Reformation of the nuclear envelope following mitosis in chinese hamster ovary cells

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Reformation of the nuclear envelope following mitosis in chinese hamster ovary cells
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Conner, Gregory E., 1950-
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Thesis:
Thesis (Ph. D.)--University of Florida, 1978.
Bibliography:
Includes bibliographical references (leaves 83-87).
Statement of Responsibility:
by Gregory E. Conner.
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Typescript.
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Vita.

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REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS











By

GREGORY E. CONNER












A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY







UNIVERSITY OF FLORIDA

1978














ACKNOWLEDGEMENTS

I would like to thank Dr. Kenneth D. Noonan, my graduate

supervisor, for his endless support and encouragement during the

course of my graduate studies.

I would also like to thank the members of my supervisory

committee, Drs. C. M. Allen, C. M. Feldherr, E. M. Hoffman, and

T. W. O'Brien for their help and advice throughout this research

project. I am particularly grateful to Dr. Feldherr for his assis-

tance in obtaining the various micrographs. I would also like to
express my thanks to Dr. Allen for his assistance with thin layer

chromatography.














TABLE OF CONTENTS

ACKNOWLEDGEMENTS. ... .. ii
LIST OF TABLES. ................... .... v
LIST OF FIGURES . vi
ABBREVIATIONS USED. . ... .. vii
ABSTRACT. . ... viii
CHAPTER I-INTRODUCTION. ... 1
NE Structure . ... .. 1
Membranes of the NE. . 3
Pore Structures. . ... .. 3
Proteinaceous Lamina ................. 4
Association of Chromatin with NE 5
Isolation and Composition of NE. 5
Isolation Procedures . 5
Composition . 7
Morphological Changes in the NE
During Cell Cycle . 9
Objectives . .12
CHAPTER II-ISOLATION AND CHARACTERIZATION ... .13
Materials and Methods. .................... 13
Materials. . .. 13
Cell Culture . 13
Isolation of Nuclei and NE ... .14
Isolation of Plasma Membrane .... 15
Chemical Assays ................... ...16
Enzyme Assays................... .. .16
Electron Microscopy. . .. 17
SDS Polyacrylamide Disc Gel
Electrophoresis (PADGE). 17
Results . .18
Isolation of Nuclei. .................. 18
Morphology of the Purified Nuclear
Fraction . 22
Isolation of NE .. ... ... 26
Morphology of the Purified NE Fraction 26
Recovery of Isolated Fractions 28
Characterization and Purity of Nuclear
and NE Fractions . 28
Enzyme Activity. ... .... .......... ..30
Chemical Composition . 32
Peptide and Glycopeptide Composition .. 34
CHAPTER III-REFORMATION OF THE NUCLEAR ENVELOPE
DURING MITOSIS . ... ... .36
Materials and Methods. .................... 36
Materials. . 36
Cell Culture and Synchrony .. 36









Labeling of Cell Cultures. ... 37
Isolation of Nuclei and NE .... 38
Determination of Specific Activities .. 38
Determination of Precursor Pool
Equilibration Rates . 39
Measurement of Nuclear Surface Areas .... 40
Polyacrylamide Gel Electrophoresis .... 40
Results . .. .. 41
Cell Synchrony ... ... .. ... ... ... 41
Peptide Composition of the NE during
Different Phases of the Cell Cycle .... 43
Dilution of [3H] Leucine Labeled NE. ... 44
Incorporation of the [3H] Leucine
into NE Protein. . 53
Dilution of [3H] Choline Labeled NE ... 59
Dilution of [32p] Orthophosphate
Labeled NE Phospholipid. ... 61
Incorporation of [32p] Orthophosphate
into NE Phospholipid .. .. .. .. .. ...... .64
Rate of Precursor Pool Equilibration .. 65
Changes in Nuclear Surface Area. ... 75
CHAPTER IV-DISCUSSION . ... .. 77
APPENDIX--DERIVATION OF THE EQUATION FOR CALCULATING
THE PROPORTION OF M/G1 NE SYNTHESIZED DE NOVO
DURING THE G2-M-G1 TRANSITION ............... 82
BIBLIOGRAPHY. . .. 83
BIOGRAPHICAL SKETCH .... ..... 88
















LIST OF TABLES

I. Recovery of Protein, DNA, and
Phospholipid in Subcellular Fractions.

II. Enzyme Activities of Subcellular
Fractions ...............

III. Chemical Content of Subcellular Fractions.

IV. Dilution of [3H] Leucine Labeled NE
Protein. ............... .

V. Dilution of Labeled NE Phospholipids .


. 29


. 31

. 33


. 47

. 60














LIST OF FIGURES

1. Diagrammatic Representation of NE
Structure .


2. Flow Diagram of Nuclei and NE Isolation
Procedures. . .. 19

3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. . .. 23

4. Electron Micrograph of the Purified Nuclear
Fraction. . ... .... .25

5. Electron Micrograph of the Purified NE
Fraction. . .. .... .27

6. Coomasie Blue Stained Profile of Sub-
cellular Fractions. . .35

7. Cell Cycle Parameters in Synchronized
CHO Cells . 42

8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells 45

9. Dilution of [3H] Leucine Labeled NE
Protein ............... .. ..... 50

10. SDS Gel Electrophoresis of NE Peptides
Isolated after Removal of [3H] Leucine
at the G2/M Boundary. . 52

11. Incorporation of [3H] Leucine into
NE Protein. . ... 55

12. SDS Gel Electrophoresis of [3H] Leucine
Pulse Labeled NE Peptides 58

13. Dilution of [32p] Labeled NE Phospholipid 63

14. Incorporation of [32p] Labeled NE Phos-
pholipid . 67

15. [3H] Leucine Pool Dilution. ... 70

16. [32P] Phosphatidic Acid Pool Dilution 74














ABBREVIATIONS USED

BME $-Mercaptoethanol

CHO Chinese hamster ovary

DNase I Deoxubonuclease I

EDTA ethylene diaminetetraacetic acid

EM electron microscopy

ER endoplasmic reticulum

HU hydroxyurea

LSC liquid scintillation counting

M mitosis

NE nuclear envelope

PBS phosphate buffered saline

PMSF phenylmethylsulfonyl fluoride

RER rough endoplasmic reticulum

RNase A Ribonuclease A

SDS sodium dodecyl sulfate

TM 50mM Tris, pH 7.6, 2.5mM MgC12










Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



REFORMATION OF THE NUCLEAR ENVELOPE
FOLLOWING MITOSIS IN CHINESE HAMSTER OVARY CELLS

By

Gregory E. Conner

August 1978

Chairman: Kenneth D. Noonan
Major Department: Biochemistry

A technique for isolating nuclei and nuclear envelopes (NE) from

Chinese hamster ovary (CHO) cells has been developed. This technique

does not depend on the use of detergents to solubilize contaminating

chromatin. In this procedure NE are prepared from purified nuclei by

nuclease digestion and subsequent high salt-sucrose gradient centrifu-

gation. The nuclei and NE fractions are free of significant contamina-

tion by other subcellular organelles as judged by electron microscopy

and enzymatic analysis. Electron microscopic observation of the puri-

fied NE clearly demonstrates that the isolated material retains both a

double bilayer and the pore complexes observed in intact nuclei,

strongly suggesting that the isolation procedure described in this

dissertation permits the recovery of "intact" NE. Biochemical analysis

of the isolated nuclei and NE shows the NE fraction to be composed

primarily of protein and phospholipid, while containing only small

amounts of DNA and RNA. Examination of the peptide composition of the

NE fraction by SDS polyacrylamide gel electrophoresis reveals a very

complex coomasie blue staining profile with prominent bands in the

55,000 to 75,000 dalton molecular weight range.








Employing this isolation technique I have examined the breakdown

and reformation of the NE during a limited stage (late G2, M, and early

G1) of the replicative cycle in synchronized populations of CHO cells.

