STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN GOSSYPIUM HIRSUTUM (L.)

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Authors Kestler, Daniel Paul, 1948-

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KESTLER, Daniel Paxil, 1948- STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN GOSSYPIUM HIRStTIUM L..

The University of Arizona, Ph.D., 1974 Chemistry, analytical

Xerox University Microfilms, Ann Arbor, Michigan 48106

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT IN

GOSSYPIUM HIRSUTUM L.

by

Daniel Paul Kestler

A Dissertation Submitted to the Faculty of the

COMMITTEE ON GENETICS CGRADUATE)

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 4 THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my direction by Daniel Paul Kestler . . , STUDIES ON THE DNA CONTENT OF A PLASTID MUTANT entitled

IN GOSSYPIUM HIRSUTUM L. be accepted as fulfilling the dissertation requirement of the degree of Doctor of Philosophy k- ?JU& )issertation Director Date 7

After inspection of the final copy of the dissertation, the following members of the Final Examination Committee concur in its approval and recommend its acceptance:

dJU 7y fi>P. Ee^w-jU, f W

This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allow­ able without special permission, provided that accurate ac­ knowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manu­ script in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: DEDICATION

This manuscript and work which it represents is dedicated to the memories of John Paul Kestler and Rose

Ann MacDonald Kestler.

iii ACKNOWLEDGMENTS

The author wishes to express his gratitude:

to Dr. Frank Katterman for many helpful suggestions during the course of the research and the preparation of this manuscript.

to Dr. John Endrizzi for use of mutant cotton seeds and greenhouse facilities.

to Dr. Kaoru Matsuda and Dr. William Bemis for advice in the preparation of this manuscript.

to Dr. Wayne Ferris for use of the electron micro­ scope and laboratory facilities.

to Mr. Glenn Lipman for preparation of the electron micrographs of leaf sections and DNA.

iv TABLE OF CONTENTS

Page

LIST OF TABLES vii

LIST OF ILLUSTRATIONS viii

ABSTRACT xi

INTRODUCTION . , 1

LITERATURE REVIEW 3

Light Microscopic Evidence of Nucleic Acids in Chloroplasts 3 Evidence of Nucleic Acids in Chloro­ plasts from Electron Microscopy ...... 4 Radioisotopic and Autoradiographic Evidence of Nucleic Acids in Chloro­ plasts 5 Evidence of the Existence of Chloroplast Nucleic Acids from Nucleic Acid Bio­ chemistry 6 Mitochondrial DNA of Higher Plants 22 Uniparental Inheritance in Higher Plants . . 22

MATERIALS AND METHODS 29

Isolation of Chloroplasts 29 Light Microscopic Examination of Purified Chloroplast Preparations 32 Isolation of Nuclei 33 Isolation of Chloroplast DNA 33 Isolation of Nuclear DNA 36 Preparation of Denatured and Denatured- Renatured Nuclear and Chloroplast DNA .... 37 Preparation of DNA for CsCl Analytical Buoyant Density Centrifugation . . 37 DNA Preparation for Electron Micrographs . . 39 Preparation of Leaf-Sections for Electron Microscopy 39

v vi

TABLE OF CONTENTS (Continued)

Page

RESULTS 41

Analysis of Cotton Chloroplastic and Nuclear DNA in Mutant and Wild Type Tissue 41 Summary of Results from Buoyant Density Studies of Cotton Nuclear and Chloro- plast DNA 62 Chloroplast Preparations Examined by Electron Microscopy 63 Electron Microscopic Study of Isolated Chloroplast DNA 73

DISCUSSION 75

SUMMARY 86

APPENDIX A: EVIDENCE THAT THE CHLOROPLAST MUTANT OF COTTON IS A TRUE CYTOPLASMIC MUTANT 88

LITERATURE CITED , 91 LIST OF TABLES

Table Page

1. Base composition (moles percent) of ribosomal RNA from some higher plants 13

2. Chloroplast and nuclear DNA buoyant density components of selected higher plants 16

3. List of higher plants with uniparental- maternal transmission of their plastids .... 24

4. Cotton chloroplast and nuclear DNA buoyant density data 45

5. Percentages of cotton chloroplast DNA from CsCl buoyant density analysis .... 50

6. Cross pollinations between variegated plants and normal green plants 89

7. Typical segregation ratios observed in the F^ generation from individual crosses of variegated-A mutant plants X normal green plants 90

vii LIST OF ILLUSTRATIONS

Figure Page

1. Green, yellow-green mosaic, and white- green mosaic leaves from Gossypium hirsutum L 30

2. Typical absorption profile of purified white tissue cotton chloroplast DNA 43

3. Typical absorption profile of purified green tissue cotton chloroplast DNA 44

4. Microdensitometer tracing of cotton chloroplast DNA from green leaf tissue .... 46

5. Microdensitometer tracing of cotton chloroplast DNA from yellow leaf tissue and yellow cotyledon tissue 47

6. Microdensitometer tracings of cotton chloroplast DNA from white leaf tissue 48

7. Comparisons of microdensitometer tracings of analytical centrifugations of chloroplast DNA from white, yellow and green tissue 52

8. Microdensitometer tracings of cotton nuclear DNA from green leaf tissue and white leaf tissue 53

9. Microdensitometer tracings of cotton chloroplast DNA from green tissue in comparison to cotton nuclear DNA from green tissue 54

10. Microdensitometer tracings of cotton chloroplast DNA from white tissue in comparison to cotton nuclear DNA from white tissue . , . . 55

viii ix

LIST OF ILLUSTRATIONS (Continued)

Figure Page

11. Microdensitometer tracings of cotton chloroplast DNA from yellow leaf tissue with a slight amount of nuclear con­ tamination in comparison to cotton nuclear DNA from green leaf tissue 56

12. Microdensitometer tracings of heat de­ natured and heat denatured-renatured nuclear DNA . , . 57

13. Microdensitometer tracings of heat de­ natured and heat denatured-renatured chloroplast DNA from green tissue 58

14. Microdensitometer tracings of chloro­ plast DNA which had heat denatured- renatured chloroplast DNA from white tissue 59

15. Microdensitometer tracings comparing heat denatured and heat denatured- renatured chloroplast and nuclear DNA from green leaf tissue 61

16. Electron micrograph of green leaf tissue chloroplast 64

17. Electron micrograph of green leaf chloroplasts 65

18. Electron micrograph of chloroplasts of green leaf tissue 66

19. Electron micrograph of yellow leaf tissue chloroplast 67

20. Electron micrograph of yellow leaf chloroplast 68

21. Electron micrograph of chloroplast of yellow leaf tissue 69

22. Electron micrograph of white leaf tissue chloroplast 70 X

LIST OF ILLUSTRATIONS (Continued)

Figure Page

23. Electron micrograph of white leaf chloroplast 71

24. Electron micrograph of chloroplast of white leaf tissue 72

25. Electron micrograph of yellow tissue chloroplast DNA shadowed with platinum- palladium 74 ABSTRACT

Chloroplast DNA was examined in a mutant strain of

Gossypium hirsutum L. The nature of the mutant was such that it exhibited uniparental inheritance and was therefore considered a cytoplasmic chloroplast mutant. The mutant exhibited three different conditions in its leaf tissue.

The leaves were green (normal), yellow, white, or sectored with green and white or green and yellow. DNA was examined from chloroplast fractions derived from the green, white, and yellow leaf tissue. The chloroplast DNA from green leaf tissue had a major component at 1.697 gm/cc and a minor component at 1.706 gm/cc. The chloroplast DNA from yellow leaf tissue exhibited a similar pattern to that of the green tissue except that the yellow tissue chloroplast

DNA had a greater amount of 1.706 DNA than did the former tissue. White tissue chloroplast DNA had three components.

The 1.706 DNA was the major component while 1.697 and 1.719 were lesser components. Nuclear DNA was examined from white and green leaf tissue. Both nuclear DNAs had buoyant den­ sities significantly lower (1.692-1.693) than any of the components of chloroplast DNA of cotton.

Chloroplasts were examined from the green, yellow, and white tissue. Green tissue chloroplasts contained

xi xii normal thalykoid and grana development, while yellow tissue chloroplasts lacked grana. White tissue chloroplasts lacked grana and thalykoid development and appeared as empty vesicles. These findings on mutant chloroplasts are consistent with other studies of internal morphology of chloroplasts from cytoplasmic mutants. INTRODUCTION

Chloroplast DNA has been examined in a number of higher plants. Buoyant density values and other valuable

parameters such as kinetic complexity, base ratios of the

DNA degradation products, and analytical complexity have

been determined for chloroplast DNA. Comparisons between

chloroplast and nuclear DNA components of various plants

based on the above parameters have been made. In addition to chloroplast DNA characterization, much work has been

performed on identifying mutant chloroplasts and their

respective morphological and biochemical deficiencies.

Work has been carried out in examining the nucleic acids

of mutant chloroplasts. Accounts of reduction in 70s ribo-

somes in mutant leaf tissue have been reported in tobacco

and cotton. Chloroplast DNA has been shown to exhibit a

different buoyant density pattern in mutant tissue of

tobacco as compared to chloroplast DNA from green leaf

tissue.

In the present study, the buoyant density of leaf

cotton chloroplast DNA has been analyzed. This is the first

reported data on cotton chloroplast DNA. In addition,

mutant yellow and white tissue chloroplast DNA were analyzed

1 for buoyant density determinations. Nuclear DNA was also examined for buoyant density determinations in order to compare and contrast the patterns obtained from nuclear and chloroplast DNA. In conjunction with studying the chloroplast DNA from yellow, white, and green leaf tissue in the cotton mutant, chloroplasts from these tissue types were examined in the electron microscope and the findings were compared to other reports on the morphology of mutant tissue chloroplasts. LITERATURE REVIEW

Light Microscopic Evidence of Nucleic Acids in Chloroplasts

The presence of nucleic acids in plant chloroplasts has been discerned by a number of researchers using a variety of techniques. Chiba (1951) recorded that chloro­ plasts of Salangenella savatieri, Tradescantia flumenensis, and Rhoes dicolor were stained by the Feulgen method and by methyl green. When the leaves were treated with hot tri­ chloroacetic acid, which eliminates nucleic acids, the staining was not observed. In addition, chloroplasts were also stained with pyronin. The staining was not observed if the leaves had been pretreated with hot trichloroacetic acid or ribonuclease. Chiba concluded that chloroplasts contain DNA and possibly RNA. Metzner (1952) reported that chloroplasts of Agapanthus were stained with methyl green and pyronin, and by the Feulgen method. He also observed that pretreatment with hot trichloroaceitc acid left the chloroplasts unstained by all of these methods. Yoshida

(1962), staining Elodea chloroplasts with pyronin, found that staining was decreased if the cells were first treated with ribonuclease, which suggested the presence of RNA.

However, other researchers were unable to detect chloroplast

3 4 staining by the Feulgen method (Littau 1958; Vosa and Kirk

1963); different techniques were needed to confirm or negate the presence of nucleic acids in chloroplasts.

Evidence of Nucleic Acids in Chloroplasts from Electron Microscopy

Through studies conducted with the electron micro­ scope, evidence was gathered to support the precocious con­ clusion that chloroplasts contain nucleic acids. Granules

100-150 8 in diameter were noted in electron micrographs of Chlamydomonas chloroplasts. The granules had visible similarities to ribosomal particles of the cytoplasm and it was suggested that these granules were ribosomes.

Similar ribosomes-like particles were observed in electron micrographs of spinach (Murakami 1963), maize (Jacobson,

Swift and Bogorad 1963), and oats (Gunning 1965).

Ris and Plaut (1962), studing cells of Chlamydomonas moewusii that had been post-fixed with uranyl acetate (a chemical that binds to nucleic acids), discovered the presence of fibrils 25-30 8 in diameter in the chloroplasts.

In cells they pretreated with deoxyribonuclease, the fibrils were no longer visible. On the basis of this work, in which similarity between chloroplast fibrils and DNA fibres in the cytoplasm of bacteria was found, Ris and Plaut proposed that chloroplast fibrils contain DNA. Later, Ris and Plaut found 25 X fibrils in the chloroplasts of selected 5 higher plants. Similar findings in higher plants have been reported by other researchers using electron microscopic techniques (Kislev, Swift and Bogorad 1965; Gunning 1965).

The evidence gathered from electron microscopic studies lends credence to the hypothesis that chloroplasts contain nucleic acids in the form of both RNA and DNA.

Radioisotopic and Autoradiographic Evidence of Nucleic Acids in Chloroplasts

Autoradiography and radioisotopic labeling provided further evidence that nucleic acids were present in chloro­ plasts. Brachet (1958) showed that after exposing the algae 3 Acetabularia to tritiated thymidine ( H-thymidine), radio­ activity could be detected in the chloroplasts. Other workers reported similar experimental results in Spirogyra

(Stocking and Gifford 1959) and in the fern Ceratopteris thalictroides (Gifford 1960). Wollgiehn and Mothes (1964) 3 . . reported that in leaf discs of tobacco, H-thymidine was incorporated into the chloroplasts; the radioactivity was lost upon treatment with deoxyribonuclease, but was not affected when treated with ribonuclease. These results offered strong support for the presence of DNA in the chloroplasts. Confirmational reports were accounted in studies of wheat (Kislev, Swift, and Bogorad 1965), and in three species of marine algae (Steffenson and Sheridan

1965). Autoradiographic methods have provided evidence of 6

DNA in protoplasts of the fern Pteridium aquilinum (Bell

1961). Through microscopic examination of autoradiographs from the fern preparations, most of the cytoplasmic 3 H- thymidine was shown to be associated with the protoplasts and mitochondria.

