mutant me’ with a wild—type
chNA representing this polymorphism group, it was first
necessary to identify and recover plants carrying the
appropriate wild-type plastome. To this end, seeds were

germinated to reisolate line Cz which had been used for the

52

Figure 3.2. .Plastome.flatator induced Plastome I mutants and
their sibling wild-types fall into two distinct classes.
BamHI digested chNA from wild-type lines D, Cr and from
plastome mutants 2.7, ,pm8, and p.11 was run on a 0.63
agarose gel. Plastome III chNA also digested with BamHI
was used as a comparison. DNA was Southern blotted onto
nitrocellulose and probed with 32P nick translated total
chNA from wild-type line D.

53

m
HI D 09% 2T0?

~23kb
- 9.4
E - 6.6
-
Itl I81
.uw
12:7: -4-3
lad
rd 05::
; 1:!!-

 

 

’”ri;u.. 3.2

54
initial studies conducted by Sears in Duesseldorf (refer to
Figure 2.1). It was hoped that these plants would be the
correct wild-type control since they resulted from self-
crossing a plant known to be a sibling of the plant on which
the p18 mutation arose (Figure 2.1). chNA from mutant p18
and wild-type line C: were isolated using the modified whole
cell DNA method. chNAs were digested with frequently
cutting enzymes (Figure 3.3A) The DNAs were blotted to
nitrocellulose filters and were hybridized using the
variable Bam 3b fragment as a probe (Figure 3.38). No major
alteration in chNA between mutant pma' and wild-type Cz
could be detected. Some degradation of chNA of wild-type

line Cr is evident (BamHI digestion in Figure 3.3).

55

Figure 3.3. Comparison of chNA between mutant pmfl'and its
sibling wild-type line C2 using restriction endonuclease
digestion and Southern hybridization. Panel A: Digestion
of n.8, wild-type line C2, and wild-type line C: with MspI,
HaeIII, and BamHI. DNA was electrophoresed on a 23 agarose
gel with marker 1 (M1). Panel B: Autoradiogram of the gel
from panel A using 32P nick-translated Bam 3b insert from
plasmid pOj118 as the hybridization probe.

56

 

Q0

0;

0d

ofi

06

ax_N

63 m .03 m .03 m.
Ea Ea Ea
:1 Sum 38: .22:

m 6 No m
Ea Ea

HI Eem Hoot

Ho so

     

6 «o m :2
Es

a. as:

Figure 3.3

57

3.3. DISCUSSION

Mutants me’ and meI, as well as the other mutants
resulting from the activation of the plastome mutator gene
have different phenotypes. In all of the mutants studied to
date, except for one, n.7, no specific lesions have been
identified. The experiments described in this chapter
indicate that chNA of mutant p.11 has the same restriction
endonuclease patterns as its sibling wild-type line C1.
This mutation p.11, therefore, is most likely the result of
a point mutation which does not give rise to a RFLP for the
restriction enzymes used in this study.

Mutant me’shows essentially the same chNA restriction
endonuclease patterns as wild-type line Ca. In Figure 3.3,
there is a hint that the third largest band hybridizing to
the probe has a slower mobility and thus a larger size than
the equivalent band of wild-type-line 02. However, careful
examination of the gel revealed that all of the bands in
lane 2 run slightly faster than the bands in lane 1. These
differences are thus .due to an artifact of the
electrophoresis and do not represent a physical
characteristic of the DNAs. Some factors which are known to
affect mobility of DNA in gel electrophoresis include:
Quality of the DNA, salt concentration and imperfections in
the gel (Sealey and Southern 1982, and Maniatis et a1.

1982). A comparison of cloned DNAs of this fragment from

58
mutant pm8'to its sibling wild-type line C2 was not possible
due to the difficulty of obtaining a clone of Bam 3b from
line C2 (discussed more extensively in chapter 4).

The discovery that our pm line contained two different
plastid types caused us to trace back a pedigree for the
origin of these plant lines (Figure 2.1). The pedigree
starts with a pm pm plastome I line derived from the
original E-15-7 plant described by Epp (1973). Seeds
resulting from self-crossing of this line were sent by Epp
to Stubbe (University of Duesseldorf) and were placed in the
field in two different years: 1976 (S 76/119a) and 1978 (#
78/311). The plants in both of these years were self—
crossed. It is not known how many plants were used in this
set of self-crosses in either season. Seeds from the
76/119a line were planted in 1980 and given the designation
80/959. Seeds from line 78/311 were planted in 1980 and
1981 and were designated 80/960 and 81/124 respectively.
The plants in this F2 generation, grown in two different
field seasons, gave rise to sectors containing mutant
plastids from which the Sears lab pm'mutants were derived.
It is this F2 generation to which we can trace back the two
different wild-type plastomes which we have observed.

However, since all of the plant lines can be traced back
to a single plant (Epp’s original pm pm plastome I plant) it
is possible that the two plastid types could have been
present from the start of the pedigree. It has been

demonstrated that different branches of the same plant may

59

contain different plastid types when plastome mutations
occur and sort out during subsequent cell divisions (Stubbe
and Herrmann 1982, Epp 1972, 1973). Possibly, the chNA
differences resulted from neutral mutations caused by the pa
gene or by the original EMS mutagenesis. The possibility
also exists that the two distinct plastid types appeared
first in the F2 generation resulting from chNA alterations
caused by the action of the plastome mutator. (Figure 2.1).
Because it is not known how many plants in lines 76/119a and
78/311 were used in self crosses to produce the F2
generation and because seeds for those plant lines are no
longer available, it is impossible to say at which point the
two different plastid types arose. Line Cl was recovered
from a limited number of seed packets from the crosses of
81/124. Each seed packet represents a different plant from
the field. With such a limited sample size, it would be
premature to conclude that the 81/124 line was
homoplastidic.

Wild-type line D is from a different stock maintained in
Duesseldorf for many years by Stubbe, but both the Cornell
and Duesseldorf lines have apparently descended from
Cleland’s original 0. johansen strain (Stubbe, personal
communication). The differences between chNAs of lines D
and C may be due to spontaneous variation, but it should
also be noted that line D had never been through a
mutagenesis treatment. As mentioned previously, mutagenesis

of the Cornell line could have caused the chNA alterations.

60
The remainder of the thesis shows the characterization of
the regions of variability in the cloned Bam 3a and Ben 12

from the wild-type chNAs.