Using [ H] leucine as a precursor for protein and either [ 3H] choline

or [32P] orthophosphate as precursors for phospholipid, I have shown

that a minimum of 60% of the early G1, NE protein and a minimum of 50%

of the early G1 NE phospholipid were present during the proceeding G2

phase of the cell cycle and were reutilized in the reformation of the

NE which occurs during late M and early G1. Pulse label studies

employing [3H] leucine or [32P] orthophosphate indicate that a burst

of NE biosynthesis occurs in early G1. Autoradiographic examination

of NE peptides isolated during these pulse label and label dilution

studies shows neither preferential loss nor preferential biosynthesis

of specific NE peptides during the G2 to M or M to G1 transition. My

evidence suggests that all the peptides of the NE, which I can resolve

in one dimensional gel electrophoresis, are synthesized during this

portion of the cell cycle. Examination of NE peptides by one dimension-

al gel electrophoresis does not highlight any reproducible changes in

NE peptide composition which can be correlated with specific phase of

the cell cycle.














CHAPTER I
INTRODUCTION

The nuclear envelope (NE) is a complex subcellular organelle

which exists at the nuclear periphery and separates the genetic

machinery of the eucaryotic cell from other cellular components. During

mitosis in many plant and animal cells, the NE becomes morphologically

indistinct and reappears after daughter chromosome separation is com-

plete. The studies which will be described in this dissertation are

directed towards understanding the mechanisms) by which this large

membranous organelle disassembles and restructures during mitosis.

Before presentation of these studies, I will briefly discuss the cur-

rently available information concerning NE structure, composition and

breakdown and reformation during mitosis.

NE Structure

The structure of the NE has been probed in great detail by many

light and electron microscopic (EM) studies (for reviews see Feldherr,

1972; Kay and Johnston, 1973; Kessel, 1973; Franke, 1974; and Franke

and Scheer, 1974). The NE (shown diagramatically in Figure 1) is com-

posed primarily of two lipid bilayer membranes which contain pore

structures. Electron microscopic observation suggests that these

bilayer membranes are continuous at the pore structures leaving apparent

gaps when viewed in transverse sections. These pore complexes do con-

tain internal structure and may be involved in nuclear-cytoplasmic

exchange of molecules (Feldherr, 1972). The outer surface of the outer
















ONM-


'PL

INM-
\\ \\\ \ 1 ^ V *' 8:* r iHC










Figure 1. Diagrammatic representation of NE structure.
ONM outer nuclear membrane; INM inner
nuclear membrane; PS pore structure; PL -
proteinaceous lamina; HC heterochromatin.







nuclear membrane is often studded with ribosomes in a manner similar to

rough endoplasmic reticulum (RER) and occasionally has been observed to

be continuous with RER (Watson, 1955). The inner aspect of the inner

nuclear membrane appears to be in close association with a proteinaceous

lamina (Fawcett, 1966) and to be intimately associated with hetero-

chromatin (i.e. dense inactive chromatin).

Membranes of the NE

The membranes of the NE, when visualized by EM, are most fre-

quently found to be 60-80 A in thickness. The two membranes are normally

separated by 100-700 A except at the pore structures where they appear

to be joined, thus forming a perinuclear cisternum. The frequent local-

ization of ribosomes and the occasional continuity of the outer membrane

with RER indicates the strong morphological similarity between membranes

of the NE and RER. As will be discussed later, the enzymatic and bio-

chemical composition of the NE serves to strengthen the resemblance of

these two membranous organelles. Currently no data are available which

conclusively demonstrate that components of the NE arise from RER or

that components of the RER arise from those of the NE.

Recent studies by Feldherr et al. (1977) and Virtanen (1977,1978)

and Virtanen and Wartiovaara.(1976) suggest that the nuclear membranes

possess a property of sidedness similar to that of other cellular mem-

branes. Specifically these authors have reported that the carbohydrate

containing portions of NE glycopeptides are located on only one side,
the cisternal side, of each nuclear membrane.

Pore Structures

When pore structures of the NE are visualized by EM in transverse

sections, they appear as gaps (150 A 700 A wide) at which the nuclear








membranes seem to be fused (Figure 1). When visualized tangentially,

the pore structures appear as complexes of 8 or 9 granules arranged as

an annulus. The central zone of the pore structure is less electron

dense than the surrounding annular material and may coincide with the

gaps seen in transverse sections. The annular pore complexes have

an overall diameter of 1000 2000 A suggesting that the pore structure

extends beyond the gaps visualized in transverse sections possibly

overlapping part of the nuclear membranes. Electron dense granules

can occasionally be seen in the interior of the pores. Some data have

been obtained which suggest that these granules are material in transit

between nucleus and cytoplasm. Many authors have suggested that the

pore complexes function in the control of nucleo-cytoplasmic communi-

cation (for review see Feldherr, 1972).

Proteinaceous Lamina

In some cell types the inner aspect of the inner nuclear membrane

appears to be associated with an electron dense material. This layer,

variously called the dense lamella (Kalifat et al., 1967), zonula

nucleum limitans (Patrizi and Poger, 1967), and the fibrous lamina

(Fawcett, 1966), ranges in thickness from 2800 A in amoeba to 150 200 A

in vertebrate cell types. This layer, apparently proteinaceous in com-

position (Stelly et al., 1970), appears to be tightly associated with

the interphase NE, in that it may copurify with NE during subcellular

isolation procedures. The exact function of this proteinaceous lamina

has not been determined, however Stelly et al. (1970) have suggested

that it is responsible for the shape and rigidity of the nucleus.








Association of Chromatin with NE

Examination of the NE by EM has demonstrated a close association

of heterochromatin with the inner side of the inner nuclear membrane.

Based on technical difficulties associated with removing trace amounts

of DNA from purified NE, several authors have suggested that the asso-

ciation of DNA with the NE may play a functional role in DNA replica-

tion (e.g. O'Brien et al., 1972). Currently no solid evidence is

available which can be used to support this contention in vertebrate

cells (Franke, 1974).

Isolation and Composition of NE
Subcellular fractionation has proved to be one of the most pro-

ductive techniques employed by cell biologists.to probe the composition,

function, and biosynthesis of various components of the cell. Many

procedures for isolation of nuclei and NE from whole tissues have been

reported (for reviews see Franke, 1974; Kasper, 1974). To our know-

ledge, however, no large scale procedure for isolation of biochemically

characterized NE from cultured cells has been described. Procedures

which purport to isolate structural components from nuclei of cultured

cells (the so-called nuclear matrix [Hodge et al., 1977] or nuclear

ghost [Riley et al., 1975]) have been reported. These isolation

methods, however, are relatively harsh removing most of the lipid

bilayers of the nuclear membranes. These procedures appear to isolate

a nonmembranous portion of the NE which, it has been suggested, is

comprised of the proteinaceous lamina and pore complexes (Dwyer and

Blobel, 1976).

Isolation Procedures

Isolation of NE is attained by disruption of purified nuclei and

subsequent centrifugal separation of membranous components from the








remaining elements of the nucleus (e.g. nucleoli, chromatin, and soluble

proteins). Because separation of NE from other nuclear components is

usually dependent on the membranous characteristic of this organelle,

the purified nuclei from which NE are to be isolated, must be prepared

in a fashion which retains both nuclear membranes and yet removes other

contaminating organelles of the cell. Such nuclear isolation procedures

have been described and are usually modifications of the technique of

Chauveau et al. (1956). The lack of a well characterized preparation

of NE from cultured cells is most probably due to difficulties in iso-

lation of large quantities of "clean" nuclei which contain both nuclear

membranes. The use of detergents to remove cytoplasmic contamination

from nuclei most likely strips the nuclei of the lipid bilayers of the

nuclear membranes.

Disruption of nuclei for isolation of NE is usually accomplished

by mechanical and/or chemical treatments. The most frequently used

mechanical techniques are sonication (e.g. Franke, 1967; Kashnig and

Kasper, 1969) and hypotonic shock (e.g. Zbarsky et al., 1969; Kartenbeck

et al., 1971). High concentrations of MgCl2 (Berezney et al., 1970;

Monneron et al., 1972), sodium citrate (Bornens, 1968; Kashnig and

Kasper, 1969), NaCl and KC1 (Franke et al., 1970; Matsuura and Ueda,

1972), and heparin (Bornens, 1973,1978) have been shown to disrupt

nuclei and aid in solubilization of chromatin during NE isolation.