Through autoradiographic techniques RNA has also been detected in chloroplasts. Segments of leaves from 3 etiolated corn seedlxngs were shown to incorporate H~ cytidine into their etioplasts (Jacobson, Swift, and Bogorad

1963). The treating of leaf sections with ribonuclease removed the label: thereby suggesting that the latter had been incorporated into etioplast RNA. Radioisotopic labeling and autoradiographic studies support the hypothesis that chloroplasts contain DNA and RNA.

Evidence of the Existence of Chloroplast Nucleic Acids from Nucleic Acid Biochemistry

The development of suitable isolation procedures provided evidence for the existence of DNA and RNA in chloroplasts. Initially, extractions of chloroplasts in­ volved the use of aqueous methods and low speed differential centrifugation, Utilizing these techniques many researchers obtained values between 1.0 and 4.0 percent nucleic acid content per dry weight of chloroplasts (Kirk and Tilney-

Bassett 1967). The disadvantage of the differential centri- fugation method of isolating chloroplasts is high nuclear 7 contamination (Weier and Stocking 1952). As a result pro­ cedures were introduced which combined differential and discontinuous density gradient centrifugation to obtain purified chloroplast preparations (Wildman, Hongladarom, and Honda 1962; Jagendorf 1955; James and Das 1957; Kirk

1963). Using the improved procedures values of 0.17 per­ cent DNA and 0.12 percent RNA per dry weight of chloroplast were obtained (Orth and Cornwell 1963). These lower values

attained by density gradient extraction reflect a decrease

in nuclear contamination not achieved using only low speed

differential centrifugation. The purity of chloroplasts

prepared by density gradient centrifugation has been

qualitatively measured by electron microscopic examination

of chloroplast fractions. These examinations have re­

vealed only a small amount of nuclear contamination (Kung

and Williams 1969).

Use of aqueous methods for preparation of chloroplast

nucleic acids is often inefficient, since chloroplasts iso­

lated by these methods are leaky. Kahn and Von Wettstein

C1961) and Jacobi and Perner (1964) reported that many

chloroplasts which were isolated by aqueous extraction

methods lost their outer membrane and the accompanying

electron dense protein material of the stroma. Spencer

and Whitfeld (1969) recovered DNA after differential cen­

trifugation of chloroplasts in hypotonic media, which. 8 suggests that the DNA was tightly bound to the chloroplast inner membrane. The major loss of nucleic acids by aqueous methods of extraction appears to be in the form of RNA.

The discontinuous gradient centrifugation, however, does permit studies on intact chloroplasts since the intact organelles were shown to band in the gradient, whereas broken chloroplasts remained near the top (Leech 1966).

In order to amend the loss of chloroplast nucleic acids through aqueous extraction, some investigators have implemented non-aqueous methods (Smillie 1963; Stocking

1959; Wells and Birnstiel 1969). Values of 1.0-2.5 percent

RNA of the dry weight of chloroplasts were obtained by several workers using a non-aqueous procedure for isola­ tion of chloroplast RNA (Heber 1963; Rupple 19641. DNA content of chloroplasts isolated by a non-aqueous method has been reported to be 0.4 percent of the dry weight of the chloroplasts in spinach; this is in close agreement with values obtained by density gradient extraction of chloroplast DNA (Biggins and Park 1964). Wells and Birnstiel

(1969), using aqueous and non-aqueous preparations of chloroplast DNA, recorded no nuclear contamination in the chloroplast DNA extracted by either method. Both aqueous, with discontinuous gradient centrifugation, and non-aqueous methods of DNA isolation in chloroplasts have yielded comparable results. However, total nucleic acid values are 9 significantly different between the two methods. This difference may reflect the higher efficiency of non-aqueous isolation procedures over that of the aqueous discontinuous gradient method in recovery of RNA. On the other hand the difference in nucleic acids recovered from chloroplasts isolated by these methods may reflect nuclear contamination in the non-aqueous preparation. The low values of chloro- plast DNA recovered by the non-aqueous method of extrac­ tion argues against the latter possibility.

Nucleic acids isolated from chloroplasts have been analyzed extensively in higher plants. Lyttleton (1962) examined plastid RNA isolated from spinach leaves and sub­ sequently discovered two classes of ribosomes that sedi- mented at different rates in the analytical ultracentrifuge.

The components had sedimentation coefficients of 68 and 83s; the 68s component was found to associate solely with the chloroplast fraction, and the 83s component was found to associate solely with the cytoplasm. In addition ribosomes which have a sedimentation coefficient of 62s have been isolated from pea seedling chloroplasts (Sissakian et al.

1965). In spinach and tobacco leaves, other accounts of sedimentation coefficients of 70s for chloroplast ribosomes, and 80s for cytoplasmic ribosomes, have been noted (Spencer

1965; Spencer and Whitfeld 1966; Boardman, Francki, and

Wildman 1965). 10

Isolation of RNA from the 70s ribosomes of Euglena gracilis chloroplast fractions has been accomplished by disc gel electrophoresis: the ribosomes yielded RNA com­ ponents with sedimentation values of 23s and 16s (Scott and Smillie 1969). Chloroplast ribosomes of Euglena separate into 50s and 30s subunits when they are subjected to low magnesium concentrations (Svetailo, Philippovich and Sissakian 1967). Chloroplast RNA with sedimentation coefficients of 23s and 16s have also been isolated from higher plants; the RNA base ratios have subsequently been determined.

Nucleic acid synthesis in chloroplasts has been implied by the radioisotopic studies discussed earlier.

Direct evidence of DNA and RNA synthesis in isolated chloro­ plasts has been introduced by Tewari and Wildman (1967) and Spencer and Whitfeld (1967, 1969). In spinach it has been shown that RNA, synthesized in the chloroplasts, hybridizes with chloroplast and nuclear DNA (Spencer and

Whitfeld 1967),

In tobacco, RNA-DNA hybridization has been carried out between chloroplast ribosomal RNA, chloroplast DNA, and nuclear DNA; high levels of hybridization were noted in both cases. Tewari and Wildman (1970) testing chloroplast

DNA for hybridization with cytoplasmic ribosomal RNA, showed that less than 0.05 percent hybridization took place. 11

In Euglena gracilis, Scott and Smillie (1967) demonstrated that chloroplast ribosomal RNA hybridized with 1.0 percent of the chloroplast genome. They argue that a level of 1.0 percent hybridization constitutes one gene for RNA of 70s ribosomes in the chloroplast genome.

Stutz and Rawson (1970) have reported, by means of hy­ bridization experiments, that Euglena chloroplast DNA does not have complementary base sequences with 80s ribo­ somal RNA. It has been noted in Euglena that 23s and 16s chloroplast ribosomal RNA are coded by separate cistrons in the chloroplast DNA (Scott 1969).

It was recorded that a significant level of hy­ bridization took place when chloroplast ribosomal RNA was hybridized with nuclear DNA (Tewari and Wildman 1967,

1970). In order to avoid the possibility of chloroplast ribosomal RNA hybridizing with chloroplast DNA contamination associated with the nuclear DNA, Tewari (1971) conducted experiments which showed the hybridization of chloroplast ribosomal RNA with highly purified tobacco nuclear DNA,

It thus, appears that chloroplast ribosomal RNA from higher plants has complementary cistrons in the nuclear genome as well as in the chloroplast genome. Chloroplast DNA does not appear to have cistrons complementary to RNA from 80s ribosomes.

Chloroplast and cytoplasmic ribosomal RNA base ratios have been cited for Euglena and Chlorella (Rawson 12 and Stutz 1969; Scott and Smillie 1967; Oshil and Hase

1968). Large differences in base ratios were seen between the 80 and 70s ribosomal RNA in both of these organisms.

In contrast, little difference is seen between base ratios for ribosomal RNA of higher plants. Table 1 records data for base ratios of 70 and 80s ribosomal RNA components of some higher plants (Rossi and Gualerzi 1970).

Tewari and Wildman (1970) have reported that com­ plementarity exists between chloroplast DNA and chloroplast transfer RNA. The hybridization attained in the study corresponded to a genetic informational potential of the chloroplast genome coding for 20 to 30 transfer RNA species.

Chloroplast DNA was also shown to hybridize with messenger

RNA derived from the chloroplasts (Tewari and Wildman 1967,

1970; Spencer and Whitfeld 1967).

Buoyant density and base ratio analysis of chloro­ plast DNA followed initial reports of the presence of DNA fibrils in electron micrographs of chloroplasts. Chun,

Vaughn, and Rich (1963), examined the chloroplast DNA of spinach and beets, and obtained essentially three components separable by CsCl buoyant density centrifugation: a major component with buoyant density of 1.695 gm/cc and two minor components with buoyant densities of 1.706 and 1.719 gm/cc.

Since their studies, much research has been executed in analyzing buoyant density values of various algae and higher Table 1. Base composition (moles percent) of ribosomal RNA from some higher plants.*

UMP GMP CMP AMP

cyt. (Total) 22.0 + 0.2 31.7 + 0.4 24.1 + 0.1 22.2 + 0.3 Ivy chlor. (Total) 22.1 + 0.3 34.1 + 0.3 21.6 + 0.4 23.4 + 0.3

cyt. (Total) 23.7 + 0.3 30.9 + + 22.3 + 0.1 jjcutuce 0.3 23.1 0.2 chlor. (Total) 19.6 + 0.1 33.3 + 0.2 21.9 + 0.1 24.2 + 0.1

cyt. (Total) 21.5 + 0.3 32.0 + 0.1 24.4 + 0.4 22.1 + 0.2 LaDiJa^c chlor. (Total) 19.8 + 0.2 32.6 + 0.2 23.0 + 0.3 24.5 + 0.3

cyt. (Total) 22.7 + 0.4 32.2 + 0.2 23.6 + 0.3 21.5 + 0.5 Turnip chlor. (Total) 19.8 + 0.1 33.1 + 0.3 22.7 + 0.3 24.4 + 0.4

Spinach cyt. (Total) 23.3 + 0.3 31.7 + 0.3 23.5 + 0.4 21.6 + 0.2 ( 25 S)* 22.8 + 0.2 32.5 + 0.1 23.4 + 0.1 21.3 + 0.3 ( 17 S)* 23.4 + 0.4 30.5 + 0.4 23.5 + 0.3 22.6 + 0.3 chlor. (Total) 20.0 + 0.2 32.4 + 0.3 23.4 + 0.2 24.2 + 0.3 ( 22 S)* 19.0 + 0.4 34.8 + 0.2 21.7 + 0.2 24.5 + 0.1 ( 15 S)* 22.4 + 0.2 30.6 + 0.2 24.0 + 0.2 23.0 + 0.3

Beet cyt. (Total) 23.4 + 0.1 31.4 + 0.2 23.2 + 0.3 22.0 + 0.4 ( 25 S) 23.2 + 0.3 32.5 + 0.4 23.1 + 0.2 21.2 + 0.4 ( 17 S) 23.4 + 0.3 30.3 + 0.5 23.5 + 0.4 22.8 + 0.6 chlor. (Total) 20.3 + 0.4 33.6 + 0.5 22.3 + 0.2 23.7 + 0.5 ( 22 S) 18.7 + 0.1 35.2 + 0.5 21.5 + 0.3 24.6 + 0.3 ( 15 S) 22.9 + 0.2 30.9 + 0.2 23.2 + 0.3 22.8 + 0.1

*Values are means of eight determinations plus or minus standard error. The exact S values for cytoplasmic and chloroplast RNA's in 0.01 M Table 1. (Continued)

NaCl at 20 yg/ml were 25.0 ± 0.2; 16.7 ± 0.3; 22.0 ± 0.3; 15.5 ± 0.2, re­ spectively (average of three determinations) determined by analytical ultra- centrifugation in a Beckman Model E centrifuge equipped with U.V. optics. Table in Rossi and Gualerzi (1970). plants. DNAs and the results of these studies are as­ sembled in Table 2,

The data of Table 2 clearly shows that buoyant density values for chloroplast DNA of the examined algae are lower than those of the corresponding nuclear DNA,

Chloroplast DNA buoyant density values of higher plants encompass a wider range. Minor high buoyant density values of 1.706 to 1.719 found in chloroplast DNA preparations of several workers (Table 2) are often questioned as to their true origin. In general there seems to be three different interpretations of what is bonafide chloroplast DNA.

Several researchers assign buoyant density values of approxi­ mately 1,706 gm/cc to chloroplast DNA (Green and Gordon

1967; Bard and Gordon 1969; Chun, Vaughn and Rich 19631; others assign only buoyant density values of 1.697-1.700 gm/cc to chloroplast DNA (Kung and Williams 1969; Wells and Birnsteil 1969; Tewari and Wildman 1970).

Data from Suyama and Bonner's (1966) study on buoyant densities of plant mitochondrial DNA reveals the average buoyant density of higher plant mitochondrial DNA to be 1.706 gm/cc. The similarity of mitochondrial DNA to those of high density bands found in chloroplast prepara­ tions increases the problem of assigning the high density values to chloroplast DNA. Kirk (1971) and Tewari (1971) have reevaluated the available data on buoyant density Table 2. Chloroplast and nuclear DNA buoyant density components of selected higher plants.