CHAPTER 4
COMPARISON OF CLONED chNA RESTRICTION ENDONUCLEASE
FRAGMENTS BamHI 31) AND 12 FROM MILD-TYPE PLASTOME I LINES OF

OINOI'HIRJ HOOKER! STRAIN JOHANSEN

61

62
4.1. INTRODUCTION

The original intention of my thesis was to investigate
chNA alterations in plastome mutants of Oenotbera induced
by the plastome mutator gene characterized by Epp. Although
the characterizations described in chapter 3 indicated that
no major DNA changes could be correlated to the mutation
events, the restriction fragment analyses did suggest that
two DNA fragments in the BamHI pattern were variable.
Differences between chNAs of lines D and C may be due to
naturally occurring variation. The variation in fragment
sizes may be similar to that which is seen when Oenotbera
plastome types I - V are compared (Gordon et a1. 1982). The
purpose of this part of the study was to locate the BamHI
variable fragments on the physical map of the Genotbera
chNA, to characterize the areas of variability between the
wild-type lines Ci, Ca and D, and to investigate any

possible homology between the two regions.

63

4.2. RESULTS

4.2.1. CLONING.

Characterization of the areas of interest in the chNA
require workable amounts of DNA. The best way to obtain
sufficient quantities is by cloning. The cloning of Bam 12
fragment from wild-type line C1 has been described in
chapter 3. chNA from wild-type line Ca was prepared using
the protoplast method, and band Bam 12 was cloned from this
line also. The BamHI 3b band was also cloned as described
in chapter 2, using a modification of the Hanahan
transformation procedure. Bam 3b is larger than Bam 12 and
thus required a higher transformation efficiency. Clones
and subclones are listed in Table 4.1.

Plasmid DNAs were isolated using the physical shearing or
the chemical lysis method as described in chapter 2.

Table 4.1. Recombinant plasmids with chNA inserts from
Oenotbera johansen. The number given for the BamHI fragment
refers to the relative size of the band in the chNA
restriction pattern generated by the enzyme.

Plasmid Clone BamHI Plant
Name Fragment Line
pOjll9 12 D
pOjo4 12 Cl
pOejl 12 C2
pOj118 3b D

pOjo6 3b Cr

64

During the pursuit of the Bam 3b clones, colony
hybridizations were used to screen for the wild-type line Cl
clone, using the Bam 3b insert DNA from plasmid pOj118.
Following this initial screen, the identity of the correct
clone was confirmed by a comparison of BamHI + EcoRI
digestions of plasmid pOj118 and Southern hybridization.
The Bam 3b clone from line C: was obtained at a very low
frequency of one out of 3,000 recombinant colonies screened.
In contrast, Bam 3b did not appear to be difficult to clone
from line 0: Out of six clones carrying BamHI fragments of
this size (7.5 kb), one contained the 3b fragment, while
five carried the 3s fragment. It is not clear why the Bam
3b fragments of wild-type lines Cr and Ca have been
difficult to clone. Screening for the fragment Bam 3a was
carried out at the same time 3b was being sought. These two
fragments are the same size and were co-purified from
agarose gels. Colony screening for Bam 3a has in three
consecutive experiments consistently detected over 75_
positive signals per 300 colonies, whereas Bam 3b gave no
positive hybridization except to the positive controls. It
could be that the insert DNA may be in some way toxic to the
host bacterial cell. The DNA may have contained sequences
that are more recombinogenic in lines C1 and 02 than they
are in line D. In order to try to eliminate the possibility
of such a problem, rec A“ host B.co]i HB101 was used, but

without success. The problem also may be due to the quality

65
of the DNA (Maniatis et a1. 1982). This reason is unlikely
because of the relative ease with which other clones have

been obtained using the same procedures.

4.2.2. LOCATION OF THE BAMHI 3b AND 12 VARIABLE REGIONS ON

THE PLASTOME I chNA.

A physical map of chNA from 0. bookeri was constructed
by Gordon et a1. (1981, 1982) using the infrequently cutting
enzymes SalI, KpnI, and PstI. These enzymes create 10 to 13
fragments in Cenotbera chNA, while BamHI creates
approximately 65 fragments. Because BamHI creates so many
fragments, no complete BamHI map of chNA is yet available
for Oenotbera. Thus, in order to locate the Bam 3b and 12
fragments on the chNA physical map, it was necessary to
prepare a Southern blot having chNA digested with Sell,
KpnI, or PstI. .

The Bam 3a and 3b fragments had already been localized on
the chNA by Ellen Johnson. The Bam 3b probe prepared from
pOj118 hybridized to PstI band 1, Sell bands 5 and 7, and
KpnI band 5 as indicated in Figure 4.1. This result
demonstrated that Bam 3b is located within the large single
copy region.

In separate experiments, I hybridized the Ban 12 1.6 kb
subcloned fragment and a smaller piece of the subcloned 1.6

kb fragment (a 280 bp restriction fragment from pOj119a

 

66

Figure 4.1. Physical map of Oenotbera bookerj strain
johansen plastome I. Adapted from Gordon et a1 (1981,
1982), using chloroplast gene names proposed by Hallick and
Bottomley (1983) listed below.

Gene Protein

rbcL large subunit of RUBISCO

atpA alpha subunit of coupling factor

ath beta subunit of coupling factor

atpE epsilon subunit of coupling factor

ath I subunit of coupling factor

atpH proton-translocating subunit of coupling factor
petA cytochrome f

petB,D cytochrome b6, subunit 4 of cyt. b6f complex
psbA 32 kilo dalton herbicide binding protein
psbB 51 kilo dalton chlorophyll a binding protein

psaA PSI p700 chlorophyll a appoprotein

67

.mm.~u ax"

 

 

 

 

 

axmr.au a
$202 ”and czoL
m
m ~_om w
a
ac x m m
\ o_ m 1
NH Eom\l r a” 0" \NH Eom
m m a
m anoms.mmz m an
m_ m of
02..

m mm A" a.e

m3 \\a2

m
a m m r m
9. r
\\ m
Am pawn IWHVII g a
.....ll one
if
H wZOchnd

 

zmeGIOfi .mhm HanOOI .0

Figure 4.1

68
which defines the variable region) to a Southern blot of
total chNA from line Cl digested with BamHI, SalI, KpnI,
PstI, and MspI (Figure 4.2B). These two probes both
hybridized to Pat! bands 4 and 5, Sell bands 2 and 6, and
KpnI bands 1 and 2. Their positions are indicated on the
physical map (Figure 4.1). The SalI, KpnI and PstI
fragments which hybridized to the probes span the border
between the inverted repeat and the large single copy region
of the chNA molecule. Figure 4.2 also shows the
hybridization pattern of the probes to chNA fragments
produced by more frequently-cutting enzymes BamHI and MspI.‘
The band which hybridizes to the pOjll9 probe is double
molar. These results suggest that the entire Bam 12
fragment including the variable region is part of the

inverted repeat (Figure 4.1.).