Deoxyribonuclease I (DNase I) has often been utilized to reduce the

viscosity of ruptured nuclei and to reduce the level of DNA contamina-

tion in isolated NE fractions (e.g. Berezney et al., 1970; Kay et al.,

1972).








After disruption of nuclei and dispersion of chromatin, differ-

ential or isopynic centrifugation is then employed to separate NE from

the remaining nuclear components (Kasper, 1974).

Composition

Franke (1974) and Kasper (1974) have extensively reviewed the

chemical and enzymatic composition of NE fractions isolated from

tissue. The NE is comprised primarily of protein and phospholipid and

has generally been described as a protein rich organelle. Reported

protein values generally vary between 60% and 75% of the total membrane

mass while phospholipid values range from 20% to 30% of the total

membrane mass (see Kasper, 1974). Phosphatidyl choline is the pre-

dominant lipid species of NE followed by phosphatidyl ethanolmine,

phosphatidyl serine and phosphatidyl inositol (Keenan et al., 1970,

1972; Khandwala and Kasper, 1971). Some neutral lipids such as

cholesterol and free fatty acids have also been reported in NE

fractions (Kleinig, 1970; Keenan et al., 1970, 1972).

Almost all isolated NE fractions contain small quantities of

DNA and RNA which vary in amount depending on the preparative pro-

cedure. Those procedures which do not strip the outer membrane of

ribosomes normally result in RNA values which are higher than those

obtained after NE isolation procedures which employ citrate or

MgC12. Small residual amounts of RNA associated with isolated NE

may reflect a functional component of the pores (Monneron and Bernhard,

1969; Dhainaut, 1970). The extent of DNase I treatment and the con-

centration of salt used in NE isolation procedures appear to affect

the quantity of DNA associated with the NE fraction.








Carbohydrates have been detected in isolated NE fractions by

chemical analysis. Kashnig and Kasper (1969) have reported that rat

liver NE contains 3%-4% carbohydrate which is made up of mannose,

glucose, and galactose. These results have been confirmed by

Franke (1977).

The enzyme composition of isolated NE has been reported to be

different by different workers although in most instances it appears

to closely resemble that of the microsomal membranes. Cytochemically

glucose-6-phosphatase (Orrenius and Ericsson, 1966; Rosen, 1969, 1970;

Leskes et al., 1971) and Mg2+ dependent ATPase (Klein and Afzelius,

1966; Yasuzumi and Tsubo, 1966) have been localized to the NE of a

variety of mammalian cell types. Typically these enzymes are present

and occasionally purified in isolated NE fractions (Franke, 1974).

The enzymatic resemblance of the NE and microsomes is strengthened by

the presence of NADH-cytochrome c reductase in NE. This activity is

not, however, increased by application of drugs (e.g. phenobarbital)

as is the enzymatic activity in microsomes (Kasper, 1971). NADPH-

cytochrome c reductase (another constituent of microsomal membranes)

has also been reported by many authors to be present in isolated NE of

liver and in NE of avian erythrocytes (Zentgraff et al., 1971), a cell

which is practically devoid of endoplasmic reticulum. The lack of

drug induction of NE NADH-cytochrome c reductase and the presence

of NE NADPH-cytochrome c reductase in ER depleted cells (avian erythro-

cytes) strongly suggests that these enzymes also do not represent ER

contamination of the isolated NE.








The cytochrome oxidase content of NE remains a controversial issue.

Several authors have reported the presence of cytochrome oxidase or

cytochrome aa3 spectrum in isolated NE (Zbarsky et al., 1969; Berezney

et al., 1972; Matsuura and Ueda, 1972; Jarasch and Franke, 1974).

Jarasch and Franke (1974) interpret their results as mitochondrial con-

tamination while Berezney et al. (1972) suggest that cytochrome oxidase

is a NE enzyme. Extensive discussions of this issue can be found in

Jarasch and Franke (1974) and Berezney et al. (1972).

At the present time, enzymatic analysis can not accurately deter-

mine NE enrichment or contamination by mitochondria or microsomes. Only

plasma membrane contamination can be accurately quantitated by use of

enzymatic markers because several plasma membrane specific enzymes

(e.g. Na+, K+, stimulated ATPase and 5' nucleotidase) have not been

reported to be present in any NE fraction. When evaluating the enrich-

ment or purity of NE fractions, it is necessary to rely heavily on the

presence of NE specific morphological features (pore structures and

double membranes) and the lack of other organellar structures such as

those of mitochondria or unbroken nuclei.

Morphological Changes in the NE
During Cell Cycle

Continuously growing cells proceed through a cycle of biochemical

and morphological events which reproducibly repeat in a fixed order

after each cell division. The events of this cell cycle have been

extensively studied (for review see Mitchison, 1971). The cell cycle

can be divided into four segments -- G1 phase, S (DNA synthetic) phase,

G2 phase, and mitosis. G1 phase is defined as that time segment which








exists between completion of cell division and initiation of DNA syn-

thesis, G2 phase is defined as that time segment which exists between

completion of DNA synthesis and initiation of cell division.

A variety of techniques are available which will induce individ-

ual cells of a culture to traverse the cell cycle in unison. These

synchronization techniques have been employed by cell biologists to

study the biochemical and morphological events which characterize

particular segments of this cycle and which are "hidden" by the random

nature of an untreated cell culture.

Mitosis is perhaps the most carefully studied morphological event

of the cell cycle. The sequence of events of mitosis is traditionally

divided into several phases based on early morphological descriptions.

Briefly, mitosis consists of four phases: prophase, metaphase, anaphase,

and telophase. The onset of prophase is marked by chromosome conden-

sation at the inner nuclear membrane. The nuclear envelope becomes

very irregular, taking on a "wave-like" appearance in the region of the

centrioles. Centriole movement to opposite sides of the nucleus (i.e.

the poles) and chromosome separation occur very early in prophase.

The nuclear envelope begins to dissociate at the poles and microtubules

of the forming spindle apparatus enter into the nucleoplasm. The

nuclear envelope breakdown proceeds toward the equatorial plane and in

late prophase the condensed chromosomes begin to assemble in the com-

pleted spindle (metaphase). At this point the NE is completely dis-

rupted. After a short pause the chromosomes separate at the centromere

into daughter chromatids and the chromatids begin moving toward the

poles anaphasee). The nuclear envelope begins to reform as this







movement begins. Rows of elongated membranous cisternae appear at

the polar aspect of the spindle. As the chromatids begin to fuse into
a chromatin mass, the row of cisternag becomes juxtaposed and extends

further toward the equatorial constriction (cytokinesis and telophase).
The NE is completely reconstructed before the finish of cytokinesis
which marks the end of the mitotic cycle.
Unfortunately very little is known concerning the fate of the

NE during mitotic breakdown nor is there good evidence relating to the
origin of the components which reform the NE in late M and early G1.

Similarly the mechanisms) whereby the NE is disassembled at the mitosis
and reformed in late mitosis have not been elucidated.
It has been suggested by a number of workers (Porter and Machado,

1960; Moses, 1964; Robbins and Gonatas, 1964; Murray et al., 1965;
Brinkley et al., 1967) that following NE breakdown, fragments of NE

mingle with and become indistinguishable from the endoplasmic reticulum

(ER). Furthermore, it has been argued that components of the ER are
utilized in reformation of the G1-NE (Porter and Machado, 1960; Moses,
1964). Other authors have suggested that the NE, or components of the

NE, persist through mitosis as distinct entities, biochemically differ-
ent from the ER, and that these components are specifically reutilized
to reform NE at the completion of M (Erlandson and De Harven, 1971;
Maruta and Goldstein, 1975; Maul, 1977). Finally some workers in the
field have suggested that the NE which reappears at the end of M is a
product of de novo synthesis of all the NE components (e.g. Jones,
1960).
The multiplicity and diversity of hypotheses which have been pre-
sented to explain NE breakdown and reformation during M are probably








due to the variety of animal and plant species in which the predom-

inantly microscopic work was performed and to the fact that the evidence

available concerning NE breakdown and reformation consists almost

entirely of morphological studies which suffer from the difficulty of

positively identifying cytoplasmic components which might be NE frag-

ments released during mitosis.