Buoyant Density of DNA (gm/cm^)

Nuclear Chloroplastic Species Reference Major Minor Major Minor

Acetabularia 1.714 1.703 1.722 Green et al. (1967) mediterranea

Chlorella ellipsoida 1.716 1.695 1.711 Iwamura (1960)

Chlamydomonas reinhardi 1.723 1.695 1.715 (a) Leff et al. (1963) (c) (b) Chun et al. (1963) (c) Chiang and Sueoka (1967)

Euglena gracilis 1.707 1.686 1.691 Edelman et al. (1964)

Antirrhinum major 1.697 1.709 Green and Gordon (1967) (snapdragon)

Vica faba 1.696 1.696 Kung and Williams (196.9) (broad bean) 1.695 1.697 Wells and Birnstiel (1969) Beta vulgaris 1.695 1.705 1.719 Chun et al. (1963) (beet)

Beta vulgaris var. cicla 1.689 1.700 Kislev et al. (1965) (Swiss chard) Table 2. (Continued)

Buoyant Density of DNA (cm/cm ) Nuclear Chloroplastic Species Reference Major Minor Major Minor

Spinacia oleracea 1.695 1.705 1.719 Chun et al. (1963) (spinach) 1.694 1.696 Whitfeld and Spencer (1968) 1.694 1.697 Wells and Birnstiel (1969) 1.695 1.696; Bard and Gordon (1969) 1.706

Nicotiana tabacum 1.696 1.706 1.711 Green and Gordon (19 65) (tobacco) 1.697 1.702 Tewari and Wildman (1966) 1.697 1.697 Whitfeld and Spencer (19685 1.697 1.700 Tewari and Wildman (1970) 1.698 1.700; Jaworski (1969) 1.707

Phaseolus vulgaris 1.694; 1.694; Beridze, Odintsova and (bean) 1.703 1.703 Sissakian (1967)

Brassica rapa 1.692 1.695 Suyama and Bonner (1966) (turnip")

Ranunculus repens 1.697 1.703 Green and Gordon (1967)

1.695; 1.700; Jaworski (19 69) (tomato) 1.706 1.707

1.697; 1.699; Jaworski (1969) (pumpkin) 1.707 1.706

Tagetes patula 1.692 1.702 1.707 Green and Gordon (1967) (marigoId) 18 studies in higher plants and each independently concludes that 1.706 DNA found in higher plant chloroplast prepara­ tions originates from mitochondrial contamination. Kirk

(1971) argues that most chloroplast DNA from higher plants should have a buoyant density of 1.697 giu/cc corres­ ponding with a 38 to 40 molar-percent GC content.

This buoyant density value is found in many higher plants as a major component of chloroplast as well as nuclear DNA (.see Table 2). Kirk (1971) states, that in many cases, nuclear and chloroplast DNA are not distin­ guishable by their native buoyant density values. Lyttleton and Peterson (1964) present data which supports Kirk's conclusion. Having isolated chloroplasts and nuclei from tobacco leaves, and then extracting total DNA from the leaf tissue, the two researchers were able to demonstrate only one peak of buoyant density 1.698 gm/cc. Thus, they con­ cluded that nuclear and chloroplast DNA can share the same buoyant density in higher plants. Other studies arriving at similar conclusions are those of: Whitfeld and Spencer (1968), Wells and Birnstiel (1969), and Kung and Williams (1969).

A third interpretation drawn from buoyant density studies of higher plants is that chloroplast DNA contains both a low, i.e., 1.697-1.700 gm/cc density component, and a high, i.e., 1.706 gm/cc density component (Bard and Gordon 1969; Jaworski, Whitfeld and Siegel 1969). Higher buoyant density components such as the 1.719 buoyant density band associated with beet chloroplast DNA ex­ tractions (Chun, Vaughn, and Rich 1963) have been linked by critics to be bacterial contamination (Kirk 1971).

A possible chloroplast origin of this high density band will be discussed later.

Other measurable characteristics of chloroplast

DNA are: temperature melt profiles, denaturation- renaturation kinetics, chromatographic base ratios, and denaturation, denaturation-renaturation buoyant densities.

A number of workers have found close similarities between temperature melt profiles of chloroplast DNA and those of nuclear DNA. Kung and Williams (1969) found Tm values for

Vica faba chloroplast and nuclear DNA of 86.5°C and

86.3°C respectively. Tewari and Wildman (1970) detected

Tm values of 85°C for chloroplast DNA and 84°C for nuclear

DNA in tobacco.

Wells and Birnstiel (1969) examined denaturation- renaturation kinetics of lettuce chloroplast and nuclear

DNA. They discovered a more rapid and complete renatura- tion pattern in the chloroplast DNA, as 70 percent of the original hyperchromicity was obtained after renaturation.

Rapid and slow reannealing components of lettuce chloro­ plast DNA were found, with kinetic complexities (molecular 20 size of the DNA based on renaturation rates of the DNA) 6 8 of 3 x 10 daltons and 1,2 x 10 daltons respectively.

Because the faster renaturing base sequences are of very low kinetic complexity they are present in the chloroplast genome more frequently than the slower reannealing com­ ponents.

Tewari and Wildman (1970) have determined denatura- tion-renaturation kinetics for tobacco chloroplast DNA.

They found no evidence of two components in the chloro-

g plast genome, and they arrived at a value of 1.12 x 10 daltons for the molecular weight of chloroplast DNA.

g This value is similar to the 1.2 _x 10 daltons value found in the slow component of lettuce chloroplast DNA, Tewari and Wildman (1970) also noted higher rates, and amounts, of reannealing in chloroplast DNA than in nuclear DNA.

Chromatographic base ratio analysis of chloroplast

DNA has been performed by several scientists. Whitfeld and Spencer (1968) determined the base ratios of nuclear and chloroplast DNA in tobacco and spinach. In the nuclear

DNA of both plants, approximately 30 percent of the cyto- sine was found in the 5-methyl form. No 5-methyl cytosine was detected in the chloroplast DNA of either plant.

Similar findings were recorded by other researchers who examined yarious chloroplast and nuclear DNA base ratios 21 (Brawerman and Eisenstadt 1964; Lyttleton and Peterson

1964; Ray and Hanawalt 1964; Tewari and Wildman 1966).

Denaturation-renaturation studies have shown that chloroplast DNA of higher plants renatures rapidly and approaches close to its native buoyant density; whereas, nuclear DNA of higher plants does not extensively renature

(Tewari and Wildman 196 6; Whitfeld and Spencer 1968; Bard and Gordon 1969; Wells and Birnstiel 1969),

In summary, biochemical examination of chloroplast nucleic acids and their associated complexes reveals dif­ ferences, as well as similarities, to corresponding nuclear components, Chloroplast ribosomes and ribosomal RNA are smaller than cytoplasmic ribosomes and ribosomal RNA.

The major component of chloroplast DNA has a buoyant density of approximately 1.697 gm/cc, while nuclear DNA of higher plants can possess buoyant densities of higher, lower, or similar value. Chloroplast DNA has also been reported to have high buoyant density satellites in addition to the main buoyant density band. Rapid and complete renatura- tion is a property of chloroplast DNA not often shared by its nuclear counterpart. Denaturation-renaturation studies O reveal a kinetic complexity of 10 daltons for chloroplast

DNA; an analytical complexity (i.e., amount of DNA per 9 per chloroplast) of 10 daltons has also been shown. Wells and Birnstiel (1969) resolved the discrepancy between values of analytical and kinetic complexity of the chloroplast

DNA by argueing for a high level of redundancy in the

chloroplast genome. In contrast, the main fractions of the

nuclear genome seems to be much more heterogeneous in

nature than chloroplast DNA.

Mitochondrial DNA of Higher Plants

Characteristics of mitochondrial DNA in higher

plants have been examined by Suyama and Bonner (1966) and

Wells and Birnstiel (1969). Both studies revealed a

buoyant density of 1.706 gm/cc for native mitochondrial

DNA. A rapid renaturation to native buoyant density was

reported by Wells and Birnstiel (1969) for mitochondrial

DNA in lettuce. For a general review of mitochondrial

DNA, the reader should consult Borst, Kroon and Ruttenberg

(1966).

Uniparental Inheritance in Higher Plants

Uniparental plastid inheritance in plants, under

the control of plastid genomes, most often remains remote

from the direct influence of nuclear genomes. A noted

exception to this fact is the Iojap system in maize

(Rhoades 1955). Uniparental inheritance was discovered

by Correns (1908). since that time, many examples of this

mode of inheritance have been the focus of study for plant

breeders. Kirk and Tilney-Bassett (1967) list, in tabular 23 form (see Table 3), examples of known uniparental inheri­ tance in higher plants. Sager (197 2) also discusses this phenomena, with emphasis on the unicellular alga Chlamy- demonas. Often uniparental inheritance in higher plants occurs when mutation in the plastid's genome results in a partial or total loss of color in the normally green tissue.

The mutant phenotype is undetectable until a sig­ nificant number of cell divisions have taken place. When these are completed, whole tissue areas can be recognized

as differing from the normal green color. In higher

plants, uniparental plastid mutations range in color from

off-green to white. Progeny segregation for traits, through the maternal line, is a genetic criteria for uni­

parental inheritance. Therefore, using reciprical crosses

as a tool of genetic analysis, uniparental inheritance

can be preliminarly determined in one or two generations.

A number of generations of true breeding are required to

distinguish maternal effects from uniparental inheritance.

Biparental inheritance in higher plants has been noted in

extrachromosomal plastid inheritance (Kirk and Tilney-

Bassett 1967).

Ultrastructure analysis of uniparental plastid

mutants in higher plants has been explored. Schmid, Price

and Gaffron (1966), through use of the electron microscope, Table 3, List of higher plants with uniparental-maternal transmission of their plastids.

Demonstration Mixed Cells

Plant Cytological Inferred by Authors Dates Observation Breeding: Seedlings

Dicotyledons

Antirrhinum majus No Yes Baur 1910, 11a No Yes Scherz 1927 No No Gairdner, Haldane 1929 Yes Yes Maly, Wild 1956 Wild 1958, 60 Yes Yes Hagermann 1960, 61a 65 Dobel, Hagemann 1963 Arabidopsis thaliana Yes Yes Robbelen 1962 Arabis albida No No Correns 1919b Aubrietia graeca No No Correns 1919b Aubrietla purpurea No No Correns 1919b Beta vulgaris No No Stehlik 1921 Munerati 1928, 42 Capsicum annuum No Yes Dale 1930 Hutchins, Youngner 1952 Epilobium hirsutum Yes Yes Michaelis 1935-:1965 (numerous mutants) Hydrangea hortensis No No Chittenden 1926 Lactuca sativa No No Whitaker 1944 Table 3. (Continued)

Demonstration Mixed Cells

Plant Cytological Inferred by Authors Dates Observation Breeding: Seedlings

Lycopersicum esculentum No No Schlosser 1935 (Solanum lycopersicum) Mesembryanthemum No No Correns 1919b cordifolium Mimulus quinquevulnerus No Yes Brozek 1923, 26 Mirabilis jalapa No Yes Correns 1909a, 09] Nicotia"na colossea No No Honing 1927 Nicotiana tabacum Yes No Woods, DuBuy 1951a No No Burk, Grosso 1963 Yes Yes Burk, Stewart, Dermen 1964 Yes Yes Wettstein, Eriksson 1965 Petunia violacea No No Terao, U 1929 No Yes Pandey, Blaydes 1957 Pharbitis nil No Yes Miyake, Imai 1935 Imai 1936b Pisum sativum No Yes deHaan 1930 Primula sinensis Yes Yes Gregory 1915 Primula vulgaris No No Chattaway, Snow 1929 Stellaria media Yes Yes Correns 1922, 31a Funaoka 1924 Trifolium pratense No Yes Nijdam 1932 Viola tricolor No Yes Clausen 1927, 30

to U1 Table 3. (Continued)

Demonstration Mixed Cells

Plant Cytological Inferred by Authors Dates Observation Breeding: Seedlings

Monocotyledons

Avena sativa No Yes Akerman 1933 Avena sativa X No Yes Love, Craig 1936 sterilis Chlorophytum comosum No Yes Collins 1922 X elatum Chlorophytum elatum No Yes Pandey, Blaydes 1957 Hordeum vulgare No No Robertson 1937 Hosta japonica Yes Yes Yasui 1929 Sorghum vulgare No Yes Karper 1934 Triticum vulgare No Yes Umar 1943 No Yes Pao, Li 1946 Zea mays No Yes Randolph 1922 Anderson 1923 No Yes Demeree 1927 Zirkle 1929

Adapted from Kirk and Tilney-Bassett (1967).

ro 2 fixation was experienced in sections of the yellow leaf tissue.

Kohel and Benedict (1971) demonstrated electron micrographs of normal green and mutant white chloroplasts from a uniparental plastid mutant in cotton. Their findings revealed significant lower rates of CC^ fixation in yellow and white leaf tissue of the mutant as compared to normal green leaf tissue. In the green leaf chloroplasts normal thalykoid and grana were seen; in the white chloroplasts, only vesicles with no internal structure could be detected.