69

Figure 4.2. Southern Hybridization of Bam 12 insert DNA to
restriction endonuclease digested Oenotbera plastome I

chNA. Panel A: Restriction endonuclease digestion of
chNA from line C1 digested with BamHI, SalI, KpnI, PstI,
and MspI. Samples were electrophoresed on a 0.63 agarose
gel and transferred to nitrocellulose. Panel B:

Autoradiogram of the gel from panel A probed with 32P nick
translated 280bp Hian fragment from line D (pOj119a).

70

9 N ID
5; - .
a "3 T "3
0:9
’4?- , O
100' - w“.
- \&E‘
«:9 ’05:- I“
o
6’— I

 

Figure 4.2

71
4.2.3. CONSTRUCTION OF A PHYSICAL MAP FOR THE DAM 12
FRAGMENT OF THE THREE MILD-TYPE PLASTOME I LINES USING

RESTRICTION ENDONUCLEASES.

In order to determine if the variation in fragment size
'was due to creation of new restriction endonuclease sites,
discrete insertion/deletion events, or a dispersed
alteration of sequences, restriction endonuclease digestion
experiments were performed utilizing the cloned DNA
described in Table 4.1.
4.2.3.1. Preliminary Mapping of the Entire 3.0 kb BamHI 12
Fragment.

Since the insert in plasmid pOj119 is the largest of the
three, it was used in preliminary screening of restriction
endonucleases. The first set of restriction endonucleases
which were tested recognize 6 bp sequences and generate a
small number of bands (0 to 3) from the vector pBR322. This
helped simplify the initial mapping process and allowed me
to begin to define a more discrete region of variability.
Since the insert chNA was cloned into the BamHI site of
pBR322, plasmid pOj119 was first digested with BamHI in
order to release the insert from the vector, and was then
digested with one of each of the restriction endonucleases
shown in Table 4.2. Those enzymes which were shown to
digest the pOj119 insert DNA were then used in digestion

experiments of the line Cl Bam 12 insert plasmid DNA.

72'

Table 4.2. Restriction endonucleases tested for digestion
of the Bam 12 insert DNA. Enzymes which showed no digestion
of the insert were tested for and demonstrated activity on
phage lambda DNA.

Restriction O of insert
endonuclease fragpents
EcoRI 3

Clal 3

BglII 2

PvuII no digestion
NruI no digestion
KbaI no digestion

XhoI no digestion

73
(The Bam 12 fragment of Line C2 was in the process of being
cloned and was not yet available for comparison at this
point.) Only one sub-fragment differed in mobility in every
comparison (Table 4.3).

These results indicated that a discrete region of the Bam

12 fragment varied between the two lines. To facilitate
fine-mapping with enzymes which cut more frequently, I
decided to subclone the region of variability.

The variable region is contained entirely within a 1.6 kb
fragment liberated during the BamHI/EcoRI digestion. The
entire Bam l2 fragment contains two EcoRI sites. In order
to be able to determine the cut sites which define the 1.6
kb subfragment, it was necessary to find the orientation of
that fragment within the insert. To determine if it is
internal (defined by two EcoRI sites) or if it is located at
an end of the insert (defined by one EcoRI site and one
BamHI site).

In order to be able to define these fragments, a series
of end-mapping experiments was carried out. End-mapping
experiments take advantage of the known locations of
restriction sites on the vector DNA since the entire vector
has been sequenced (Maniatis et a1. 1982, BRL reference
catalogue). The restriction site for EcoRI on plasmid
pBR322 is located at base 0 (4362). The single BamHI site
is located at base 376. Insertion of the BamHI chNA

fragment into the vector results in a duplication of that

74

Table 4.3. Single and double digestions of Bam 12 inserts
from pOj119. A 3 indicates the band which contained the
variable region when compared to the same digestions of
pOjo4.

Restriction 3 of approximate
endonuclease fragments size (kb)

EcoRI 3 1.603
0.95
0.75

ClaI 3 1.503
1.40
0.10

BglII 2 1.803
1.40

.403
.95
.56
.19
.10

EcoRI/ClaI 5

OOOOH

.403
.40
.20
.10

BglII/Clal 4

OOHH

.603
.95
.60
.15

EcoRI/BglII 4

GOOD-0

75
single BamHI site. Using this information, and combining it
with restriction endonuclease digestions, the orientation of
the insertion and the definition of the and fragments could
proceed.

Plasmids pOj119 and pOjo4 were digested with EcoRI alone.
Fragments located at the end of the insert,would have pBR322
DNA attached. Determination of the orientation of the ends
was possible because of the uneven amount of pBR322 DNA on
either side. The left and would have an additional 376 bp
of pBR322, the right and would have about 4000 bp. The
plasmids pOj119 and pOjo4 were also digested with both EcoRI
and BamHI for comparison. The results are shown in Table.
4.4. The resulting map is shown later in the chapter in
Figure 4.8, panel A.

The variable region is contained within a 1.6 kb
EcoRI/BamHI fragment which was convenient for subcloning
using pBR322. Subcloning was necessary to simplify the fine
mapping of the variable region. The BamHI 12 1.6 kb
subfragments were subcloned into plasmid pBR322 as described
in chapter 2. The subclones use the same clone name with

the addition of an "a” (pOj119a, pOjo4a, pOejla).

76

Table 4.4. End mapping experiments. Plasmids pOj119, pOjo4
were digested with EcoRI or both EcoRI and BamHI for
determination of the orientation of the variable region and
the and fragments. A 3 indicates the region which varies in
size when pOj119 and pOjo4 are compared. In the double
digestion, vector pBR322 bands are indicated by parentheses.
Fragments which change in size and hence are located at the
ends are underlined. L = left end, R = right and. (Figure
4.8,A).

restriction 3 of approximate
endonuclease fragments size (kb)
EcoRI 3 5.70 R 3

1.27 L

0.70
BamHI/EcoRI 5 4.00 (pBR322)

1.60 R 3

0.90 L

0.70

0.376 (pBR322)

77
4.2.3.2. Fine mapping of the variable region of Bam 12.
Insert DNA was isolated from the plasmids pOj119a,
pOjo4a, and pOejla and was digested with a number of
restriction endonucleases (summarized in Table 4.5).