Objectives

The main objective of the studies presented in this dissertation

is to investigate the mechanism whereby the NE disassembles and reforms

during mitosis. To examine biochemical changes and the biosynthesis

of NE during the cell cycle it is necessary to develop a procedure to

use with cultured cells which would permit the isolation of "clean"

NE containing both lipid bilayers of the nuclear membranes as well as

the proteinaceous lamina.

In Chapter II of this dissertation I will report a preparation

technique which permits the isolation of nuclei from Chinese hamster

ovary (CHO) cells without the use of reagents (such as detergents) which

might disrupt membranes. The isolated nuclear fraction has been used

to purify sufficient quantities of NE to allow both biochemical and

EM studies of this organelle.

In Chapter III of this dissertation I will present a biochemical

investigation of the breakdown and reformation of the NE during mitosis

in CHO cells. Using radioactive precursors of protein and phospho-

lipid I have studied the reutilization and biosynthesis of NE in the

late G2-M-early G1 portion of the cell cycle.













CHAPTER II
ISOLATION AND CHARACTERIZATION

Materials and Methods

Materials

All tissue culture materials were purchased from Grand Island

Biological Company (Grand Island, New York). Tissue culture plastic-

ware was obtained from Corning Glass Works (Corning, N.Y.). Ultra-

pure sucrose, [methyl-3H] thymidine, and [5-3H] uridine were obtained

from Schwarz-Mann Division, Becton, Dickinson and Company (Orangeburg,

New York). Deoxyribonuclease I (DNase I) and ribonuclease A (RNase A)

were purchased from Worthington Biochemical Corporation (Freehold, New

Jersey) and were shown to be protease-free via the assay of Tomarelli

et al. (1949). [Methyl-3H] choline chloride was obtained from Amer-

sham Searle Corporation (Arlington Heights, Illinois). All reagents

for electrophoresis were purchased from Bio-Rad Laboratories (Rockville

Centre, New York). All other reagents were obtained from Scientific

Products (Ocala, Florida).

Cell Culture

Chinese hamster ovary cells (originally obtained from Dr. Kenneth

Ley, Sandia Laboratories, Albuquerque, New Mexico) were maintained at

370C in suspension culture in Ham's F-10 nutrient medium, supplemented

with 10% (v/v) calf serum and 5% (v/v) fetal calf serum. Cell density

was monitored daily and maintained between 1.2 x 105 4 x 105 cells/ml.








Isolation of Nuclei and NE

A flow chart of this procedure is presented in Figure 2. All

procedures were carried out at 00C unless otherwise noted. For each

NE preparation 3- 4 x 109 cells were harvested, washed twice with phos-

phate buffered saline (PBS, pH 7.2), and resuspended at 2 4 x 107

cells/ml in a homogenization buffer containing 10mM Tris (pH 7.6),

10mM KC1, and 10mM EDTA. The cells were allowed to swell for 15 min

and then lysed (in 15 ml batches) by 5-7 strokes in a Dounce homogen-

izer fitted with a tight pestle. Homogenization was continually

monitored by phase contrast microscopy to assure maximum cell lysis

with minimum nuclear breakage.

Immediately following lysis, the homogenate obtained from each

batch of cells was diluted with 2 volumes of 65% (w/w) sucrose, 50mM

Tris (pH 7.6), 7.5mM MgCl2, and kept on ice until homogenization of

the entire cell sample was completed. The diluted homogenate was cen-

trifuged in a Sorvall HB-4 rotor for 30 min at 4,936 x gmax. The

resultant crude nuclear pellet was resuspended in 24 ml of 65% (w/w)

sucrose, 50mM Tris (pH 7.6), 2.5mM MgC12 (65% sucrose-TM). Aliquots

of the crude nuclear pellet were overlayed with 6 ml of 60% sucrose-TM,

12 ml of 55% sucrose-TM, 6 ml of 50% sucrose-TM and 7 ml of 30% sucrose-

TM to form a discontinuous sucrose gradient. The gradient(s) was

subsequently spun for 1 hr at 72,100 x g in a Beckman SW27 rotor.

Nuclei banding at the 65%-60% and 60%-55% sucrose interfaces were

removed, combined, and diluted to 50% sucrose with TM buffer. Using

an SW27 rotor (72,100 x gmax for 1 hr) these nuclei were sedimented

through another discontinuous gradient containing 12 ml of 55% sucrose-

TM, 3 ml of 60% sucrose-TM and collected onto a cushion of 3 ml 65%








sucrose-TM. Material caught on the 65% sucrose-TM cushion was removed,

diluted to 10% sucrose with distilled water, and pelleted in an HB-4

rotor at 1020 x gmax for 5 min. This pellet is designated the purified

nuclear fraction.

In order to bring about nuclear lysis the purified nuclei were

resuspended in 1 ml of 10mM Tris (pH 8.5), containing O.1mM MgCl2.

After 20 min in 10mM Tris, O.1mM MgCl2, DNase I and RNase A were each

added to a final concentration of lO00g/ml and the incubation continued

for 20 min at 22C. Following lysis (which could be monitored with

phase optics) four volumes of 60% (w/w) sucrose, 50mM Tris (pH 7.6),

500mM MgC12 were added to the lysed nuclei. The solutions were

thoroughly mixed and the suspension placed in the bottom of a Beckman

SW41 centrifuge tube. A linear 20%-45% (w/w) sucrose gradient con-

taining 50mM Tris (pH 7.6) and 500mM MgCl2 was formed on top of the

sample and the gradients spun for 2 hr at 153,244 x gmax in a Beckman

SW41 rotor. Nuclear envelopes banded at 1.19 g/cc (35% sucrose in

500mM MgCl2). The purified NE's were diluted with 50mM Tris (pH 7.6)

500mM MgCl2 and then pelleted in a Beckman fixed angle 40 rotor for

30 min at 130,766 x gmax. The NE's were subsequently resuspended in

10% sodium dodecyl sulfate (SDS), 10mM Tris (pH 8.5), 1% B-Mercapto-

ethanol if they were to be analyzed by electrophoresis, or in 10%

sucrose, 10mM Tris (pH 7.6), 0.5mM MgC12 if chemical or enzymatic

analysis was to be performed on the sample.

Isolation of Plasma Membrane

Plasma membrane was isolated by a slight modification (McClure

and Noonan, 1978) of the aqueous two phase polymer technique of Brunette

and Till (1971).








Chemical Assays

All protein determinations were performed according to the pro-

cedure of Lowry et al. (1951). Phospholipid phosphorus (Rouser et al.,

1966) was measured in chloroform-methanol extracts (Rouser and

Fleischer, 1967) of various subcellular fractions. Phospholipid

content of the various fractions was extrapolated from the phosphorus

determination by multiplying the phosphorus value by a factor of 25.

Cholesterol was determined according to Glick et al. (1964). In

order to determine the RNA and DNA content of the NE fraction, cells

were grown for five generations in the presence of O.5lCi/ml [3H]

thymidine or 0.4pCi/ml [3H] uridine prior to preparation of NE. The

specific radioactivity of DNA or RNA (cpm/pg nucleic acid) was deter-

mined from the cell homogenate by liquid scintillation counting and

spectrophotometric assay. DNA content was determined by the diphenyl-

amine assay (Burton, 1956) while RNA content was determined by the

orcinol (I-San Lin and Schjeide, 1969) assay of extracts prepared

according to Fleck and Munro (1962). The specific radioactivity

obtained for DNA and RNA was then used to determine the relative

nucleic acid content of nuclei and NE.