Shumway and Weier (1967) examined the chloroplasts of the

Iojap mutant of maize. Through the use of electron micro­ graphs, they showed that the white tissue chloroplasts were devoid of internal development while normal thalykoids and grana comprised the green tissue chloroplasts.

With use of sedimentation centrifugation, Katterman and Endrizzi (1973) indicated that white tissue of a plastid mutant in cotton possesses a low 70s ribosome content, as contrasted with relatively higher amounts of 70s ribosomes 28 in yellow and green tissue. Their conclusions conflict somewhat with the findings of Kohel and Benedict (1971) in a similar white leafed cotton mutant. The latter workers argue for the necessity of functional 70s ribosomes in the white tissue, as a significant amount of enzymatic activity takes place via photosynthesis. Wildman, Lu-Liao and Wong-

Staal (1973) found an intermediate level of 70s ribosomes in a white mutant of tobacco. They also reported inter­ mediate values for 23s and 16s ribosomal RNA in mutant as compared to normal green leaf tissue. Wong-Staal and Wildman

(1973) recorded a difference in analytical buoyant density data for chloroplast DNA in mutant and normal tissue of tobacco.

Proof of the existence of uniparental plastid inheritance in higher plants has been offered. Cytological as well as biochemical evidence has been presented citing differences in the chloroplasts and their related components of cytoplasmic mutants in higher plants. MATERIALS AND METHODS

Isolation of Chloroplasts

Tender leaves 3 to 6 centimeters in diameter and 3 to 5 weeks of age were taken from greenhouse grown cotton plants (Figure 1). These plants were a cytoplasmic mutant strain of Gossypium hirsutum (see Appendix A) cultivar

Deltaphine Smooth Leaf which contained green, yellow, and white leaf tissue (Katterman and Endrizzi 1973). Chloro- plast fractions were prepared from the three different types of leaf tissue. The leaves were washed in deionized water and patted dry with paper toweling. Peioles and midveins were removed and the leaf lamella were chopped with razor blades adapted to a General Electric electric knife in

Spencer's media at a ratio of 3:1 (i.e., Spencer's media to gram weight of leaf) (Whitfeld and Spencer 1968). The media was added gradually as the leaf lamella became mascerated.

After grinding the leaf preparation to a pulpy consistency, the mascerate was filtered through a double layer of Mira cloth (Chicopee Mills, Inc.). The filtrate was then centri- fuged at 3,000 rpm for 10 minutes in a Sorvall model RC2-B refrigerated centrifuge (5°C) using an SS-34 rotor. All cen' trifugations were carried out with this equipment at the above temperature unless further noted. The supernatant

29 30

Figure 1. Green, yellow-green mosaic, and white- green mosaic leaves from Gossypium hirsutum L. 31 was discarded and the pellet, designated the crude chloro­ plast pellet, was resuspended in Honda's media (Honda,

Wildman and Hongladarom 1962).

The proportion of Honda's media to crude chloroplast pellet was such that the pellet derived from 45 grams of leaf lamella, was resuspended in 15 ml of Honda's media.

The resuspended crude chloroplast pellet was layered onto discontinuous sucrose gradients made up of 5 ml of 20 per­ cent (weight : volume) sucrose in Honda's media, layered onto 10 ml of 45 percent (weight : volume) sucrose in

Honda's media, which in turn was layered onto 10 ml of 60 percent (weight : volume) sucrose in Honda's media (Whitfeld and Spencer 1968). Five ml of the crude chloroplast pellet, derived from 45 gms of leaves, was layered on each gradient such that the total crude chloroplast pellet was layered on three gradient tubes. The gradients were then spun at

23,000 rpm for 2 hours in a Beckman Model L-2 Ultra-

Centrifuge, equipped with a S.W. 25.1 centrifuge head. The centrifugation, as well as all other extraction procedures for isolating chloroplasts, was carried out in the cold at approximately 5°C, After centrifugation, 2 bands were visible at the 20-45 percent interface and at the 45-60 percent interface. These bands were green in the case of chloroplast extractions from green leaf tissue and they were brown to yellow in color when white or yellow leaf 32 tissue was used for isolation of chloroplasts. A brown and white pellet was observed at the bottom of the centrifuge tubes regardless of the type of leaf tissue used for chloroplast isolation. The 2 bands from any particular chloroplast isolation were independently collected with a

Pasture pipette. The top and bottom bands were then diluted with 2 volumes of Spencer1s media and centrifuged for 10 minutes at 10,000 rpm. The supernatant was dis­ carded and the pellet was centrifuged once more to remove excess liquid. The pellets were then used for light micro­ scopic examination and chloroplast DNA extraction.

Light Microscopic Examination of Purified Chloroplast Preparations

Purified chloroplast fractions from both the 20-45 percent and the 45-60 percent interface were examined under phase microscopy. Both fractions of the green tissue chloroplasts contained large amounts of intact chloroplasts.

The lower fraction contained a visible amount of nuclear fragments, while none were observed in the upper fraction.

The two fractions of chloroplasts from yellow leaf tissue contained a predominant amount of intact chloroplast. A significant amount of broken chloroplasts were seen in both fractions and the lower band contained a visible amount of

broken nuclei. The white tissue chloroplast fractions contained a far greater amount of broken chloroplasts than 33 either the yellow or green tissue fractions. The lower fraction was also visible contaminated with broken nuclei.

Isolation of Nuclei

The pelleted material, composed primarily of nuclei, was resuspended In Spencer's media which had 0.02 M CaC^ substituted for 0.01 M MgC^. Triton X-100 (Rohm and Haas} was added to this suspension to a final concentration of

5 percent (volume : volume) (Whitfeld and Spencer 19681.

The nuclear suspension was centrifuged at 3,000 rpm for 10 minutes. Resuspensions of the nuclear preparations and subsequent centrifugations were carried out until the nuclear pellets were gray in color. The nuclear pellets were then ready for DNA extraction.

Isolation of Chloroplast DNA

The purified pellet, recovered from the top band of the discontinuous sucrose gradients, was resuspended in 2 ml of Spencer

0.01 M MgC^. Bacillus subtilus temperature resistant a-amylase was suspended in SSC (sodium saline citrate) at a concentration of 10 mg/ml. One ml of this reagent was added to the resuspended purified chloroplast pellet derived from the green leaf tissue and then incubated for 30 minutes at 37°C. The suspension was centrifuged at 4,000 rpm for 34

10 minutes. The purified chloroplast pellet from green leaf tissue was then ready for DNA extraction. In the case of yellow or white tissue chloroplast fractions a-amylase treatment prior to DNA extraction was omitted.

Purified chloroplast pellets were resuspended in

2 ml of a solution containing 0.2 M EDTA (disodium ethylene- diaminetetraacetate); 0.05 M Tris pH 8.0 and 15 percent

Triton X-100 (volume : volume). Sodium perchlorate was added to the suspension at a final molarity of 1.5.

The mixture was incubated at 50°C for 15 minutes and then shaken gently with an equal volume of chloroform- isoamyl alcohol (25 : 1 volume : volume) for 30 minutes on a wrist shaker. The mixture was centrifuged at 7,000 rpm for 10 minutes. After collection of the aqueous phase, the interphase and lower chloroform-isoamyl phase was discarded.

The aqueous phase was transferred to h inch dialysis tubing and dialyzed against 1 liter of 0.1 SSC at approxi­ mately 5°C for 6 hours. The DNA solution was adjusted with

CsCl to a final density of approximately 1.700 gm/cc and a final volume of 2.1 ml by the formula of Vinograd and

Hearst (1962):

Weight % 25oQ (CsCl) = 137.48— 1.38.11 (1/p) where

p = density (gm/cc) of CsCl solution at 25°C. 35

Ethidium bromide was added to a final concentration of 100 yg/ml. This was accomplished by adding 10 yl of a solution of 0.02 gm/ml of ethidium bromide suspension to the DNA solution. The DNA solution was then placed into Beckman S.W.

50.1 centrifuge tubes and 2.0 ml of CsCl solution of density

1.45 gm/cc; previously adjusted to contain 100 yg/ml ethidium bromide was layered onto the initial DNA solution to make a step gradient CBrunk and Leick 1969). A small amount of light mineral oil was then layered onto the top of the gradient to prevent evaporation of the high salt solution.

The tubes and contents were centriguged in a Beckman Model

L-2 Ultracentrifuge for 14 hours at 40,000 rpm at 18°C.

After centrifugation, the gradient was examined under a black light of wave lengths 290 to 400 nm (namometers).

Near the center of the tube, a white fluorescent band was observed. The band, containing DNA, was removed with a 22 gauge needle and syringe and then shaken with an equal volume of water saturated isoamyl alcohol in order to eliminate the ethidium bromide. This procedure was re­ peated several times with fresh portions of isoamyl alcohol before the DNA solution was placed into a H inch diameter dialysis bag and dialyzed against 1 liter of 0.1 SSC at approximately 5°C for 24 hours with frequent changes in the dialyzing fluid. The chloroplast DNA was then ready for analysis, Isolation of Nuclear DNA

Purified nuclear pellets were resuspended in 5 ml of 0.1 M EDTA with 0.05 M Tris (pH 8.0) to which 2.5 per­ cent SDS (sodium dedocyl sulfate) was added. Sodium per- chlorate was added to a final molarity of 1.5 and the suspension was incubated for 20 minutes at 37°C. The solution was shaken with an equal volume of chloroforro- isoamyl alcohol (.24 : 1 volume : volume) for 30 minutes at room temperature and then centrifuged for 10 minutes at 7,000 rpm. The aqueous phase was separated, and com­ bined with two volumes of ice cold ethanol. The crude precipitating DNA was immediately spooled with a glass rod and redissolved in 3.0 ml of SSC. Heat-treated pancreatic ribonuclease (Marmur 1961) was added to the solution at a final concentration of 50 yg/ml and the solution was in­ cubated at 37°C for 20 minutes. This enzymatic solution was then shaken with an equal volume of chloroform-isoamyl alcohol as previously described. Having been shaken for

10 minutes, the suspension was centrifuged at 7,000 rpm for

15 minutes. The aqueous phase was retained and then com­ bined with 2 volumes of ice cold ethanol. Semi-purified

DNA was quickly spooled with a glass rod and redissolved in 2 ml of SSC; one volume of 2.5 M phosphate buffer (made by mixing 20 volumes of 2.5 M I^HPO^ and 1 volume of 33 percent phosphoric acid) was added (Bellamy and Ralph 1968}. 37 To this mixture, one volume of 2-methoxyethanol was added,

and the solution was shaken vigorously for 3 minutes.

Following this treatment, the solution was centrifuged for

5 minutes at 10,000 rpm. At the end of the centrifugation,

the clear upper phase, which contained DNA, was retained

for final purification. Final purification of the nuclear

DNA was accomplished by adding enough sodium acetate to

bring the solution to 0.3 M and then slowly adding 0.52

volumes of isopropyl alcohol at room temperature. The

DNA formed a white fiberous percipitate which was spooled

with a glass rod. It was then dissolved in SSC and dialyzed

overnight against 1 liter 0.1 SSC at approximately 5°C.

Preparation of Denatured and Denatured-Renatured Nuclear and Chloroplast DNA

DNA was heated at 100°C for 10 minutes in SSC. Ali-

quotes were taken immediately and frozen; other samples

were adjusted to 2.0 SSC and incubated for 120 minutes at

60°C. The samples were then ready for buoyant density

studies.

Preparation of DNA for CsCl Analytical Buoyant Density Centrifugation

Sample DNA was taken at a concentration of approxi­

mately 2 yg/0.6 ml to which was added approximately 2.0 yg

of .Micrococcus lysodikticus. Nine-tenths of a grain of

optical grade CsCl was added to this mixture and adjusted 38 to a final density of 1.71 gm/cc by means of a Bosch and

Lomb refTactometer with a refractive index at room tem­ perature of 1.400 (Mandel, Schildkraut and Marmur 1968).

The DNA sample was then centrifuged in a Spinco

Model-E Analytical Centrifuge at 44,000 rpm for 20 hours at 20°C using an AN-D rotor. Pictures were taken using

U.V. optics and Kodak commercial film. The negatives of the banded DNA samples were traced on a Beckman Analytrol and relative buoyant densities were calculated by the method of Sueoka (.1961). The equation used for calculating buoyant density is as follows:

2 2 P = PQ — 0.00892 (r — r ) gm/cc.

where

PQ = density of the marker (Micrococcus lysodikticus

DNA has a buoyant density of 1.731 gm/cc in CsCl)

r = distance of the marker DNA from the axis of rota- o tion

r = distance of the sample DNA from the axis of rota­

tion

0.00892 = 4.2 (w2) (10~10)

and

w = speed of rotation of the rotor in rpm. 39

DNA Preparation for Electron Micrographs

One to 2 yg of DNA was taken in 0.2 ml of 0.1 SSC, and 20 pi of cytochrome c (20 yg/ml) was added. In addi­ tion, one volume of 5 molar ammonium acetate and 10 yl of 1 percent isopropyl alcohol was added to the DNA solu*- tion. The solution was then gently passed down a glass incline slide onto a hypophase of glass distilled water in a Langamier trough (Wong and Wildman 1972). The DNA was picked up on carbon stabilized copper grids coated with formbar, and washed for 10 seconds in 95 percent ethanol.