Size differences were estimated for the three variable

reSions by a digestion of the subclone plasmid DNAs with
HincII, shown in Figure 4.3. In order to accurately measure
the differences between the DNAs, digestion products were
compared to size standards run on the same gel. These
differences were estimated to be 95 bp between line D and
line Cr, 76 bp between line C1 and 02, and 170 bp between
line D and C2. The HincII digestion also allowed
localization of the single HincII site of the 1.6 kb insert
on the physical map (Table 4.6) using the same reasoning as
in the plasmid DNA end-mapping experiments.

The smallest restriction endonuclease fragment containing
the variable region in pOj119a (line D) is a 280 bp Hian
fragment shown in Figure 4.4. The corresponding bands were
calculated to be approximately 185 bp in pOjo4a (line Cl and

109 bp in pOejla (line 02).

Table 4.5. Single digests of the 1.6 kb insert of subclone
pOj119a. An 3 indicates the variable fragment; no digestion
indicates lack of restriction site for that enzyme within
the insert DNA. The activity of enzymes which did not out
the insert DNA was confirmed by digesting plasmid pBR322
DNA.

restriction 3 of approximate
endonuclease fragments size b

Hian 11 443
2803

180

160

138

104

72

4(50

Tan 10 6103
295

240

178

135

110

4(50

MaeIII 4 6203
460
320
180

RsaI 2 15003
115

ClaI 2 15153
100

HincII 2 10503
' 550

DraI no digestion
HaeIII no digestion
Mspl no digestion

ScrfI no digestion

79

Figure 4.3. Restriction endonuclease digestion and agarose
gel electrophoresis of plasmid DNAs from pOj119a, pOjo4a,
pOejla enabling the calculation of the sizes of the BamHI 12
subclone variable bands. Plasmid DNAs containing the 1.6kb
Bam 12 subfragments from the three wild-type lines D
(pOj119a), Cr (pOjo4a), Ca (pOejla) and pBR322 (P) were
digested with DraI or HincII. The DNA was electrophoresed
on a 0.83 agarose gel with markers 1 (M1) and 3 (Ma).

80

Dra I ' Him: 12'

 

F IF l
M, 0 0.02:3 0 c.02P M3

 

Figure 4.3

81

Figure 4.4. Restriction endonuclease double digestions of
the Dam 12 1.6kb subclones. 1.6kb insert DNA from wild-
type lines D (pOj119a) C1 (pOjo4a) and C2 (pOejla) were
digested with Hian (H), Hian + AluI (Al) and Hian + MboI
(Mb) and electrophoresed on 103 PAGE with markers 3 (Me) and

4 (Me).

82

H H+Al H+ Mb
M3? 0. 02"0 c. cg '0 c. 02'M4

 

Figure 4.4

83

As summarized in Table 4.5, digestion of the 1.6 kb
inserts with Hian revealed at least 11 bands (Figure 4.4).
Digestion with Tan revealed at least 10 bands (Figure 4.5).
Digestion with MaeIII revealed 4 bands (Figure 4.6) and
digestion with RsaI revealed 2 bands (Table 4.5).‘ To begin
assigning map locations to the subfragments, a series of
end-mapping experiments was conducted. These experiments
were based upon the same reasoning as the end-mapping
experiments of the entire 3.0 kb insert with the following
modifications. Because the enzymes used in the fine mapping
experiments generate many fragments in the vector as well as
in the insert, a large portion of the vector was physically
removed by selecting a fragment after digestion of the
plasmids with Sell and PstI followed by gel electrophoresis.
These two enzymes, which each cut the vector once without
cutting the insert, liberate the insert DNA with vector DNA
on both sides (refer to Figure 4.8, panel B). The EcoRI end
has an additional 748 bp attached and the BamHI end has an
additional 276 bp of pBR322 DNA attached. When this DNA is
subsequently digested with another restriction endonuclease
and compared to insert DNA lacking the vector DNA ends, but
digested with the same enzyme, two fragments should differ
in size. This difference should correspond to the original

subfragment of the insert plus the piece of attached vector

84
DNA. The amount of attached DNA is calculated using the
published information containing the location of restriction
sites in pBR322 (Maniatis et a1. 1982, BRL reference
catalogue).

This set of end-mapping experiments was performed using
the enzymes Hian, Tan, MaeIII, and RsaI. Results of these
digestions are shown in Table 4.6. Figure 4.5 shows a gel
of the Hian and Tan digestions. The bands with altered
mobility were identified and then assigned an end position
on the map (Figure 4.8, panel B).

End cut sites were assigned for MaeIII and RsaI in the
same manner. These bands were also placed on the map, as
shown in Figure 4.8, panel B.

The end-mapping digestions with Hian and Tan
demonstrate the bands at the ends do not contain the
variable region, since the variable band does not change its

size whether insert alone or insert plus vector ends is cut.

85

Table 4.6. End mapping for comparison of the 1.6 kb inserts
from all three lines with and without attached vector DNA.
The inserts alone and the inserts plus pBR322 SalI and PstI
vector ends were digested with the enzyme indicated to yield
a number of fragments. Sizes are shown for fragments from
pOj119a. Bands from vector pBR322 are indicated by
parentheses. A 3 indicates the variable band and underlined
bands indicate fragments located at an end. L = left side
and R = right side of the physical map (Figure 4.8, panel
B).

Restriction aprox. size aprox. size
endonuclease 1.6 kb PstI/Sell
Hian 19_ R 999 L
280 3 999 R
999 L 280 3
138 138
104 104
72 72
4<50 4<50
Tan 6103 6103
295 999 L
240 400 (pBR322)
13131: $79.11
135 295
199_R 240
135
4<50 4<50
MaeIII 999 L 3 999 L 3
460 999 R
999 R 460
180 205(pBR322)
180
170(pBR322)
RsaI 1500 L 3 1800 L 3
11.5.3 .6223
240(pBR322)
HincII 1050 R

 

86

Figure 4.5. Restriction endonuclease digestions of Bam 12
1.6kb subclone inserts to define the and fragments. 1.6kb
insert DNA (i) and insert DNA with the asymmetric vector
ends (v) from the three wild-type lines D (pOj119a), Cr
(pOjo4a), and C2 (pOejla) were digested with Hian (H), Tan
(T) or both and run on 103 PAGE with marker 2 (M2).
Fragments located at the ends of the insert in one Hian
digestion experiment and one Tan digestion experiment are
indicated by arrows. Bands which are the result of partial
digestion are indicated with ”p”’s.