Enzyme Assays

Glucose-6-phosphatase activity was determined according to

Franke et al. (1970) with the inorganic phosphate release being mea-

sured according to Fiske and Subbarowe (1925). 5'-Nucleotidase was

assayed via the technique of Widnell and Unkless (1969) with the

inorganic phosphate released being measured by a modification of the

technique described by Chen et al. (1956). Succinate dehydrogenase








was assayed according to Veeger et al. (1969). Cytochrome c oxidase

was assayed according to Smith (1955).

Electron Microscopy

Nuclei and NE's were fixed (00C, 2 hr) in 1.8% glutaraldehyde,

8% sucrose, 50mM phosphate (pH 7.6), and 2.5mM MgC12. Both the nuclei

and NE fractions were postfixed in the same buffer containing 1%

osmium tetroxide (22C, 1 hr). Samples were sequentially dehydrated

in increasing concentrations of ethanol and finally embedded in Spurr's

low viscosity medium (1969). Thin sections were stained with uranyl

acetate (Watson, 1958) and lead citrate (Venable and Coggeshall, 1965)

and then examined in an Hitachi AS8 electron microscope.

SDS Polyacrylamide Disc Gel Electrophoresis (PADGE)

All electrophoretic analyses were performed in 1.5mm thick slab

gels according to the procedure of Laemmli (1970). The gels used were

composed of a 5.6% acrylamide stacking gel overlaying a linear 7.5%

to 12.5% acrylamide gradient separation gel. The acrylamide ratio to

bis-acrylamide was maintained at 37.5:1 in both the stacking and

running gel. Purified NE's were solubilized by boiling at 1000C for

5 min in 10% SDS, 10mM Tris (pH 8.5), 1% B-mercaptoethanol (BME).

Plasma membrane was solubilized by resuspension and boiling in 2% SDS,

62.5mM Tris (pH 6.8), 10% glycerol, and 0.1% BME ("sample buffer").

Lysed nuclei, sampled after DNase I and RNase A digestion, and whole

cell homogenates were solubilized by boiling in one volume of 2x
"sample buffer." Each of the samples were then exhaustively dialyzed

against "sample buffer" containing 0.1% 6-mercaptoethanol and O.ImM

phenylmethylsulfinyl fluoride (PMSF). Inclusion of PMSF in the sample








buffer is essential since we have found that NE's dissolved in sample

buffer lacking PMSF are subject to extensive proteolysis, possibly

as the result of the co-purification of an intrinsic protease with

the purified envelopes (Carter et al., 1976).

Following electrophoretic separation of the membrane peptides

and glycopeptides, the slabs were fixed for 30 min in 10% trichloro-

acetic acid and then allowed to equilibrate overnight in 25% ethanol,

8% acetic acid. The following day the gels were stained with Coomasie

brilliant blue according to Weber and Osborn (1969) and finally

destined in 25% ethanol, 8% acetic acid.

Results

Isolation of Nuclei

The primary goal which I set for myself prior to isolating NE's

was that the purified NE's contain both the inner and outer membrane

bilayer as well as the pore complex and be free of contaminating cyto-

plasm or nucleoplasm. In order to achieve this goal, a nuclear

fraction had to be isolated which itself contained both the inner and

outer nuclear membranes and was free of substantial cytoplasmic con-

tamination. The nuclear isolation procedure which I have employed in

my work has been described in detail in the Materials and Methods

section and is presented diagrammatically in Figure 2.

In many of the nuclear isolation procedures previously published

workers have used nonionic detergents to remove cytoplasmic components

(Muramatsu, 1970) from the nuclei. Unfortunately the use of such

detergents on isolated nuclei from rat liver has been shown to remove

almost all of the nuclear membrane phospholipid and much of the protein




















Supernatant


inder of 55%-60% and
adient 60%-65% sucrose
SCARD) interfaces


rI-


60%-65% sucrose
interface


Dounce homogenize in hypotonic buffer
at 2-4 x 10/ cells/mi. Dilute with
2 volumes 65% sucrose, 50mM Tris
(pH 7.6), 7.5 mM MgC12.


Spin 4936 x gmax' 30 min
Resuspend in 65% sucrose TM and place
at the bottom of a SW27 gradient.


Spin 72,100 x gmax, 1 hr

Remove nuclei from interfaces.
Dilute to 50% sucrose TM, and place
in a SW27 gradient.

Spin 72,100 x gmax 1 hr


Remove nuclei from 60%-65% sucrose inter-
face, dilute with distilled water to 10%
sucrose


Cells


homogenate

I-----


\u.


pellet


Rema
gra
(DI!


Remainder of
gradient
(DISCARD)











pellet


pellet -



Figure 2. Flow Diagram of Nuclei and


Supernatant
(DISCARD)


Spin 1020 x gmax' 5 min


Purified Nuclear Fraction
Lyse in 10mM Tris (pH 8.5), 0.1mM MgC12,
nuclease digest, dilute with 60% sucrose,
50mM Tris (pH 7.6), 500mM MgC12 and place
at the bottom of a SW41 gradient.


Spin 153,244 x gmax' 2 hr



Remove NE (band at 1.19 g/cc). Dilute
with 50mM Tris (pH 7.6), 500mM MgCl2.


Spin 130,766 x gmax' 30 min


Purified NE Fraction


NE Isolation Procedure


Supernatant
(DISCARD)








(Aaronson and Blobel, 1974). For this reason I avoided the use of any

detergents in my nuclear isolation procedure. Instead, I developed

homogenization conditions which minimized nuclear clumping and adhesion

of cytoplasmic material to the nuclei. Specifically I found that

immediate dilution of the cell homogenate with 65% (w/w) sucrose, 50mM

Tris (pH 7.6), 7.5mM MgCl2 (Figure 2) maintained the nuclei in a

dispersed state. Isolation of "clean" nuclei from such an homogenate

could then be readily accomplished by density gradient centrifugation

if the nuclei were maintained under the salt and pH conditions outlined

in Figure 2 and handled with care so as to avoid premature lysis.

In the procedure which I developed (Figure 2) the whole cell

homogenate is diluted with 65% sucrose in 50mM Tris (pH 7.6), 7.5mM

MgCl2, and spun for 30 min at 4,936 x gmax. This differential centri-

fugation serves to collect nuclei in a pellet while leaving most other

membranous organelles in the supernatant. This crude nuclear pellet

is subsequently subjected to two sequential centrifugations on

sucrose step gradients (Figure 2) in which purified nuclei band at

the 60-65% sucrose interface. The purified nuclei are subsequently

diluted with distilled water (to give a final sucrose concentration

of 10%) and then collected as a pellet by a 5 min, low speed, differ-

ential centrifugation step (Figure 2).

Three precautions must be taken to ensure the isolation of a

"clean" nuclear fraction from CHO cells. (a) Homogenization must

be performed at pH 7.6 in the absence of divalent cations. (b)

Homogenization, resuspension, and mixing of fractions must be done

very carefully so as to avoid premature lysis or shearing of the








nuclei. Breakage of the nuclei at early stages in the isolation pro-

cedure inevitably results in nuclear clumping with concomitant

entrapment of cytoplasmic and nucleoplasmic contaminants. (c) Homog-

enization, resuspension, and mixing must be carefully monitored to

insure oneself that the nuclei are adequately dispersed. Failure to

achieve a relatively homogeneous mixture of nuclei inevitably results

in failure of the subsequent centrifugation steps to remove cytoplasmic

contaminants.

Morphology of the Purified Nuclear Fraction

As can be seen in the phase contrast micrograph presented in

Figure 3, nuclei purified via the procedure outlined in Figure 2 are

intact, contain distinct nucleoli, and show few cytoplasmic "tags."

Electron micrographs of the purified nuclear fraction show no obvious

contamination of the nuclei with mitochondria, RER, vesicles or large

sheets of plasma membrane (Figure 4A). Although rough or smooth

microsomes are not immediately detectable in the nuclear fraction,

it must be pointed out that the outer nuclear membranes of some nuclei

are blebbed. It is possible that these blebs could represent micro-

somal contamination. However it is more likely that the blebs simply

represent swelling of the outer nuclear membrane. A higher magnifi-

cation micrograph (Figure 4B) of nuclei isolated in the "purified

nuclear pellet" (Figure 2) clearly demonstrates that both the inner

(arrow) and outer (double arrow) nuclear membranes are present on

the isolated nuclei and that very few ribosomes are attached to the

outer membrane. Furthermore, the isolated nuclei have obvious pores

(triple arrow) associated with them.