This procedure was followed by blot drying on filter paper.

The DNA was shadowed on a rotary turntable with platinum- palladium. Specimens were examined in a Phillips EM 200 electron microscope.

Preparation of Leaf-Sections for Electron Microscopy

Leaf-sections from green, yellow, and white leaves were fixed with 5 percent gluteraldehyde in 0.1 molar phosphate buffer (Sorenson buffer), for 7 hours by the method of Schraid, Price and Gaffron (1966). The leaves were then washed overnight in Sorenson's buffer and treated with

2 percent OsO^ in phosphate buffer for 4 hours. The samples were then dehydrated in cold absolute ethanol, followed by three 5 minute changes in propylene oxide. The leaves were then embedded in epon, stained with Reynolds lead 40 citrate, and sectioned with a diamond knife on a Porter-

Blume microtome. The sections were then examined in a

Phillips -200 electron microscope. RESULTS

Analysis of Cotton Chloroplastic and Nuclear DNA in Mutant and Wild Type Tissue

Chloroplast DNA was found to be much more difficult to extract in green leaf tissue than from the more fragile yellow and white leaf tissue. Light microscopic examination of the various preparations (see Materials and Methods) revealed a significant amount of starch granules associated with the green tissue chloroplasts, while in mutant tissue a much smaller amount of starch granules were found as­ sociated with the chloroplast fraction. The greater tissue strength and larger amount of starch associated with the green leaves may contribute to the low recoveries of chloroplast DNA, Incubation of chloroplast fractions from green tissue with a-amylase, and increasing the time of shaking the chloroplast preparation with chloroform-isoamyl alcohol, did in fact lead to higher yields of chloroplast

DNA. For this reason these treatments were incorporated into extraction procedures for chloroplast DNA extraction from green leaf tissue. In other respects, the extraction procedures were similar to those used for obtaining chloro­ plast DNA from mutant yellow and white leaf tissue. Chloro­ plast DNA was obtained from green (gr. cDNA), yellow (yw.

41 cDNA), and white (w. cDNA) leaf tissue. The different chloroplast DNAs banded in ethidium bromide CsCl gradients and were visible under black light, as were control samples of M, lysodikticus and cotton nuclear DNA. Chloroplast

DNA ranged between 10 and 15 yg/ml for each extraction

(45 grams of green leaf tissue) as measured by optical density values at 260 nm.

Typical absorption spectra of chloroplast DNA from white and green leaf tissue are shown in Figures 2 and 3 respectively. Characteristically, chloroplast DNA from cotton show unusually high absorption at 230 nm, which may be due to the presence of gossypol. During extraction of cotton chloroplasts, a light brown color was observed in the supernatant after the initial differential centri- fugation. Since gossypol is present in significant amounts in glanded cotton it is believed that it could account for the high absorption at 230 nm in cotton chloroplast DNA preparations,

Three separate chloroplast DNA extractions from white tissue, and two separate chloroplast DNA extractions each from the yellow and green tissue, were analyzed for buoyant density values. Table 4 records the buoyant density values of cDNA obtained in each of the studies. Figures 4f

5, and 6 show densitometer tracings of photographs taken during analytical centrifugation of the respective cDNAs 0.5

0.4

-p>i •H U) C 0.3

0.1

220 230 240 250 260 270 280 290 300 (A) Nanometers

Figure 2. Typical absorption profile of purified white tissue cotton chloroplast DNA.

oj 220 230 240 250 260 270 280 290 300 (X) Nanometers

Figure 3. Typical absorption profile of purified green tissue cotton chloroplast DNA. 45

Table 4. Cotton chloroplast and nuclear DNA buoyant density data.

GC Number Density Mole Independent Component gm/cc Tissue % Isolations

(A) Low 1.697 White 3

1.697 Green 1

1.697 Yellow 1

1.698 Green 1

1.698 Yellow 1

X±2s— = 1.697+0.0004 37.75 7 (Total)

(B) High I. 1.706 White 2

1.705 White 1

1,706 Yellow 2

1.706 Green 1

1.705 Green 1

X+2s— = 1.70610.0003 46.94 7 (Total)

CC) High II. 1.719 White 3

X+2s- = 1.719+0.0000 60.24 3 (Total

(D) Nuclear 1.692 Green 1

1.693 White 1

X = 1.6925 36.22* 2 (Total)

_ *Calculated with Kirk's (1967) correction factor "Buoyant Density + °-003 ^cc' 46

Marker DNA

Green Tissue Chloroplast DNA

Green Tissue Chloroplast DNA

1.697 1,731 1.706

Figure 4. Microdensitometer tracing of cotton chloroplast DNA from green leaf tissue.

The analytical centrifugations were made in a Beckman Model E Ultra-Centrifuge at 20 C and 44,000 rpm for 20 hours. 47

Marker DNA

Yellow Tissue Chloroplast DNA (leaf)

Yellow Tissue Chloroplast DNA (cotyledon)

1.697 1,731 1.706

Figure 5. Microdensitometer tracing of cotton chloroplast DNA from yellow leaf tissue and yellow cotyledon tissue.

The analytical centrifugations were performed as in Figure 4. 48

Marker DNA

White Tissue Chloroplast DNA

White Tissue Chloroplast DNA

White Tissue Chloroplast DNA

1.719 1.697 .731 1.706 Figure 6. Microdensitometer tracings of cotton chloroplast DNA from white leaf tissue.

The analytical centrifugations were performed as in Figure 4. 49 in CsCl. A high density component of 1.706 gm/cc, and a low density component of 1.697 gm/cc were observed for the cDNA of all three tissues. In addition a 1.719 gm/cc com­ ponent was observed in cDNA preparations from white tissue.

Comparison of the buoyant density patterns of' chloroplast DNA in white, yellow and green tissue reveals a surprising phenomena. The majority of chloroplast DNA in white tissue (49.7 percent) has a buoyant density of

1.706 gm/cc; minor buoyant density components are at 1.719

(21 percent) and 1.697 (29.3 percent) gm/cc respectively.

Yw. cDNA, extracted from either cotyledon or leaf tissue, shows a major component (68.25 percent) of buoyant density

1.697 gm/cc, and an intermediate amount of DNA of buoyant density 1.706 gm/cc (31.75 percent). No 1.719 cDNA com­ ponent was found associated with yw. cDNA.

DNA from green tissue was found also to have a major band (74.8 percent) of buoyant density at 1.697 gm/cc and a shoulder (25.2 percent) at buoyant density

1.706 gm/cc. A 1.719 gm/cc component was not found as­ sociated with gr. cDNA. Table 5 shows the relative pro­ portions of components of the gr., yw., and w. cDNA. The proportions were determined by weight of tracings on paper of each particular peak relative to the weight of all the cDNA components of the given sample. Comparison of DNA buoyant density profiles of chloroplast DNA extracted from 50

Table 5. Percentages of cotton chloroplast DNA from CsCl buoyant density analysis.

Buoyant Density Tissue Sample Band Percentage DNA*

White 1 1.719 20 1.706 55 1.697 25

White 2 1.719 21 1.706 50 1.697 29

White 3 1.719 22 1.706 44 1.697 34

Green 1 1.706 26.3 1.697 73.7

Green 2 1.706 24.1 1.697 75.9

Yellow 1 1.706 31.8 (cotyledon) 1.697 68.2

Yellow (leaf) 2 1.706 31.7 1.697 68.3

Green tissue chloroplast DNA X percent (1.697) = 74.8 Green tissue chloroplast DNA X_percent (1.706) = 25.2 Yellow tissue chloroplast DNA X percent (1.697) = 68.25 Yellow tissue chloroplast DNA_X percent (.1.706) = 31.75 White tissue chloroplast DNA X percent (1.719) = 21 White tissue chloroplast DNA X percent (1.706) = 49.7 White tissue chloroplast DNA X percent (1.697) = 29.3

•Percentages of DNA were determined by weighing paper tracings of the particular buoyant density components for any given sample. 51 the three tissues is seen in Figure 7. Similar results were recorded by Wong-Staal and Wildman (1973) in studies involving a cytoplasmic mutant of tobacco.

In order to determine if cotton nuclear leaf DNA and chloroplast DNA share any common buoyant density values nuclear DNA of white and green leaf tissue was subjected to analytical centrifugation and subsequent buoyant density analysis. Nuclear DNA (nDNA) from both sources shows the presence of only one component. The buoyant density of nDNA from the green tissue is 1.692, whereas that of the white tissue is 1.693 (Figure 8). The difference of 0.001 gm/cc buoyant density between these two nuclear DNAs is probably insignificant. Differences in the buoyant densities of chloroplast DNA and nuclear DNA are clarified by Figures

9 and 10 which compare the densitometer tracings of green leaf nuclear and chloroplastic DNA and white leaf nuclear and chloroplast DNA. The densitometer tracing in Figure 11 contrasts yellow leaf chloroplast DNA, which has a small amount of nuclear contamination, with green leaf nuclear

DNA,

Samples of denatured, denatured-renatured nuclear and chloroplast DNA were studied to see if chloroplast DNA renatures more completely than nuclear DNA. Figures 12, 13, and 14 show densitometer tracings of buoyant density cen­ trifugation of denatured and denatured-renatured DNA from: 52

Marker DNA 1.719 1.697

White Tissue Chloroplast DNA

Yellow Tissue Chloroplast DNA

Green Tissue Chloroplast DNA

1.731 1.706

Figure 7. Comparisons of microdensitometer tracings of analytical centrifugations of chloroplast DNA from white, yellow and green tissue.

The analytical centrifugations were performed as in Figure 4. 53

Marker DNA

Green Tissue Nuclear DNA

White Tissue Nuclear DNA

1,731 1.692-1.693

Figure 8. Microdensitometer tracings of cotton nuclear DNA from green leaf tissue and white leaf tissue.

The analytical centrifugations were performed as in Figure 4. 54

Marker DNA

Green Tissue Chloroplast DNA

Green Tissue Nuclear DNA

1.706 I 1.692 1.731 1.697

Figure 9. Microdensitometer tracings of cotton chloroplast DNA from green tissue in comparison to cotton nuclear DNA from green tissue.

The analytical centrifugations were performed as in Figure 4. 55

1.697 Marker DNA

White Tissue Chloroplast DNA

White Tissue Nuclear DNA

I 1,731

Figure 10. Microdensitometer tracings of cotton chloroplast DNA from white tissue in comparison to cotton nuclear DNA from white tissue.

Differences in buoyant densities for the two types of DNA are seen. The analytical centrifugations were performed as in Figure 4. 56

Marker DNA

Yellow Tissue Chloroplast DNA

1.706

Green Tissue Nuclear DNA

1.692 1.731 1.697

Figure 11. Microdensitometer tracings of cotton chloroplast DNA from yellow leaf tissue with a slight amount of nuclear contamination in comparison to cotton nuclear DNA from green leaf tissue.

The analytical centrifugations were performed as in Figure 4. 57

Marker DNA

Denatured Green Tissue Nuclear DNA

Denatured-Renatured Green Tissue Nuclear DNA

|' 1.706 1,731 1.711

Figure 12. Microdensitometer tracings of heat de­ natured and heat denatured-renatured nuclear DNA.

The analytical centrifugations were performed as in Figure 4, 58

Marker DNA

Denatured Green Tissue Chloroplast DNA

1.712

Denatured-Renatured Green Tissue Chloroplast DNA

1,731 1.700

Figure 13. Microdensitometer tracings of heat de­ natured and heat denatured-renatured chloroplast DNA from green tissue.

The analytical centrifugations were performed as in Figure 4. Marker DNA

Denatured White Tissue Chloroplast DNA

Denatured-Renatured White Tissue Chloroplast DNA

1.712 1.731 1.702

Figure 14. Microdensitometer tracings of chloro­ plast DNA which had heat denatured-renatured chloroplast DNA from white tissue.

The analytical centrifugations were performed as in Figure 4. 60 green leaf tissue nuclei, green leaf tissue chloroplasts, and white leaf tissue chloroplasts. Denaturation- renaturation buoyant density differences are greater for those of gr. cDNA and w. cDNA than those obtained for cotton nuclear DNA. This greater difference found in chloroplast DNA preparations reflects either a greater amount of redundancy or a greater amount of a singular type of DNA found in chloroplast DNA in relation to the heterogeneity associated with DNA from a nuclear source.

Figure 15 compares the densitometer tracing of denatured- renatured green tissue chloroplast DNA with that of denatured-renatured green tissue nuclear DNA; one can visualize from this figure that denatured-renatured chloro­ plast DNA comes closer to its native buoyant density than does the corresponding nuclear DNA.

Calculations for GC content from buoyant density bands of nuclear and chloroplast DNA are shown in Table 5.

Kirk's (1967) correction factor for obtaining GC content of plants which contain 5-methyl cytosine was employed in the analysis. The equation which relates buoyant density values to GC content follows (Schildkraut, Marraur and Doty,

1962); 61

Marker DNA

Denatured-Renatured Green Tissue Nuclear DNA

Denatured-Renatured Green | Tissue Chloroplast DNA

1.731 1.706

Figure 15. Microdensitometer tracings comparing heat denatured and heat denatured-renatured chloroplast and nuclear DNA from green leaf tissue.