87

D C C

 

2
'HHTHIVHHTHllH HTH1
T T T T T T
r—l.l—.l'. Il—ll .Iml. IMF—I l.—ll. ”—1
VI lIVVVI IIVVVII VMz

 

 

Figure 4.5

88

The Tan digestions were plagued with partial digestion,
which occurred more frequently than with the other enzymes.
Partial digestion is indicated by the appearance of faint
bands in the gels (Figure 4.5 and Figure 4.6). These
partial bands made the Tan digestion difficult to
interpret. Tan is sensitive to methylation when the
guanosine in the recognition site is methylated, TCGl°ATC
(Manitis et a1. 1982 and BRL techline, personal
communication). Although chNA is not methylated,
restriction endonuclease digestion experiments were
performed on cloned chNA which had been isolated from Me+
bacterial cells. -

One particular Tan partial band appeared in every digest
to different degrees, and is indicated with ”p”’s in Figure
4.5. This band is approximately 110 bp smaller than the
largest band. By comparing this band with the other two
subclones, I could determine that this partial band contains
part of the variable region (compare Figure 4.6 Tan
digestions below band 1). This partial band indicates the
presence of a Tan site 110 bp from one side of the largest
Tan fragment, probably on the right side of the variable
region (Figure 4.8 panel B), because double digestion with
Tan and MaeIII did not cut this 500 bp fragment (Figure
4.6).

Using the information generated from the end—mapping

experiments, restriction sites were assigned to the physical

 

 

89

Figure 4.6. Single and double digestions of the Bam 12
1.6kb insert DNAs. Cloned 1.6kb insert DNAs from wild-type
lines D (pOj119a), Cr (pOjo4a), and 02 (pOejla) were
digested with Tan (T), MaeIII (M)), HincII (Hc), or
combinations of the enzymes. DNA was electrophoresed on 83
PAGE with markers 2 (M2) and 4 (M4).

90

D C/ Cg
l T M lfi' M II T M l
M4T M MHchT M MHchTMMHchM4M2

 

 

 

Figure 4.6

91

map (Figure 4.8, panel B). Analysis with these enzymes
showed that the variable region is contained within a 620 bp
MaeIII fragment which was mapped to the EcoRI end of the 1.6
kb insert. Hian produced too many restriction fragments to
allow for direct placement of the 280 bp variable fragment
on the physical map. Assignment of this fragment was then
undertaken in a more indirect manner.

The next set of mapping experiments was done with double
digestions using combinations of Hian, Tan, HincII and
MaeIII. This allowed determination of the order of some of
the Hian, Tan and MaeIII fragments. Figure 4.6 shows
digestions using combinations of MaeIII, Tan and HincII.
Examination of these digestions shows that HincII, which
cuts the insert only once has its restriction site within
the largest MaeIII band. In the MaeIII/HincII double
digestions, the 620 bp MaeIII fragment is cleaved into
fragments of 475 bp and 145 bp. A comparison of the same
digestions of all three subclones (compare between lines 0,
C1, and Cz of Figure 4.6) reveals that the region of
variability is located on the larger of these subfragments.

Double digestion experiments with Hian and HincII are
shown in Figure 4.7. It was hoped that since HincII digests
the 1.6 kb fragment only once, a double digest with HincII +
Hian would be able to show which of the Hian fragments
contains the HincII site. That result could have helped in

the assignment of the 280 bp Hian fragment on the map.

 

92

Figure 4.7. Single and double digestions of the Bam 12
1.6kb insert. 1.6kb insert DNAs from wild-type lines D
(pOj119a), C1 (pOj4a), and 02 (pOejla) were digested with
Hian (H), HincII (He), or both, and electrophoresed on 103 4
PAGE with Markers 2 (M2) and 4 (M4).

 

93

D C, 02
l pg’IIll +1 I l +1 ”l
M2 M4H Ho Ho H Ho Ho H Hc Hc M4

lfiz

 

*piQ... 4.7

94

However, the bands in the Hian and Hian + HincII lanes of
Figure 4.7 looked identical. It could not be determined
which Hian fragment is cut with HincII. Examination of
recognition sequences of the two enzymes revealed
overlapping cut sites. It is possible, then, that the right
Hian border of the variable region also contains a HincII
site. (This is considered more thoroughly in the
discussion.)

On the left side of the variable region, Hian and Tan

sites map very close to each other. Examination of Hian
and Tan° recognition sequences also have revealed
overlapping cut sites. The variable region, therefore is

located between a 280 bp Hian/Tan, Hian/HincII fragment.
In an attempt to cut into the variable region, double
digestions using Hian with either AluI or MboI were
performed. Figure 4.4 shows that these two additional
enzymes did not cut into the variable region. Since MboI
and AluI did not cut into the variable region, no further

mapping using these enzymes was pursued.

95

Figure 4.8. Restriction endonuclease map of the cloned
BamHI 12 fragment. Restriction endonuclease map generated
from restriction endonuclease digestions is shown in two
parts. Panel A: Map of the BamHI 12 3.0kb cloned region.
Panel B: map of the 1.6kb variable region with bordering
pBR322 DNA from the PstI site on the left and from BamHI to
its SalI site on the right. The variable regions are shown

as blocked areas. The Tan site determined by partial
digestion is indicated in parentheses. The main map is of
DNA from pOj119 and pOj119a. Corresponding areas of

variation are shown as blocks below the variable region of
pOj119 and pOj119a. All other areas are of the same size in
all three plasmids. An asterisk indicates a restriction
site with two possible locations.

 

96

23000 L

...... I.
.3 u a .0: .23. .l.

s s
..w as

 

   

 

 

Figure 4.8

97
4.2.4. CONSTRUCTION OF A PHYSICAL MAP OF BAM 3b DNAs FROM

PLASMIDS pOj118 AND pOjoS USING RESTRICTION ENDONUCLEASES.

Since the insert DNA of plasmid pOj118 appeared to be
larger than the insert DNA from pOjoB, insert DNA from
pOj118 was used in preliminary screening of restriction
endonucleases. The first set which was tested consisted of
6 bp recognizing enzymes which generate a small number of
fragments from vector pBR322 DNA. The insert DNA was
released from the vector by initial digestion of the plasmid
with BamHI. The DNA was then digested with one of each of
the restriction endonucleases shown in Table 4.7.

Those enzymes which were shown to digest the pOj118
insert DNA were subsequently used in digestion experiments
of insert DNA isolated from plasmids pOj118 and pOjo6.
Table 4.8 lists the endonucleases used, the number of
fragments they generated and the sizes .of pOj118 and pOjoB
fragments. In the comparison of the two clones, which
represent two of three ‘of the plant lines compared
previously, the initial digestions revealed two nearly
compensating variable regions within the Bam 3b fragment.
These regions have been designated "A” and ”B”. "A" is
contained within a 2.0 kb BamHI/EcoRI fragment and is larger
in pOj118, (line D). Region ”B” is contained within a 1.4kb
BglII(HindIII)/BglII fragment and is larger in pOjoS (line

01).