***'' *.. ^D B
7 'i-.;; l
HetA-*''^Pf


Figure 3. Phase Contrast Micrograph of the Purified
Nuclear Fraction. (x1340)


~~9;r ~~


~ii*i r*


























Figure 4. Electron Micrograph of the Purified Nuclear Fraction
Purified nuclei were fixed, embedded, and stained as
described in Materials and Methods.

A. Representative section through "purified nuclear
fraction" (x3775).

B. High magnification EM of two randomly chosen nuclei
with the attention being directed to the nuclear
membranes (x24,350) (') outer bilayer; (T) inner
bilayer; C( ) pores.





25








Isolation of NE

The NE isolation procedure described in the Materials and Methods

section is a combination and modification of the procedures published

by Kay et al. (1972) and Monneron et al. (1972) for the isolation of

NE from rat liver nuclei.

As outlined in Figure 2, the NE isolation procedure begins with

the "purified nuclear fraction." In the isolation method used the

purified nuclei are lysed in 10mM Tris (pH 8.5) containing O.ImM MgCl2.

Incubation of the nuclei in this hypotonic buffer for 20 min at 0C

produces a very viscous solution which is subsequently incubated with

DNase I and RNase A for 20 min at 22C. Addition of the respective

nucleases immediately reduces the viscosity of the lysed nuclei.

Following nuclease digestion, the material is diluted with 4 volumes

of 60% sucrose, 500mM MgC12 (to remove residual chromatin [Monneron

et al., 1972]) in 50mM Tris (pH 7.6) and the mixture vortexed.

During the ensuing centrifugation (SW41 rotor at 153,244 x gmax'

2 hr), NE float up into the gradient leaving nucleic acid, soluble

proteins of the nucleoplasm, and unlysed nuclei in the dense load

zone. Under the centrifugation conditions used, the NE fraction bands

in the gradient at a density of approximately 1.19 g/cc. The NE are

subsequently removed from the gradient and pelleted (Figure 2).

Morphology of the Purified NE Fraction

Electron micrographs of sections through the purified NE frac-

tion (Figure 5) demonstrate that most, if not all, of the isolated

nuclear membranes retain the double membrane sheets (single arrow)

characteristic of the NE. Furthermore, as would be expected if one













































Figure 5. Electron Micrograph of the Purified NE Fraction

The NE fraction was fixed, embedded, and stained
as described in the Materials and Methods
section (x31,850). Regions of double membranes
(T) can be seen as well as pores in tranverse
) and tangential section (f .








were isolating intact NE's, nuclear pores can be seen in both trans-

verse (double arrow) and tangential section (triple arrow). Clearly

the presence of pores and double membranes in this purified fraction

supports its identify as a NE fraction.

Few, if any, vesicles are apparent in this preparation (Figure 5),

suggesting little or no microsomal contamination. Similarly no mito-

chondria or unbroken nuclei are observed in this "purified NE fraction."

Recovery of Isolated Fractions

Typical recoveries of protein, phospholipid and DNA obtained

during preparation of the NE are presented in Table I. Approximately

14% and 30% of the homogenate protein and DNA, respectively, are

recovered in the nuclear fraction. Based on the recovery of DNA it

appears that 30% of the total starting nuclei are isolated in the

"purified nuclear fraction" (Figure 2). My work suggests that <70%

of the starting nuclei are lost during the repeated centrifugations

(Figure 2) which I find to be necessary for preparing clean, intact

nuclei.

As can be seen in Table I the NE fraction contains 8% of the

starting nuclear protein and 50% of the starting nuclear phospholipid

while only 0.3% of the nuclear DNA is recovered in this fraction. If

one assumes that all nuclear phospholipid resides in the nuclear

membranes (Franke, 1974), one can estimate that approximately 50% of

the starting NE are isolated in the "purified NE fraction" (Figure 2).

Characterization and Purity of Nuclear and NE Fractions

In order to further confirm the relative purity of the isolated

nuclear and NE fractions, as well as to determine the composition of











Table I

Recovery of Protein, DNA, and Phospholipid in Subcellular Fractions

% of Total % of Total % of Total % of Total % of Total
Fraction Cellular DNA Cellular Protein Nuclear DNA Nuclear Protein Nuclear Phospholipid


Homogenate 100 100 -


Nuclei 30 14 100 100 100


NE 0.1 1 0.3 8 50



Recoveries were determined by assay of individual fractions sampled during the isolation
procedure.








the nuclei and NE's, I have examined the enzymatic activities and

chemical content of both fractions.

Enzyme Activity

The activity of 5'-nucleotidase was examined in homogenate,

nuclei, and NE fractions in order to evaluate possible plasma membrane

contamination of the purified NE fraction. Table II clearly shows

that little plasma membrane (as assayed by 5'-nucleotidase) is found

associated with either the nuclei or NE. Similarly, assays of

succinate dehydrogenase activity (as a marker for mitochondrial con-

tamination) indicated that neither the nuclear nor the NE fraction

has significant quantities of this enzyme associated with them

(Table II). Cytochrome c oxidase assays of homogenate, nuclei, and

NE indicated that this enzyme was a very minor component of the NE

fraction (Table II). In comparison to values obtained for purified

mitochondria, the NE fraction contains extremely low levels of this

enzyme which could reflect either minor mitochondrial contamination

(Jarasch and Franke, 1974) or that cytochrome c oxidase is a normal

component of the NE (Berezney et al., 1972).
Glucose-6-phosphatase activity, frequently reported to be a

marker of the ER (Kasper, 1974), has been cytochemically identified

in the NE of liver and purified in some NE fractions isolated from

this tissue (see Introduction). Assays of the glucose-6-phosphatase

activities of homogenate, nuclei, and NE fractions from CHO cells

(Table II) indicate that a 2 fold purification of this activity is

obtained in nuclei and NE relative to the homogenate. The lack of

further purification of this enzyme in the NE fraction relative to









Table II

Enzyme Activities of Subcellular Fractions


Homogenate Nuclei NE Mitochondria *

5'-Nucleotidase
(nmoles P04 = hydrolyzed/ 49 (6) ND (2) ND (2)
mg protein/hr)

Succinate Dehydrogenase
(nmoles succinate oxidized/ 470 (2) ND (2) ND (2)
mg protein/hr)

Cytochrome c Oxidase
(nmoles cytochrome c oxidized/ 0.7 (2) ND (2) 1.5 (2) 18.0 (2)
mg protein/min)

Glucose-6-Phosphatase
moless PO4 = hydrolyzed/ 0.41 (2) 0.75 (2) 0.80 (2)
mg protein/hr)


Each fraction was assayed as described in Materials and Methods, immediately following
isolation of NE fractions. ND -- no detectable activity. The minimum detectable activity in succinate
dehydrogenase assays was 50 nmoles succinate oxidized/mg protein/hr and the minimum detectable activity
in 5' nucleotidase assays was 3 nmoles/mg protein/hr. The minimum detectable activity in cytochrome c
oxidase assay was 0.3 nmoles cytochrome c oxidized/mg protein/min. Numbers in parentheses are the number
of assays performed. $ Digitonin treated mitochondria, isolated from bovine liver, were obtained from
Dr. T. W. O'Brien.







the nuclear fraction may be due to loss or denaturation of the

enzyme by the high salt conditions employed during the latter steps

of our isolation procedure (Kasper, 1974).

Chemical Composition

A preliminary analysis of the relative chemical composition

of the homogenate, nuclei and NE fractions are presented in Table III.

Among the points presented in Table III which should be stressed is

the fact that the "purified NE fraction" does contain small amounts

of residual DNA and RNA. Similar findings with regard to nucleic

acid composition of isolated NE have been reported in previously

published characterizations of NE from liver (Kasper, 1974) and may,

in fact, represent physiologically significant components of the NE.