The analytical centrifugations were performed as in Figure 4. 62 where

CGC) - mole fraction of guanine cytosine in the native

DNA

p = buoyant density of sample DNA

Summary of Results from Buoyant Density Studies of Cotton Nuclear and Chloroplast DNA

The results from buoyant density studies of chloro­ plast and nuclear DNA reveal the following:

(A) Chloroplast DNA from green and yellow tissue has a main buoyant density band of 1.697 gm/cc and it also has one satellite of buoyant density 1.706 gm/cc. Approximately

74.8 percent of the gr. cDNA is found in the low density component, while 25.2 percent of the DNA is associated with the 1.706 gm/cc component. In yw. cDNA 68.25 percent of the DNA is associated with the 1.697 DNA component and 31.75 percent of the cDNA is associated with the 1.706 component.

White tissue chloroplast DNA has two high density bands of

1.719 and 1.706 gm/cc respectively and a low density band at 1.697 gm/cc. The 1.719 band of w. cDNA contains 21 percent of the DNA while the 1.706 and 1.697 contain 49.7 and 29.3 percent respectively.

(B) A reversal in the relative amounts of 1.697 and

1.706 chloroplast DNA is seen between green tissue chloro­

plast DNA and white tissue chloroplast DNA. With regard to only the 1,706 and 1.697 components of w. cDNA the 1,706 63 component contains approximately 61,9 percent of the DNA, while in yellow and green tissue chloroplast DNA the 1.706

DNA component contains approximately 31.75 and 25.2 percent of the cDNA respectively.

(C) Chloroplast DNA which has been denatured and re- natured shows a greater amount of reannealing than similarly treated nuclear DNA,

(D) Cotton nuclear DNA has a buoyant density of 1.692-

1.693 gm/cc which is significantly lower than any of the components of chloroplast DNA from cotton.

(E) Components of cotton chloroplast DNA have higher

GC molar percentages than do those of the corresponding nuclear DNA,

Chloroplast Preparations Examined by Electron Microscopy

Electron micrographs were taken in order to compare chloroplasts of green, white, and yellow leaf tissue. The chloroplasts from green tissue, seen in Figures 16, 17, and 18 exhibit well developed thalykoids and grana. Figures

19, 20, and 21 show chloroplasts from yellow leaf tissue.

In these mutant chloroplasts, grana are completely lacking.

Figures 22, 23 and 24 show chloroplast-like vesicles of white leaf tissue. Both grana and thalykoid development is absent from these plastids. Katterman and Endrizzi (1973} found that chloroplasts from mutant yellow and white tissue 64

Figure 16. Electron micrograph of green leaf tissue chloroplast.

Grana and thalykoid development is found in this chloro­ plast. 65

Figure 17. Electron micrograph of green leaf chloroplasts.

Grana and thalykoid development is found in these chloro­ plasts. 66

Figure 18. Electron micrograph of chloroplasts of green leaf tissue.

Thalykoid development is found in these chloroplasts. Figure 19. Electron micrograph of yellow leaf tissue chloroplast.

Only thalykoid development is found in this chloroplast. Figure 20. Electron micrograph of yellow leaf chloroplast.

Only thalykoid development is found in this chloroplast. Figure 21. Electron micrograph of chloroplast of yellow leaf tissue.

Only thalykoid development is found in this chloroplast. 70

Figure 22. Electron micrograph of white leaf tissue chloroplast.

No grana or thalykoid development is found in this chloro­ plast. 71

Figure 23. Electron micrograph of white leaf chloroplast.

No grana or thalykoid development is found in this chloro­ plast. 72

Figure 24. Electron micrograph of chloroplast of white leaf tissue.

No grana or thalykoid development is found in this chloro­ plast. appeared approximately one half the size of chloroplasts from normal tissue, when viewed under phase-contrast light microscopy. No normal chloroplasts were seen in examina­ tion of mutant white tissue, and less than 50 percent of the yellow leaf chloroplast had normal morphology. The

physical appearance of mutant chloroplasts in this study

are consistent with the physical appearance of chloroplasts from corn (Shumway and Weier 1967) and cotton (Kohel and

Benedict 1971),

Electron Microscopic Study of Isolated ~~ Chloroplast DNA

In order to examine the condition of cDNA prepared

from CsCl gradient centrifugation electron micrographs were

taken of isolated cotton chloroplast DNA which had been

spread on a Langmuier trough, picked up with carbon

stabilized copper grids, and shadowed on a rotary turn

table with platinum polladium. Figure 25 reveals an elec­

tron juicrograph of the chloroplast DNA. Short fragments

and circles were found in the preparations. The circles

in the photograph CFigure 25) average 1.1 ym in contour

length. Nuclear and deoxyribonuclease treated chloroplast

DNA were utilized as controls. In the nuclear preparation

only long and short linear strands were seen; in the chloro­

plast DNA preparations, which had been pretreated with de­

oxyribonuclease, no DNA like fibrils or circles were observed. Figure 25. Electron micrograph of yellow tissue chloroplast DNA shadowed with platinum-palladium.

Circles are seen toward the lower right portion of the photo. DISCUSSION

Results from analysis of buoyant density data for normal and mutant cotton chloroplast DNA reveals: a major component at 1.697 gm/cc (74.8 percent) and a minor com­ ponent at 1.706 (25.2 percent) in gr. cDNA, a major com­ ponent at 1.697 gm/cc (68.25 percent) and a slightly larger minor component than that of gr. cDNA at 1.706 (31.75 percent; in yw. cDNA. In white tissue, cDNA contains three components of which the 1.706 peak is the largest

(49.7 percent) followed by the 1.697 (29.3 percent) and the 1.719 (21.0 percent) components.

Kirk (1971) concludes that chloroplast DNA in higher plants has an average buoyant density of 1.697 gm/cc; the higher buoyant density bands appearing in chloroplast DNA preparations can be attributed to mito­ chondrial (i.e;, 1.706 component) and bacterial (i.e.,

1.719 component) contamination. Table 2 shows various examples of chloroplast DNA from higher plants with buoyant density close to 1.697 gm/cc.

Many preparations of cDNA have been shown to have a second buoyant density band of 1.706 which Kirk (1971) assumes to be mitochondrial DNA since Suyama and Bonner

(1966) examined the mitochondrial DNA of higher plants

75 76 and discovered a buoyant density value of 1.706 qm/cc for several species. Because 1.706 components appear in chloroplast DNA isolates, it is important to evaluate the present criteria for assigning mitochondrial DNA buoyant density values to DNA isolated from higher plants, and provide alternate explanations for the origin of high buoyant density bands found in chloroplast DNA prepara­ tions.

Mitochondrial DNA in higher plants is extracted from seedlings by grinding the tissue and taking the pellet which results from high speed centrifugation of the super­ natant of a low speed differential centrifugation. This pellet is then treated with deoxyribonuclease to remove contaminating nuclear and plastid DNA. The DNA which is then extracted after deoxyribonuclease treatment is called mitochondrial DNA. The two required criteria for obtaining mitochondrial DNA are: isolation by high speed differential centrifugation, following low speed centrifugation, and de­ oxyribonuclease resistence. There is little doubt that intact mitochondria are resistant to deoxyribonuclease, while nuclei are not resistant; it is questionable whether chloroplasts are non-resistant to treatment with deoxy­ ribonuclease. Therefore, the DNA recovered after de­ oxyribonuclease treatment of mitochondrial preparations may contain plastid DNA. 77

Wells and Birnstiel (1969) extracted chloroplast

DNA from broad beans, lettuce, spinach and sweet peas which had been pre-treated with deoxyribonuclease. Whitfeld and

Spencer (1968) did not recover any DNA from spinach and tobacco chloroplasts, having pre-incubated them with de­ oxyribonuclease. Bard and Gordon (1969) reported that treatment of spinach chloroplasts with deoxyribonuclease prior to DNA extraction resulted in loss of both the 1.697 and 1.706 buoyant density components. They thereby at­ tributed both components to be of chloroplast origin on the basis of sensitivity to deoxyribonuclease. Wong and

Wildman (1972) reported that after treatment of chloroplasts or mitochondrial preparations with deoxyribonuclease they recovered a 1.705 gm/cc buoyant density band. The un- treat d DNA, from either mitochondria or chloroplasts, revealed identical buoyant density patterns in CsCl. This led Wong and Wildman to conclude that it is impossible to separate chloroplasts from mitochondria by discontinuous density gradient centrifugation. However, Kung and

Williams (1969), through examinations with the electron microscope of chloroplasts isolated on discontinuous sucrose gradients, found no mitochondrial contamination.

Therefore, criteria for distinguishing chloroplast DNA from mitochondrial DNA based on resistance or non-resistance to deoxyribonuclease is invalid. The buoyant density bands of 1.706 or greater which are found associated with chloroplast preparations cannot be attributed to mito­ chondria on the basis of deoxyribonuclease resistance.

Discontinuous sucrose gradients that were used in the isolation of purified chloroplasts in this study were similar to those used by Kung and Williams C1969) in their electron microscopic study of chloroplast fractions. One or two high density bands were found in all buoyant density runs involving cotton chloroplast DNA. By rejecting mito­ chondrial contamination of the chloroplast bands isolated by discontinuous sucrose gradients and by using green house grown plant material the possibility of mitochondrial and bacterial sources for the high density components found in cotton cDNA preparations can be minimized.

Wong-Staal and Wildman (1973) reported that in a veriegated mutant of tobacco, different buoyant density patterns were realized for chloroplast DNA extracted from the white leaf tissue and the green leaf tissue. In the white leaf tissue, a high density component of 1.705 gra/cc was the predominate DNA; in the green tissue a low density component of 1.700 gm/cc was the major DNA. This apparent reversal of major DNA fractions in the mutant and normal chloroplasts was explained by an increase in 1.705 DNA in the mutant tissue. Since a DNA of buoyant density

1.705 had previously been shown, on the basis of 79 deoxyribonuclease resistance, to be mitochondrial DNA, the authors implied that white tissue contained more mito­ chondrial DNA than the green leaf tissue.

In this study and analysis of chloroplast DNA of

cotton, a reversal of 1.706 and 1.697 peaks of white and green leaf preparations has been demonstrated. Thus, there

are two reports of this phenomena in different cytoplasmic

mutants of tobacco and cotton. Other possibilities beside

bacterial and mitochondrial contamination will be dis­

cussed for the origin of chloroplast DNA components.

It is possible that chloroplast DNA contains more

than one chromophore. Wells and Birnstiel (1969) found

two distinct fractions of chloroplast DNA exhibiting dif­

ferent kinetic complexities. Wong and Wildman (1972)

found small circles associated with the 1.705 DNA common

to both mitochondrial and chloroplast sources. Nass and

Ben-Shaul (1972) reported findings of 3.6 m circular DNA

molecules associated with the 1.701 gm/cc buoyant density

component of Euglena chloroplast DNA. This high density

component was found to have a high hybridization potential

with 70s ribosomal RNA and it will be discussed further in

connection with non-random shearing of cDNA. Differential

localization of DNA has been reported in wheat chloroplasts,

and this could represent discrete chromophores in these

chloroplasts. Episomes, such as those common to bacteria 80 cannot be ruled out as a source of chloroplast DNA com­ ponents, although none of these structures have been iso­ lated from chloroplasts. Thus, if chloroplasts had more than one chromophore each of these structures could contain

DNA of different buoyant density and this would lead to multiple buoyant density patterns associated with cDNA analysis.

However, circular chloroplast DNA of approximately

40 pi has been reported in spinach and peas and no small circular molecules were observed (Manning, Wolstenholme, and Richards 1972; Kolodner and Tewari 1972). It is known that fragmented DNA can reanneal to form circular DNA.

This could be a possible source of the small circles ob­ served in Euglena. Nevertheless, it can not be ruled out that chloroplast genomes have distinct and discrete DNA components in the form of both large and small circular molecules.

The nature of DNA extraction is such that the DNA is subjected to fragmentation. Pipetting, extraction with syringes, spooling and vortexing, all lead to shearing of the DNA. By very mild extraction procedures, DNA still becomes fragmented. Woodcock and Fernandez-Moran (19681 have reported large amounts of fragmented DNA from chloro­ plasts subjected to osmotic lysis. Kolodner and Tewari

(1972) reported small fragments of chloroplast DNA isolated with SDS and chloroform-isoamyl alcohol. 81

Evidence for non-random shearing of chloroplast DNA in Euglena gracilis was noted by Rawson and Haselkorn

(1973). Chloroplast DNA of buoyant density 1.685 gm/cc was subjected to a slight shearing force which decreased the average molecular weight of the chloroplast DNA from 7 6 1.25 x 10 daltons to 3.3 x 10 daltons. Buoyant density analysis of this sheared DNA yielded two components with buoyant density values of 1.685 gm/cc and 1.701 gm/cc respectively. A high density component of 1.701 gm/cc had previously been associated with DNA extracted from

Euglena chloroplasts (Stutz and Vandrey 1971; Manning et al. 1971; Nass and Ben-Shaul 1972; and Manning and

Richards 1972). Thus, the appearance of 1.701 chloroplast

DNA as a result of shearing the 1.685 DNA argues for non- random shearing of chloroplast DNA. Ribosomal RNA of chloro­ plasts from Euglena has a GC content of 51.5 percent. This corresponds to a DNA with a buoyant density of 1.701 gm/cc

(Rawson and Stutz 1969). Stutz and Vandrey (1971) have

shown in Euglena that chloroplast ribosomal RNA binds six times greater to 1.701 DNA than to 1.685 chloroplast DNA.