98

Table 4.7. Restriction endonucleases tested for digestion
of the Ben 3b insert DNA. Enzymes which showed no digestion
of the insert were tested and confirmed for their activity
on phage lambda DNA.

 

Restriction 3 insert
gpdongclease fragpents

Ach 4

BglII 3

EcoRI 7

HincII 3

HindIII 4

NruI 2

Sell 2

SphI 2

XbaI 2

Bell no digestion
ClaI no digestion
NdeI no digestion
SacI no digestion
SmaI no digestion
SstI no digestion

Xhol no digestion

99

of Bam 3b

 

 

 

Table 4.8. Digestions inserts from plasmids
pOj118 and pOjo6. "A" indicates one of the variable
regions, "B” indicates the other variable region.
Restriction. 3 of approx. size aprox. size
endonuclease fragments pOjll8 (kgl pOjo6 (kb)
Ach 4 4.00 A 3.90 A
1.90 B 1.95 B
1.80 B 1.85 B
0.45 0.45
BglII 3 5.00 A 4.90 A
1.40 B 1.45 B
1.00 1.00
EcoRI 7 2.10 A 2.00 A
1.85 1.85
1.10 B 1.15 B
0.94 0.94
0.72 B 0.74 B
0.61 B 0.62 B
0.55 0.55
HincII 3 2.90 B 2.95 B
2.70 A 2.60 A
1.90 B 1.95 B
HindIII 4 2.75 2.75
2.50 A 2.40 A
2.40 B 2.45 B
0.41 0.41
NruI 2 5.20 B 5.25 B
2.00 A 1.90 A
SalI 2 6.00 A 5.90 A
1.90 B 1.95 B
SphI 2 4.40 B 4.45 B
3.00 A 2.90 A
XbaI 2 6.10 A 6.00 A
1.40 B 1.45 B

100
Digestion with other enzymes used in this study generated
more than one fragment containing variable region B.

In order to determine the orientation of the variable
regions within the Bam 3b fragment, a series of end-mapping
experiments was performed using insert DNAs compared with
whole plasmid DNAs digested with Ach, EcoRI and HindIII.
Figure 4.9, lanes 2 - 5 shows end-mapping digestions using
EcoRI. Table 4.9 lists the sizes of pOj118 DNA sub-
fragments and the assignment of end fragments to the map in
Figure 4.11.

The end-mapping experiments demonstrated that the two
‘variable regions are located at opposite sides of the Bam 3b
fragment approximately 3.6 kb apart from each other. The
two regions have opposing types of alterations. This made
it possible to assign restriction sites for the other
endonucleases without the need for further end-mapping
experiments.

The digestions with enzymes SalI, Accl and HincII, (Table
4.8) all produced a 1.90 kb band which is larger in line C1.
Comparison of SalI, Ach and HincII recognition sequences
revealed that the sequences may overlap. The Ach 1.9 kb
restriction site was mapped to the right end of the Ben 3b
insert (Table 4.9) SalI only cuts the 3b fragment once and
defines the two variable regions. The 1.9 kb SalI fragment
is larger in line Cl and contains region B and thus

corresponds to the 1.9 kb Ach fragment.

101

Table 4.9. End-mapping for comparison of the Bam 3b inserts
from lines D (pOj118) and Ci(pOjo6). Plasmids containing
the Bam 3b inserts and the Ben 3b inserts alone were
digested with the enzyme indicated to yield a number of
‘fragments. Sizes are shown for pOj118 DNA. Bands from
vector pBR322 alone are indicated by parentheses. .End
fragments are underlined. L = left side of the insert, R =
right side of the insert. A indicates one variable region, B
indicates the other. See map, Figure 4.11.

Restriction aprox. size approx. size
endonuclease Bam 3b insert whole plasmid
Ach 4.00 A 4.00 A
1.90 R B 3.20 R B
1.90 B 2.40 L
0.45 L 1.90 B
l.60(pBR322)
EcoRI ' 2.10 L A 4.40 R
1.85 2.45 L A
1.10 B 1.85
0.94 1.10 B
0.72 B 0.94
0.61 B 0.72 B
0.55 R 0.61 B
HindIII 2.75 5.60 R B
2.50 L A 2.75
2.40 R B 2.74 L A

102

Figure 4.9. Restriction endonuclease digestions of the Dam
3b 7.5 kb inserts and plasmid DNAs. Plasmid DNA containing
the cloned Bam 3b insert was isolated from wild-type lines D
(pOj118) and C1 (pOjo6). The four lanes indicated as
digested by EcoRI show isolated insert DNA alone (i) or the
insert still contained within the plasmid vector (v). The
two lanes indicated. as digested with BglII contain
subfragments isolated from these two cloned DNAs as follows:
Insert DNAs were digested with HindIII, and the 2.4 kb
double bands which contain insert DNA of both ends of the
7.5 kb fragment was purified and digested with BglII. Bands
located within variable region A are indicated by "a”, bands
located within variable region B are indicated by "b”. DNA
was run on 13 agarose with markers 1 (M1) and 3 (Me).

103

Eco RI BglII
0 011.7 CI .097
M' v v I I I I M3

m ..r
'¥ '

       

Figure 4.9

104

As discussed in section 4.3, later in this chapter,
HincII will always recognize Sell out sites. Thus, the cut
sites of SalI and HincII which both delineate region B can
be placed at the same location. Using this information, in
conjunction with that of the location of the variable
regions, the remaining HincII sites were placed on the
physical map (Figure 4.11).

HindIII cuts the Bam 3b inserts into four fragments
(Table 4.8). The location of the end fragments has been
described above. Location of the two internal HindIII sites
was accomplished by single and double digestions of the
insert DNAs with HincII and HindIII. Figure 4.10 shows the
results of the digestions. The internal 2.75 kb band of
HindIII is not cut with HincII, whereas the other internal
HindIII band of 0.41 kb is cut by HincII. The 2.7 kb
HincII band located within region A is cut with HindIII.
This result enabled the mapping of the 0.41 HindIII band
next to the 2.4 kb HindIII band in region A. The sites for
the restriction endonucleases are indicated on the physical
map (Figure 4.11). Other restriction sites were placed on
the map in Figure 4.11 in the same manner.