It should also be noted that, not unexpectedly, if the nuclease

digestions (Figure 2) were omitted from the isolation procedure, sig-

nificantly larger amounts of DNA and RNA were recovered in the

purified NE fraction.

To confirm the membranous nature of the isolated NE fraction,

phospholipid content was determined in the homogenate, nuclei, and

NE fraction contained 200pg phospholipid/mg protein indicating that

the fraction isolated is relatively protein rich (see pg. 31 of

Fleischer and Kervina, 1974) perhaps due to the proteinaceous lamina

associated with the inner nuclear membrane. In order to demonstrate

that this relatively low phospholipid/protein ratio was not due to

incomplete extraction of phospholipid, NE's were prepared from cells

which had been maintained for 5 generations in medium containing

O.lpCi/ml [3H] choline. Ninety-seven percent of the [3H] choline











Table III

Chemical Content of Subcellular Fractions


ug DNA/mg


pg RNA/mg


pg Phospholipid/mg


pg Cholesterol/mg


Fraction Protein Protein Protein protein


Homogenate 90 220 91 ---


Nuclei 200 90 30 ---


NE 10 20 200 20


PM --- --- --- 117




Individual fractions were sampled during the isolation procedure and assayed for protein,
DNA, RNA, phospholipid or cholesterol as described in Materials and Methods. A plasma membrane
enriched fraction was prepared from CHO cells via the two-phase aqueous polymer technique of
Brunette and Till (1971).








present in the NE fraction was extracted by chloroform methanol and

accounted for in subsequent phosphorus determinations (data not

shown).

The cholesterol content of NE and plasma membrane were compared

in order to determine if there were any clear differences in choles-

terol composition between these two fractions taken from CHO cells.

As can be seen in Table III, five-fold less cholesterol (per mg pro-

tein) was found in the NE as compared to the PM. These data are in

agreement with previously published work concerning the cholesterol

content of bovine liver NE (Keenan et al., 1970).

Peptide and Glycopeptide Composition

When the purified NE fraction was examined by SDS gel electro-

phoresis (Laemmli, 1970), a complex Coomasie blue staining profile

was obtained (Figure 6) which was distinguishable from the stained

profiles obtained from whole cell homogenate, purified nuclei and

plasma membrane derived from CHO cells (Figure 6).

The major Coomasie brilliant blue staining components of the

NE fraction ranged in molecular weight from 55,000 to 75,000 daltons.

The majority of the remaining NE peptides and glycopeptides were of

a MW higher than 75,000 daltons. It should be noted (compare nuclear

with NE fraction, Figure 6) that few, if any, peptides are found in

the NE fraction which co-migrate with the histones (arrows) found

in the nuclear fraction again arguing against significant contam-

ination of the NE with DNA.
























i I











1 2 3 4 5 6




Figure 6. Coomasie Blue Stained Profiles of Subcellular
Fractions

Fractions were isolated and electrophoresed as
described in the Materials and Methods section.
Seventy-five micrograms of protein were applied
to each slot. From left to right: lane 1,
standards (phosphoryase A, 100,000 MW; bovine
serum albumin, 69,000 MW; ovalbumin, 43,000 MW;
DNase I, 31,500 MW; soybean trypsin inhibitor,
23,000 MW; and cytochrome c, 13,500 MW); lane 2,
DNase I and RNase A; lane 3, NE; lane 4, nuclei;
lane 5, plasma membrane and lane 6, whole cell
homogenate. Arrows ( ) indicate the region in
which histones migrate in these gels.














CHAPTER III
REFORMATION OF THE NUCLEAR ENVELOPE DURING MITOSIS

Materials and Methods

Materials

All tissue culture materials were purchased from Grand Island

Biological Company (Grand Island, New York). Tissue culture plastic-

ware was purchased from Corning Glass Works (Corning, New York).

[4,5-3H] L-leucine, [methyl-3H] thymidine, and ultrapure sucrose

were purchased from Schwarz Mann, Division of Becton, Dickinson and

Company (Orangeburg, New York). [Methyl-3H] choline chloride and

[32P] orthophosphate were purchased from Amersham Searle Corporation
(Arlington Heights, Illinois). All reagents for electrophoresis

were purchased from Bio-Rad Laboratories (Rockville Centre, New

York). All other reagents were obtained from Scientific Products

(Ocala, Florida).

Cell Culture and Synchrony

Chinese hamster ovary cells were grown in suspension culture

as described in Chapter II. Cells were synchronized via a modifica-

tion of the isoleucine deprivation technique first introduced by

Tobey and Crissman (1972). In our procedure suspension cultures

(usually 6 liters) were allowed to grow to stationary phase

(6-8 x 105 cells/ml) in complete media. The cells were left for 12

to 24 hr at stationary phase and then harvested and resuspended in








medium containing 10% calf serum, 5% fetal calf serum and ImM

hydroxyurea (HU). Ten hours after the addition of the medium

supplemented with HU (when the majority of the cells were arrested

at the G1/S boundary), the cultures were re-harvested and resuspended

in fresh, complete medium. Immediately upon resuspension in fresh

medium the cells began to traverse the cell cycle. Attempts to

synchronize other CHO subclones using this technique were not

successful.

In order to make comparisons between experiments, I have plotted

synchronization curves as fraction of cells divided (N-No/No) versus

time. No is the number of cells before division and N is the number

of cells at any given time during division.

Labeling of Cell Cultures

Labeling of cell cultures in preparation for isotope dilution

experiments was performed by maintaining cells for 5 generations

prior to synchronization in a particular radioactive precursor (at

the specific activity indicated) and then synchronizing the cultures

in the presence of either O.2pCi/ml [3H] leucine, O.lpCi/ml [3H]

choline, or 0.41Ci/ml [32P] orthophosphate. In order to determine

the dilution of radiolabel which occurred during the mitotic phase

of the cell cycle, the labeled cell cultures were harvested

immediately prior to entrance into M (i.e. in very late G2) and

washed twice in PBS, pH 7.2. One aliquot of cells was used to

prepare G2 NE while the remaining cells were returned to culture in

fresh media containing no radioactive precursor. After the cells had

completed M, this aliquot was used to prepare the G1 NE's.








Pulse label experiments with [3H] leucine were performed by

growing and synchronizing cultures in the absence of radiolabel.

Following release of the cells from HU, the cultures were resuspended

in media containing 3.3pg/liter leucine (25% of the leucine normally

present in F-10 medium). One hour before each sequential NE iso-

lation, an aliquot of the culture was removed from the synchronized
stock culture and [3H] leucine was added to a final concentration

of 0.2pCi/ml [3H] leucine.

Pulse labeling of cultures with [ 32P orthophosphate was per-
formed as in [ 3H] leucine pulses except that media added after

release of the cells from the HU block was complete F-10 with no
dilution of any component. [32P] Orthophosphate was added to a final

concentration of O.8pCi/ml.

Isolation of Nuclei and NE

Preparation of nuclei and NE was described in detail in
Chapter II.

Determination of Specific Activities

The specific activity of [3H] leucine labeled cell components
was determined by liquid scintillation counting (LSC) and protein

assay according to Lowry et al. (1951) after either solubilization

of the cell component in 10% sodium dodecyl sulfate (SDS), 10mM

Tris (pH 8.5), and 1% B-mercaptoethanol (BME) and dialysis for 36 hr
against 2% SDS, 62.5mM Tris (pH 6.8), 10% glycerol, 0.1% 6ME and
0.1mM phenylmethylsulfonyl fluoride (PMSF) or following precipitation

of the isolated component with 10% trichloroacetic acid (TCA). The
specific activity of [32p] or [3H] choline labeled phospholipid was








determined from phospholipid phosphorus assays (Rouser et al., 1966)

of chloroform-methanol extracts (Rouser and Fleischer, 1967) of the

specific cell component.