This shows that 1.701 cDNA may be a region of the DNA

responsible for RNA coding as well as for other high GC

coded messages.

The nature of high density chloroplast and nuclear

satellite DNA in higher plants has been examined and its 82 relationship to ribosomal RNA has been shown. Chloroplast ribosomal RNA from higher plants has a GC content of approxi­ mately 56 mole percent (Table 1); this corresponds to a region of DNA with buoyant density 1.715 gm/cc. If non- random shearing took place in cotton chloroplast DNA, and a region of the DNA that was sheared contained genes for ribosomal RNA, the corresponding sheared fraction of chloroplast DNA could have a higher buoyant density than the main band of DNA.

Jaworski (1969) reported in his study of pumpkin that a high density chloroplast DNA component (1.706 gm/cc) preferentially bound ribosomal RNA components from both 70s and 80s ribosomes. The hybridization of RNA from 70s chloroplast ribosomes to the chloroplast satellite DNA was almost four times as great as the hybridization of 80s ribosomal RNA to the high density chloroplast DNA satellite.

Matsuda, Siegel and Lightfoot (1970) reported that in pumpkin nuclear DNA there is a high buoyant density component of 1.708 gm/cc. It has been shown that this high density DNA preferentially hybridized with 70s ribo­ somal RNA, although it did show some hybridization potential for 80s ribosomal RNA. Considering this DNA as a product of non-random shearing of the nuclear DNA, the satellite species of pumpkin DNA could form from a breakage of the

DNA near a high GC region which contains the 70s ribosomal 83

RNA cistrons. In muskmelon, a similar high density nuclear satellite DNA has been found with a buoyant density of

1.719 gm/cc (Bendich and Anderson 1974). This corresponds to a GC content of 60 percent, which is very reflective of the GC content of chloroplast 70s ribosomal RNA. The high density satellite DNA preferentially hybridized 70s ribo­ somal RNA nearly three times as much as that of the main buoyant density component of muskmelon nuclear DNA. Thus, the high density satellite of muskmelon nuclear DNA could originate from non-random shearing of the nuclear DNA.

It has been noted previously (see Results) that chloroplast fractions of mutant tissue in cotton contain a much greater amount of chloroplast fragments than do chloroplast fractions isolated from normal leaf tissue.

Figures 16-23 reveal that mutant chloroplasts have greatly reduced internal development as compared to chloroplasts from normal green tissue. The lack of internal development may result in a loss of strength in the mutant chloroplasts.

Thus, DNA isolated from mutant chloroplasts could be ex­ posed to more shearing forces than the relatively protected

DNA of the green tissue chloroplasts. A greater exposure to shearing forces of mutant chloroplast DNA may lead to a larger amount of non-random shearing: therefore to a greater amount of high buoyant density bands associated with chloroplast DNA of the mutant tissue. In white 84 tissue, in which, a .maximum amount of non-random shearing may take place, a high buoyant density band of 1.706 gm/cc is the predominate species, and a second high buoyant den­ sity band of 1,719 gm/cc is also present.

In the yellow tissue, in which an intermediate amount of non-random shearing could take place, a greater amount of 1.706 chloroplast DNA is observed than in the green tissue where the lowest amount of non-random shearing would take place. Thus, a process of non-random shearing, which takes place adjacent to high GC regions of the chloro­ plast DNA, can explain the different buoyant density patterns observed in chloroplast DNA extracted from the three types of cotton leaf tissue.

Non-random shearing of DNA, which results in high density fractions, reflects the internal structure of the

DNA. A localization of GC within the DNA suggests the existence of a base or GC concentration polarity within the DNA,

This polarity may reflect regional ordering of cistrons involved in the same, or similar, biochemical processes, much like the localization of bacterial operons discovered by Jacob and Monod (1961). Many similarities have been found between organelle and bacterial systems such as: possession of 70s ribosomes and their associated

RNA components, relative genomic sizes, lack of protein complexes associated with the DNA, and rates of DNA syn­ thesis. Recently, it has been shown that ribosomal RNA of Euglena chloroplasts hybridizes almost exclusively with the heavy strand of chloroplast DNA (Stutz and Rawson 1970).

Prokaryotic genomes, with their localization of genes into operon-like systems, may reflect the genomic organization of chloroplast DNA. In turn, a localization of specific bases in the chloroplast genome may be a physical represen­ tation of higher genetic order in the chloroplast genome. SUMMARY

A cytoplasmic chloroplast mutant, and its associated chloroplast DNA, has been examined in cotton (Gossyprum hirsutum). The chloroplast DNA was found to have three different buoyant density patterns. Chloroplast DNA from the green tissue had a predominate buoyant density com­ ponent of 1.697 gm/cc and a satellite buoyant density com­ ponent of 1.706 gm/cc. Chloroplast DNA of yellow tissue had a main component of buoyant density 1.697 gm/cc, and an enriched-satellite band of buoyant density 1.706 gm/cc.

White tissue chloroplast DNA had; a main band of buoyant density 1.706 gm/cc, a large satellite of buoyant density

1.697 gm/cc, and a small satellite of buoyant density

1.719 gm/cc.

Non-random shearing of the chloroplast DNA extracted from mutant tissue could result in greater amounts of high buoyant density bands. This non-random shearing has been offered as a possible explanation of buoyant density patterns found for various components of chloroplast DNA, Cotton nuclear DNA was shown to have a significantly lower buoyant density than any of the chloroplast DNA components.

Denatured-renatured DNA of nuclear origin had a higher

86 buoyant density, and subsequently, a slower reannealing rate than that of cotton chloroplast DNA.

Electron micrographs of cotton chloroplast DNA reveal many short fragments and some small circles of DNA.

Examination of leaf tissue from green, white, and yellow leaves of cotton showed normal chloroplasts in the green tissue and chloroplasts with only thalykoid development in the yellow tissue. Chloroplasts in white tissue had no internal lamelar development as seen by electron microscopy. APPENDIX A

EVIDENCE THAT THE CHLOROPLAST MUTANT OP COTTON IS A TRUE CYTOPLASMIC MUTANT

Published evidence cites that the chloroplast mutant used in this study is a cytoplasmic mutant (Endrizzi et al. 1974). Tables 6 and 7, which have been taken from the above publication, display segregation patterns of the chloroplast mutation in cotton. A maternal line of uni- prental inheritance is observed in segregates of this mutant as seen in these two tables. Therefore, the mutant can be considered a true cytoplasmic mutant and most likely is of chloroplast origin.

88 Table 6. Cross pollinations between variegated plants and normal green plants.

Pooled results of individual crosses and the segregation of the generation. After Endrizzi et al. 1974.

Number of cross bolls, and the number of seedlings from each boll that produced: Seedlings that were Seedlings that Seedlings that were all green segregated all yellow Cross1 Number of Number of Number of Number of Number of Number of bolls seedlings bolls seedlings bolls seedlings

Green X Variegated-A** 84 1693

Green X Variegated-B*** 15 229

Variegated-A X Green 7 193 27 666 130

Variegated-B X Green 14 391

*Parent listed first in a cross in the female.

**Variegated-A = flower used in the cross had variegated bracteoles and was on variegated fruiting and vegetative branches.

***Variegated-B = flower used in the cross did not have variegated bracteoles and was on nonvariegated (green) fruiting and vegetative branches of a variegated plant. Supposedly, these flowers contain no mutant plastids. 90

Table 7. Typical segregation ratios observed in the F^ generation from individual crosses of variegated-A mutant plants X normal green plants.

Number of Plants Cross Number Green Variegated Yellow

206 32

219 19 2

179 22 9 1

101 5 18 8

229 2 8 16

207 2 29

208 28

After Endrizzi et al. 1974. LITERATURE CITED

Bard, S. A. and M. P. Gordon, 1969. Studies on spinach chloroplast and nuclear DNA using large-scale tissue preparations. Plant Physiol. 44: 377-384.

Bell, P. R., 1961. Interactions of nucleus and cytoplasm during cogenesis in Pteridium aguilinum. Proc. Roy. Soc. (London) 153: 421-432.

Bellamy, A. R. and R. K. Ralph, 1968. Recovery and purifica­ tion of nucleic acids by means of cetyltrimethyl- aramonium bromide. In Methods of Enzymology, L. Grossman and K. Moldave Eds., Vol. XII, Part B, pp. 156-160, Academic Press, New York.

Bendich, A. J. and R. S. Anderson, 1974. Novel properties of satellite DNA from muskmelon. Proc. Natl. Acad. Sci. 71: 1511-1515.

Beridze, T. G., M. S, Odintsova, and N. M. Sissakian, 1967. Distribution of DNA components of bean leaves in all fractions, Mol. Biol. 1: 122-133.

Biggins, J. and R. B. Park, 1964. Nucleic acid content of chloroplasts of spinach isolated by a non-aqueous technique. Nature 203: 425-426.

Boardman, N. K., R. I. B. Francki, and S. G. Wildman, 1965. Protein synthesis by cell-free extracts from tobacco leaves II. Association of Activity with Chloroplast Ribosomes. Biochemistry 4: 872-876.

Borst, P., A. M. Kroon, and G. J. C. M. Ruttenberg, 1966. Mitochondrial DNA and other forms of cytoplasmic DNA. In Genetic Elements Properties and Function. D. Shugar Ed., pp. 81-116. Academic Press, New York.

Brachet, J., 1958. New observations on biochemical inter­ actions between nucleus and cytoplasm in Amoeba and Acetabularia. Expl. Cell Res. Suppl. 6: 78-96.

Brawerman, G. and J. M. Eisenstadt, 1964. Deoxyribonucleic acid from the chloroplasts of Euglena gracilis. Biochim. Biophys. Acta 91: 477-485.

91 92

Brunk, C. F. and V. Leick, 1969. Rapid equilibrium iso- pycnic CsCl gradients. Biochem. Biophys. Acta 179: 136-144.

Chiang, K. S. and N. Sueoka, 1967. Replication of chloro- plast DNA in Chlamydomonas reinhardi during vegeta­ tive cell cycle: Its mode and regulation. Prod. Natl. Acad. Sci. 57: 1506-1513.

Chiba, Y. , 1951. Cytochemical studies on chloroplast I. Cytological demonstration of nucleic acids in chloroplasts. Cytologia 16; 259-64.

Chun, E. H. L., M. H. Vaughn, and A. Rich, 1963. Isolation and characterization of DNA associated with chloro­ plast preparations. J. Mol. Biol. 7: 130-141.

Correns, C., 1908. Verebungsversuche mit blass (gelb) grunen und blattrigen Sippen bei Mirabilis jalopa, Uritica pilulifera, und Lumaria annua. Zeitschrift fur Inchiktive Abstrammunzs und Vererbungslehre 1: 291.

EdeLman, M. C., C. A. Cowen, H. T. Epstein, and J. A. Schiff, 1964. Studies of chloroplast development in Euglena VII. Chloroplast-associated DNA. Pi*oG. Natl. Acad. Sci. 52: 1214-1219.

Endrizzi, J. E., F. R. H. Katterman, W. D. Fisher1, and L. S. Stith, 1974. Extrachromosoma1 inheritance of a variegated chlorophyll mutant in cotton. Egypt. J. Gen. Cytol. 3: 277-284. 3 . . Gifford, E. M., 1960. Incorporation of H-thymidme into shoot and root apices of Ceratopteris thalictroided« Am. J. Botany 47: 834-837.

Green, B. R. and M. P. Gordon, 1965. Satellite DNA's ot tobacco. Federation Proc. 24; 539.

Green, B. R. and M. P. Gordon, 1967. The sattelice DNA'a of some higher plants. Biochim. Biophys. Acta 145: 378-390.

Green, B. R., V. Heilborn, S. Limbosch, M. Boloukhera, and J. Brachet, 1967. The cytoplasmic DNA'S of Acetabularia mediterranea. Proc. Natl. Acad. Sci. 58: 1351-1358. 93

Gunning, B. E. S., 1965. The fine structure of chloroplast stroma following aldehyde osmium tetroxide fixation. J. Cell. Biol. 24: 79-93.

Heber, U., 1963. Ribonucleinsauen in den chloroplasten der blattzelle. Planta 59: 600-616.

Honda, S. I., S. G. Wildman, and T. Hongladarom, 1962. The appearance and properties of spinach chloro- plasts isolated in NaCl, sucrose, and ficoll- dextran. Plant Physiol. 37: xli.

Iwamura, T., 1960. Distribution of nucleic acids among subcellular fractions of Chlorella. Biochem. Biophys. Acta 42: 161-163.

Jacob, F. and J. Monod, 1961. On the regulation of gene activity. Cold Springs Harbor Symp. Quant. Biol. 26: 193-211.

Jacobi, G. and E. Perner, 1964. Strukturelle und bio- chemische probleme der chloroplastenisolierung. Flora 150: 209-226.

Jacobson, A. B., H. Sv/ift, and L. Bogorad, 1963. Cyto- chemical studies concerning the occurrence and distribution of RNA in plastids of Zea Mays. J. Cell Biol. 17: 557-570.