Another method was used to better define the variable
regions of the 3b fragment. Digestion with HindIII
revealed two bands of approximately 2.4 kb located at the
ends of the 3b fragment (Table 4.9 and Figure 4.10). These

two HindIII fragments contained the variable regions.

105

Figure 4.10. Single and double digestions of the 7.5kb
BamHI 3b insert. Cloned Bam 3b 7.5kb insert DNAs from wild-
type lines D (pOj118) and C1 (pOjo6) were digested with
HindIII (HIII), HindIII + AchI (Ac), HindIII + HincII (Hc),
and HindIII + SphI (Sph). The DNA was run on a 0.83 agarose
gel with markers 1 (M1) and 3 (Ma). Bands which are the

result of partial digestion are indicated with ”p"’s.

106

HH Hm HM’
Hm Ac Hc Sph

rfil jl Wlfi
mqoqoqoqofi
m

— 7‘? \Y: F '1 ?~
\~.‘.-\~» ‘_|.-

 

 

\
p..--
-‘

Figure 4.10

107

The two bands were isolated from a gel and digested with a
number of enzymes including BglII. Figure 4.9 shows the
result of a digestion of both 2.4 kb HindIII subfragments.
Region B is shown to be contained within a 1.4 kb BglII
fragment. Since the sites for BglII and HindIII map close
together, it is not known at this point which enzyme best
defines the left side of region B (refer to Figure 4.11).

To summarize, the Bam 3b fragment contains two regions of
variability. The size variations of these two regions
nearly compensate for each other. Region A is contained
within a 2.0 kb BamHI/ EcoRI fragment and can be further
defined by Ach which generates a 1.65 kb fragment with an
EcoRI end. Region B is 3.6 kb away from region A and is
defined by a 1.4 kb BglII(HindIII)/ BglII fragment (band 2
of the BglII digestions in Figure 4.10). Region A is larger
in pOj118 by approximately 100 bp, region B is larger in

pOjoS by approximately 50 bp.

108

Figure 4.11. Restriction endonuclease map of the cloned
BamHI 3b 7.5kb inserts. The restriction endonuclease map
was generated from restriction digestions. The main map is
of pOj118 insert DNA. Corresponding areas of variation are
shown as blocked areas below the variable regions. All
other regions map the same in both plasmid inserts.
Asterisks indicate restriction sites which have two possible
locations.

109

infloow

.aaFiiiJ

 

 

sumouw
e e

 

3m

Algom

mx D.~IlL
J

 

Figure 4.11

110
4.2.5. Comparison of DNA in the variable regions of Bam 3b
and Ben 12.

Since both the Bam 3b and Bam 12 chNA fragments differed
among the wild-type lines by small changes which seem to be
due to discrete insertions or deletions, it was necessary to
determine if the variable regions shared any DNA sequences.
Preliminary evidence from colony hybridizations indicated
that the two regions are not homologous.

The polyacrylamide gel in Figure 4.12 shows insert DNAs
which were digested with Hian and Tan. Replica samples
were digested and separated on a 43 Nusieve agarose gel,
followed by transfer to a nitrocellulose filter and Southern
hybridization of the DNA using the 280p variable Hian
fragment from pOj119 as the probe. No hybridization of the
probe to the BamHI 3b insert DNA was detected, whereas
hybridization to DNA from pOj119a and pOjo4a did occur (data
not shown).

These data indicate that no direct homology exists
between the two BamHI variable regions in the chNAs of the

wild-type lines examined.

111

Figure 4.12. Polyacrylamide gel electrophoresis of
restriction endonuclease digested Bam 3b and Ram 12 inserts.
Insert DNAs from wild-type lines D (pOj118, Bam 3b and
pOj119a, Ben 12) and C1 (pOjoS, Bam 3b and pOjo4a, Ben 12)
were digested with Hian. 7.5kb insert from pOj118 and
pOjo6 were also digested with Tan. DNA was run on 83 PAGE

with marker 3 (Ma).

112

Bam3b Bam/Z Bam3b
Hinf I Tan

M30, 0c. 00, 0

 

 

Figure 4.12

113
4.3. DISCUSSION
Restriction endonuclease mapping analysis of the two
variable BamHI fragments from chNA revealed small
differences between plastome I chNAs of Oenotbera bookeri

strain johansen wild-type lines D, C1, and Cz. Southern

hybridization experiments allowed for the localization of
these bands on the physical map of the chloroplast genome.
Bam 3b is located in the large single copy region in the
vicinity of the atpA, atpH and ath genes (Figure 4.1). Ben
12 is located within the inverted repeat at or near the
junction of the large single copy region (Figure 4.1).

The physical mapping of the variable fragments described
above has allowed the identification of more specific sites
of variability within those larger fragments. The results
of fine mapping the Bam 12 fragment are illustrated in
Figure 4.8, while a preliminary map for the Bam 3b fragment
has been compiled in Figure 4.11.

The enzymes Tan and Hian each cut the Bam 12 fragment
into about ten subfragments. Although the fragments at the
ends and in the variable region could be mapped, the exact
cut sites for the others have not been determined. At the
left border of the variable region, each enzyme has one cut
site very near the other. In fact, Hian and Tan have
recognition sequences which could overlap in the following

ways:

114

EinfT'GlANTC overlaps with Fe I'TlCQA

Hian'GlANZQ overlaps with Fan'TlCGA

If the DNA of this region had one of the above overlapping

sequences, initial digestion with Hian would eliminate the
Tan site, thereby making the site unavailable to the
enzyme. The second enzyme might not be able to bind and
make a cut so close to the end, but even if the second
digestion was successful, initial digestion with Tan would
leave a Hinfl cut site only one base pair from the end of
the fragment. This difference in fragment size would not be
detectable on 103 PAGE. As mentioned in the results section
(4.2.3.2.), Hian and HincII also have recognition sequences
which may occur in very close proximity. The recognition

sites are:

GlANTC HIan

0r(r or C)l(A or G)AC Erncrr

At least two possible sequences could create overlapping

restriction sites of these two enzymes:

arnfv' GlAGTC A(G)C

0 107011400 HincII

115

.EIncII'GTClG acrc

GTC GlACTC Hian

The larger BamHI fragment also provided a challenge to
restriction mapping. Three enzymes, Ach, HincII and Sell,
gave a fragment of 1.9 kb as indicated in Table 4.8 and on
the restriction map (Figure 4.11). Examination of the
recognition sequences for these enzymes indicated that they

have overlapping recognition sequences.