Jagendorf, A. T., 1955. Purification of chloroplast with glycerol density gradient centrifugation. Plant Physiol. 30: 138-143.

James, W. 0. and V. S. R. Das, 1957. The organization of respiration in chlorophyllous cells. New Phy- tologist 56: 325-343.

Jaworski, A. J., 1969. Some Physical and Functional Characteristics of Chloroplast DNA. Ph.D. Disserta­ tion, Univ. of Arizona, Tucson.

Jaworski, A., P. R. Whitfeld, and A. Siegel, 1969. Two fractions of DNA associated with chloroplasts. Federation Proc. 28: 864.

Kahn, A. and D. Von Wettstein, 1961. Macromolecular physiology of plastids II. Structure of isolated spinach chloroplasts. J. Ultrastruct. Res. 5: 555-574. 94

Katterman, F. R. H. and J. E. Endrizzi, 1973. Studies on the 70s ribosomal content of a plastid mutant in Gossypium hirsutum. Plant Physiol. 51: 1138-1139.

Kirk, J. T. 0., 1963. The deoxyribonucleic acid of broad bean chloroplasts. Biochim. Biophys. Acta 76: 417-424.

Kirk, J. T. 0., 1967. Effects of methylation of cytosine residues on the buoyant density of DNA in caesium chloride solutions. J. Mol. Biol. 28: 171-172.

Kirk, J. T. 0., 1971. Will the real chloroplast DNA please stand up? In Autonomy and Biogenesis of Mitochondria and Chloroplasts, N. K. Boardman, A. Linnane, and R. M. Smillie Eds. North Holland Publish. Co., Amsterdam.

Kirk, J. T. 0. and R. A. E. Tilney-Bassett, 1967. The Plastids. W. H. Freeman and Company. London and San Francisco.

Kislev, N., H. Swift, and L. Bogorad, 1965. Nucleic acids of chloroplasts and mitochondria in Swiss Chard. J. Cell Biol. 25: 327.

Kohel, R. J. and C. R. Benedict, 1971. Description and CO^ metabolism of abberant and normal chloroplasts in variegated cotton, Gossypium hirsutum L. Crop Sci. 11: 486-488.

Kolodner, R. and K. K. Tewari, 1972. Molecular size and conformation of chloroplast deoxyribonucleic acid from pea leaves. J. Biol. Chem. 247: 6355-6364.

Kung, S. D. and J. R. Williams, 1969. Chloroplast DNA from broad bean. Biochim. Biophys. Acta 195: 434-445.

Leech, R. M., 1966. Comparative biochemistry and compara­ tive morphology of chloroplasts isolated by dif­ ferent methods. In Symposium of Biochemistry of Chloroplast, T. W. Goodwin Ed., pp. 65-74. Academic Press, London.

Leff, J. M., M. Mandel, H. T. Epstein, and J. A. Schiff, 1963. DNA satellites from cells of green and aplastidic algae. Biochem. Biophys. Rev. Commun. 13: 126-130. 95

Littau, V. C., 1958. A cytochemical study of the chloro- plasts in some higher plants. Am. J, Botany 45: 45-53.

Lyttleton, J. W., 1962. Isolation of ribosomes from spinach chloroplasts. Expl. Cell Res. 26: 312-317.

Lyttleton, J. W. and G. B. Peterson, 1964. The isolation of deoxyribonucleic acid from plant tissue. Biochim. Biophys. Acta 80: 391-398.

Mandel, M., C. L. Schildkraut, and J. Marmur, 1968. Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. In Methods of Enzymology, Vol. XII, part B, pp. 184-195, L. Gross­ man and K. Moldave Eds., Academic Press, New York.

Manning, J. E. and 0. C. Richards, 1972. Synthesis and turn­ over of Euglena gracilis nuclear and chloroplast de­ oxyribonucleic acid. Biochem. 11: 2036-2043.

Manning, J. E., D. R. Wolstenholme, and 0. C. Richards, 1972. Circular DNA molecules associated with chloroplasts of spinach. J. Cell Biol. 53: 594-601.

Manning, J. E., D. R. Wolstenholme, R. S. Ryan, J. A. Hunter, and O. C. Richards, 1971. Circular chloroplast DNA from Euglena gracilis. Proc. Natl. Acad. Sci. 68: 1169-1173.

Marmur, J., 1961. A procedure for the isolation of deoxy­ ribonucleic acid from micro-organisms. J. Mol. Biol. 3: 208-496.

Matsuda, K., A. Siegel, and D. Lightfoot, 1970. Variability in complementarity for chloroplastic and cytoplasmic ribosomal ribonucleic acids among plant nuclear de­ oxyribonucleic acids. Plant Physiol. 46: 6-12.

Metzner, H., 1952. Cytochemische untersuchungen uber das vorkommen von wukleinsauren in chloroplasten. Biol, Zentrablatt 71: 257-272.

Murakami, S., 1963. Ribosomes in spinach chloroplasts. Expl. Cell Res. 32: 398-400.

Nass, M. M. K. and Y. Ben-Shaul, 1972. A novel closed circular duplex DNA in bleached mutant and green strains of Euglena gracilis. Biochim. Biophys. Acta 272: 130-136. 96

Orth, G. .M. and D. G. Cornwell, 1963. The isolation and composition of chloroplasts and etiolated plastids from corn seedlings. Biochim. Biophys. Acta 71: 734-736.

Oshil, Y and F. Hase, 1968. Studies on nucleic acids in chloroplasts isolated from Chlorella phecoides. Plant and Cell Physiol. 9: 69-85.

Rawson, J. R. and R. Haselkorn, 1973. Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis. J. Mol. Biol. 77: 125-132.

Rawson, J. R. and E. Stutz, 19 69. Isolation and char­ acterization of Euglena gracilis cytoplasmic and chloroplast ribosomes and their ribosomal RNA components. Biochim. Biophys. Acta 190: 368-380.

Ray, D. S. and P. C. Hanawalt, 1964. Properties of the satellite DNA associated with the chloroplasts of Euglena gracilis. J. Mol. Biol. 9: 812-824.

Rhoades, M. M., 1955. Interaction of genie and non-genic hereditary units and the physiology of non-genic inheritance. In Encyclopedia of Plant Physiology, Vol. I, pp. 19-57, W. Ruhland Ed. Springer-Verlag, Berlin.

Ris, H. and W. Plaut, 1962. Ultra-structure of DNA con­ taining areas in the chloroplasts of Chlamydomonas. J. Cell Biol. 13: 383-391.

Rossi, L. and C. Gualerzi, 1970. Non-random differences in the base composition of chloroplast and cyto­ plasmic ribosomal RNA from some higher plants. Life Sci. 9: 1401-1407.

Rupple, H. G., 1964. Uber nucleinsauren in chloroplasten von Allium porrum und Antirrhinum majus. Biochim. Biophys. Acta 80: 63-72.

Sager, R., 1972. Cytoplasmic Genus and Organelles. Academic Press, New York.

Schildkraut, C. L., J. Marmur, and P. Doty, 1962. De­ termination of the base composition of DNA from its buoyant density in CsCl. J. Mol. Biol. 4: 430-443. Schmid, G., J. M. Price and H. Gaffron, 1966. Lamellar structure in chlorophyll deficient but normally active chloroplasts. J. Microscopie 5: 205-227.

Scott, N. S., 1969. Cited In Genetic Autonomy of Extra- nuclear Organelles, p. 153. K. K. Tewari, ed. In Annual Review of Plant Physiol., L. Machlis, ed., Annual Review Inc., Palo Alto.

Scott, N. S. and R. N. Smillie, 1967. Evidence for the direction of chloroplast ribosomal RNA synthesis by chloroplast DNA. Biochem. Biophys. Res. Comm. 28: 598-603.

Scott, N. S. and R. M. Smillie, 1969. Ribosomal RNA in chloroplasts of Euglena gracilis. Current Mod. Biol. 2: 339-342.

Shumway, L. K. and T. E. Weier, 1967. The chloroplast structure of Iojap Maize. Am. J. Botany 54: 773-80.

Sissakian, N. M., I. I. Philippovich, E. N. Svetailo, and K. A. Aliyev, 1965. On the protein-synthesizing system of chloroplasts. Biochim. Biophys. Acta 95: 474-485.

Smillie, R, M., 1963. Formation and function of soluable proteins in chloroplasts. Can. J. Botany 41: 123-154.

Spencer, D., 1965. Protein synthesis by isolated spinach chloroplasts. Arch. Biochem. Biophys. Ill: 381- 390.

Spencer, D. and P. R. Whitfeld, 1966. The nature of the ribonucleic acid of isolated chloroplasts. Arch. Biochem. Biophys. 117: 337-346.

Spencer, D. and P. R. Whitfeld, 1967. Ribonucleic acid synthesizing activity of spinach chloroplasts and nuclei. Arch. Biochem. Biophys. 121: 336-345.

Spencer, D. and P. R. Whitfeld, 1969. DNA synthesis in isolated chloroplasts. Biochem. Biophys. Res. Comm. 28: 538-542. 98

Steffenson^D. M. and W. F. Sheridan, 1965. Incorporation of H-thymidine into chloroplasts DNA of marine algae. J. Cell Biol. 25: 619-629.

Stocking, C. R., 1959. Chloroplasts isolated in nonaqueous media. Plant Physiol. 34: 56-61.

Stocking, C. R. and E. M. Gifford, 1959. Incorporation of thymidine into chloroplasts of Spirogyra. Biochero. Biophys. Res. Comm. 1: 159-164.

Stutz, E. and J. R. Rawson, 1970. Separation and char­ acterization of Euglena gracilis chloroplast single stranded DNA. Biochim. Biophys. Acta 209: 16-23.

Stutz, E. and J. P. Vandrey, 1971. Ribosomal DNA satellite of Euglena gracilis chloroplast DNA. Fed. Expl. Biol. Soc. 17: 227-280.

Sueoka, N., 1961. Variation and heterogeneity of base composition of deoxyribonucleic acids: A compila­ tion of old and new data. J. Mol. Biol. 3: 31-40.

Suyama, Y. and W. D. Bonner, 1966. DNA from plant mito-^ chondria. Plant Physiol. 41: 383-388.

Svetailo, E. N., I. I. Philippovich, and N. M. Sissakian, 1967. Difference in sedimentation properties of chloroplast and cytoplasmic ribosomes from pea seedlings. J. Mol. Biol. 24: 405-415.

Tewari, K. K., 1971. Genetic autonomy of extranuclear organelles. In Ann. Rev. Plant Physiol., pp. 141-168. L. Machlis ed., Annual Reviews Inc., Palo Alto.

Tewari, K. K. and S. G. Wildman, 1966. Chloroplast DNA from tobacco leaves. Science 153: 1269-1271.

Tewari, K. K. and S. G. Wildman, 1967. DNA polymerase of isolated tobacco chloroplasts and the nature of its polymerized product. Proc. Natl. Acad. Sci. 58: 689-696.

Tewari, K. K. and S. G. Wildman, 1970. Information content in the chloroplast DNA. Symp. Soc. Expl. Biol. 24: 147-179. 99

Vinograd, J. and J. E. Hearst, 1962. Equilibrium sedimenta­ tion of macromolecules and viruses in a density gradient. In Progress in the Chemistry of Organic Natural Products, p. 418, L. Zechmeister, Ed., Springer-Verlag Press, Vienna, Aus.

Vosa, C. and J. T. 0. Kirk, 1963. The genetic material of the plastid. In The Plastids, p. 337, J. T. D. Kirk and R. A. E. Tilney-Bassett, Eds., W. H. Freeman and Company, London and San Francisco.

Weier, T. E. and C. R. Stocking, 1952. A cytological analysis of leaf homogenates I. Nuclear contamina­ tion and disorganized chloroplasts. Am. J. Botany 39: 720-726.

Wells, R. and M. Birnstiel, 1969. Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochon­ drial deoxyribonucleic acid from higher plants. Biochem. J. 112: 777-786.

Whitfeld, P. R. and D. Spencer, 1968. Buoyant density of tobacco and spinach chioroplast DNA. Biochim. Biophys. Acta 157: 333-343.

Wildman, S. G., T. Hongladarom, and S. I. Honda, 1962. Chloroplasts and mitochondria in living plant cells. Science 138: 434-435.

Wildman, S. G., C. Lu-Liao, and F. Wong-Staal, 1973. Maternal inheritance, cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tobacum. Planta 113: 293-312.

Wollgiehn, R. and K. Mothes, 1964. Uber die incorporation von ^H-thymidin in die chloroplasten-DNS von Nicotiana rustica. Expl. Cell Res. 35: 52-57.

Wong, F. Y. and S. G. Wildman, 1972. Simple procedure for isolation of satellite DNA1s from tobacco leaves in high yield and demonstration minicircles. Biochim. Biophys. Acta 259: 5-12.

Wong-Staal, F. and S. G. Wildman, 1973. Identification of a mutant in chioroplast DNA correlated with forma­ tion of defective chloroplasts in a variegated mutant of Nicotiana tobacum. Planta 113: 313-326. 100

Woodcock, C. L. F. and H. Fernandez-Moran, 1968. Electron microscopy of DNA conformations in spinach chloro- plasts, J. Mol. Biol. 31: 627-631.

Yoshida, Y., 1962. Nuclear control of chloroplast activity in Elodea leaf cells. Protoplasma 54: 476-492.