911(A or 9)(9 or T)99 Ach
g1(r or g)1(a or g);g aancrr
alrccac 5211

The regions of variability within the Bam 3b and Bam 12
fragments do not show any homology when examined using
Southern hybridizations. The regions which contain these
fragments (near the border of the inverted repeat for Bam 12
and near the ATPase genes of the large single copy region
for Bam 3b) have been shown to be sites of rearrangements
within the chloroplast genome of higher plants (Palmer
1985b, Mubumbila et al. 1984). These DNA rearrangements
are seen when chNAs from spinach, lettuce, legumes and the
anagraceae are compared.

Possibly, as more sequence data become known, including

DNA sequences from the areas of variation of the Oenotbera

116
chNA which have been mapped in this study, it will be
possible to find characteristics such as secondary
structures of the DNAs which may lend themselves to
rearrangements.

The types of alterations seen in cloned chNA from the
wild-type lines in this study suggest that
insertion/deletion events have occurred in the chNA of
Oenotbera. Similar chNA alterations have been recognized
in broader comparisons among different species within a
genus: Oenotbera, subsection Euoenotbera (Gordon et a1.
1982), Oenotbera subsection Mhnzia (von Stein and Hatchtel
1986) Bpilobium, (Schmitz et a1. 1986), Triticum and
Aegilops (Bowman et a1. 1983), Brassica (Palmer et al.
1983), legumes (Michalowski et a1. 1987), and Zea (Doebley
et a1. 1987). Some of these studies have led to the
observation that the insertions/deletions in chNA occur
more frequently near the junction of the large single copy
region and the inverted repeat region, and that these
junctions appear to be more subject to change than is the
rest of the genome (Mubumbila et al. 1984, Palmer 1985b,
Nhitfeld and Bottomley 1983 and Gordon et a1. 1982).

The insertions/deletions which were detected in the
Oenotbera plants in this study are small (approximately
100 bp or less). This observation is consistent with the
size, nature and location of the insertion/deletions which

have been described between Oenotbera plastome types I -V

117

(Gordon et a1. 1982) and for some of the other
insertions/deletions which have been found in other plants
also by restriction endonuclease analysis. Restriction
endonucleases rarely detect point mutations because they
recognize only a small subset of bases, making the
probability of detecting a point mutation very low. Thus,
the occurrence of point mutations cannot be ruled out by the
studies described here. However, point mutations cannot be
the cause of the differences which have been characterized
here since each restriction endonuclease tested gives an
identical number of fragments in all of the cloned chNAs
tested, and each one shows discrete and consistent
alterations in fragment sizes. Before restriction
endonuclease mapping and DNA sequencing techniques were
available to test the possibility, Kutzelnigg and Stubbe
(1974) hypothesized that the genetically-defined plastome
types might be further subdivided.

The differences which have been seen between chNAs occur
between wild-type lines of Oenotbera bookeri strain johansen
plastome I. Two of these wild-type lines were isolated
following Epp’s EMS mutagenesis experiments (Figure 2.1).
Thus, we do not known if the differences in the chNA are
the result of natural events or if they were caused by the
EMS mutagenesis. EMS is a chemical mutagen which is known
to produce mainly point mutations in chromosomes of

Drosopbjla (Grigliatti, 1986). These point mutations are

118

usually in the form of transitions from CC to AT in
prokaryotic DNA (Kreig, 1963), or G0 to AT and AT to G0 in
eukaryotes (Auerbach, 1976). Studies of base-specific
mutagens of nuclear genes on tomato have shown that EMS
primarily affects GC base pairs (Jain and Rent, 1966).
Areas of chromosomes rich in AT sustain less damage than do
regions containing more 60. A possible explanation of the
changes in chNA of lines C1 and 02 is that EMS is able to
cause small deletions or insertions in chNA.
Alternatively, initial treatment of the plants with the
mutagen may have introduced some GC to AT changes in the
chNA. The altered base composition of the region may have
been changed enough to create rearrangement "hot spots"
thereby making further insertions/deletions possible. One
way to start investigating the possibility is to see if
other Oenotbera plastome I species such as Oenotbera bookeri
and 0. slate contain small insertion/deletions in their
chNAs. Another test may be to perform mutagenesis
experiments and look for small changes compared to untreated
lines over time. An EMS treated line may undergo
alterations at a more accelerated rate.

Based on the information from the many studies on chNA
organization, it appears that many small changes may occur
in chNA without resulting in any obvious phenotypic
changes. This would suggest that those particular regions

do not have a coding, regulatory or structural function. It

119
may be possible that many small regions of variation are
missed by restriction endonuclease mapping. Indeed, if
region B of the Ram 3b fragment had been a bit larger, it
may have compensated for the variable region A, thus making
detection of any differences possible only at the DNA
sequence level, or through examination of chNA with a

battery of enzymes.

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120

121

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APPEND I!

133

AluI
Ach
BamHI
BclI
BglII
ClaI
DraI
EcoRI
HaeIII
HincII
HindIII
HinFI
MaeIII

MboI

134

RESTRICTION ENDONUCLEASES

AND THEIR RECOGNITION SEQUENCES
(adapted from the BRL reference catalogue)

1010?

GTlcr‘GAC

GlGATCC
TlGATCA
AlGATCT
ATlCGAT
TTTlAAA
claarcc

00100

GTPylPuAC

1110011
GIANTC
lernac

1GATC

MspI
NdeI
NruI
PvuII
RsaI
SacI
SalI
ScrfI
SmaI
SphI
SstI
T.,:
XbaI

XhoI

01000
CAlTATG
T001001
caclcrc
GTlAC
0100110
GlTCGAC
CClNGG
0001000
001r010
GAGCTIC
TiCGA
TlCTAGA

ClTCGAC

 

135
DNA MARKER BAND SIZES

Marker 1 (M1): Phage lalbda (A) DNA digested with EcoRI +
HindIII. Adapted from the BRL Reference catalogue.

Band Number Size b

21,226
5,148
4,973
3,530
2,027
1,904
1,584
1,330

983
831
564
125

HHH
NHOQQQmM-tht-I

Marker 2 (H2): BRL 123 bp ladder

Incre-enta of 123 bp from 4,182 to 123 bp.

Marker 3 (Ma): Plan-id pBR322 DNA digested with Hian
Band Number Size b

1,632
517
506
396
344
298
221
220
154
75

OCDQQOSOI-war-t

p—o

136

Marker 4 (M4): Plas-id pBR322 DNA digested with MspI

Band Nunber Size b

1 622

2 527

3 404

4 309

5 242

6 238

7 217

8 201

9 190

10 180

11 160 X 2
12 147 X 2
13 123

14 110

15 90

76

H
U)