Determination of Precursor Pool Equilibration Rates

Cell cultures were grown for 5 generations in either 0.2pCi/ml

[3H] leucine or 0.4pCi/ml [P32] orthophosphate. The cells were then

synchronized in the presence of 0.2pCi/ml [ H] leucine or 0.4jCi/ml

[32P] orthophosphate. Five to six hours after release from HU, an

aliquot of cells was taken from the culture and the initial precursor

pool specific activity was determined as described below. Immediately

after removal of this initial aliquot, the remaining cells were

washed twice with PBS, sampled again, and returned to unlabeled

medium. Each hour for the next 4 hr after being returned to unlabeled

medium, a sample of cells was removed, and the precursor pool specific

activity was determined.

To determine the size of the [3H]1eucine pool, cells were

collected on a glass fiber filter, washed quickly with 10 volumes of

ice cold PBS, and solubilized in 0.2N NaOH for 5 minutes at 700C.

The solubilized material was chilled, precipitated with 10% trichloro-

acetic (TCA) overnight at 4C and the supernatant collected by cen-

trifugation. [3H] Leucine in the supernatant (i.e. the acid soluble

pool) was determined by LSC and total cell protein was assayed

according to Lowry et al. (1951).

Phospholipid precursor pool size was based on labeled phospha-

tidic acid. [32P] Phosphatidic acid specific activity was determined

by harvesting cells via centrifugation and immediately extracting








the harvested cells with chloroform-methanol (Rouser and Fleischer,

1967). After the Folch back washes of the chloroform-methanol

extract, phosphatidic acid was found in the lower organic phase.

Phosphatidic acid was purified by two sequential separations on

silica gel thin layer chromatography using solvent system I developed

by Skipski and Barclay (1969) for acidic phospholipids. The purified

phosphatidic acid was assayed for phosphorous (Rouser et al., 1966)

and the [32P] content determined by LSC.

Measurement of Nuclear Surface Areas

Nuclear surface areas were measured by point and intersection

planimetry on photographs of thick sections of embedded whole cells.

Samples of cells were taken from a synchronized culture 5h and 8h

after release from HU. Samples were fixed in 2% glutaraldehyde, 2%

formaldehyde and 50mM PO4 = (pH 7.4) for 30 minutes at 220C. Post-

fixation was performed in 1% OsO4, 100mM PO4 = (pH 7.4) for 30

minutes at 220C. Samples were then dehydrated and embedded as

described in Chapter II. Thick sections were cut on a Sorvall micro-

tome and stained with 0.1% toludine blue 0 in 1% sodium borate for

15 seconds at approximately 2000C. Sections were photographed using

a Wild MII microscope with camera attachment. The resulting photo-

graphs were subsequently subjected to planimetry according to

Weibel (1969).

Polyacrylamide Gel Electrophoresis

All electrophoresis was performed as described in Chapter II.

Fluorography of [3H] labeled NE gels was performed according to

Bonner and Laskey (1974).








Results

Cell Synchrony

The relative synchrony obtained by the technique outlined in

the Materials and Methods section is presented in Figure 7. [3H]

Thymidine incorporation into DNA indicated that following release

from HU, DNA synthesis was initiated immediately and that the syn-

thetic phase (S pahse of the cell cycle) lasted approximately 5 hr.

A 5 hr S phase is in good agreement with the value previously obtained

by Tobey and Crissman (1972) using these same cells. Concurrent with

cessation of DNA synthesis (i.e. 5 hr after release from HU),

mitotic cells, defined by metaphase figures appeared (Figure 7). The

mitotic phase of the cell cycle was complete in approximately 4-5 hr

(i.e. occurred 5-10 hr after release from HU). During this time

frame the cell population increased by approximately 80% (Figure 7).

It must be pointed out that from Figure 7 it is apparent that the G2

phase of the CHO cell cycle synchronized by the technique outlined

in Materials and Methods is very short. The fact that mitotic

figures begin appearing as soon as DNA synthesis is terminated

(Figure 7) does not allow accurate measurement of the G2 phase of the

cell cycle. It should also be noted that the maximum mitotic index

at any time after release from HU is 25-30% of the total cell popula-

tion. Thus, during the 4-5 hr mitotic phase of these synchronized

cells a proportion of the cells are either in the preceding G2 or

have progressed into the G1 phase and are therefore coexisting with

cells in mitosis. Since I well recognize that I do not have 100% of

the cells in mitosis at any one time, I refer to the 4-5 hr time

period during which the cell population divides as the G2-M-G1



















S/ -5

S.


o 0


o z



TIME AFTER ADDITION
OF HU(HRS)









Figure 7. Cell Cycle Parameters in Synchronized CHO Cells

A suspension culture was synchronized as
described in Materials and Methods. DNA syn-
thesis was determined by pulsing duplicate 2 ml
aliquots of cells for 10 min at 370C with
2.6 pCi/ml [3H] thymidine. Labeled cells were
then washed three times with ice cold PBS, pre-
cipitated and washed three times with ice cold
10% TCA, and radioactivity in the precipitate
was determined by LSC. Cell number was deter-
mined from 4 replicate counts using a Levy-
Hausser counting chamber. Mitotic index is
expressed as percent of cells in mitosis
observed in phase microscopy. [3H] Thymidine
incorporation into TCA insoluble material 0 0;
cell density A-A; mitotic index ID-D.








transition. It is during this transitional period of the cell cycle

that the NE breaks down and reforms.

Peptide Composition of the NE during Different Phases of the Cell
Cycle

Maul et al. (1972) have reported that changes in the number of

nuclear pores per unit nuclear surface area occur as HeLa cells pro-

gress through the cell cycle. Riley and Keller (1978) have reported

major morphological changes and minor compositional changes in non-

membranous nuclear ghosts isolated from HeLa cells at various stages

of the cell cycle. Hodge et al. (1977) have also examined polypep-

tides of the HeLa cell nuclear matrix isolated from synchronized

populations of cells at various stages of the cell cycle. These

authors reported, in agreement with Riley and Keller, that minor

changes in the composition of nuclear matrix could be noted when

nuclear matrixes, isolated from cells in various phases of the cell

cycle, were compared by electrophoresis. Sieber-Blum and Burger

(1977), using CHO cells, have compared NE, isolated from synchronized

cell cultures, by electrophoresis and found no noticeable differences

in the stained gel profiles of cell cycle specific NE fractions.

In an effort to determine whether any changes occur in CHO NE

during the cell cycle which could be detected as changes in the

coomasie blue staining profile of NE peptides and glycopeptides

separated on SDS-PAGE, I have isolated NE from cells at various stages

of the cell cycle. Specifically NE were isolated from a) cells which

were in the logarithmic phase of growth (i.e. primarily G1 cells);

b) cells which had been grown to stationary phase, diluted, allowed

to reinitiate the cell cycle in fresh HU containing media and then








collected 10 hr after dilution (i.e. cells at G1/S boundary, Figure 7);

c) cells 5 hr after release from HU (i.e. cells at the G2/M boundary);

and d) cells 10 hr after release from HU (i.e. cells in early G1).

The NE's from these four distinct cell populations were then solu-

bilized in SDS as described in Materials and Methods and equal

amounts of protein applied to individual wells of 7.5 12.5% Laemmli

discontinuous SDS-PAGE. As can be seen in Figure 8, coomasie blue

staining of the various isolates showed some differences in the

overall coomasie blue stained profile of NE's isolated from the

different stages of the cell cycle. However the cell cycle related

differences shown in Figure 8 were not consistently seen from exper-

iment to experiment suggesting that these differences are of

questionable physiologic significance. It is my own bias, based on

observations drawn from a number of experiments identical to that

outlined in Figure 8, that there are no reproducible differences in

the coomasie blue stained profile of NE derived from the four phases

of the cell cycle discussed in this section of the dissertation.

Dilution of [3H] Leucine Labeled NE

Using synchronized and uniformly labeled CHO cells I have

examined the dilution of labeled NE in order to follow synthesis of

NE peptides during the G2-M-G1 transition. Such label dilution

experiments were directed at determining whether peptides present in

NE, isolated from early G1 cells (10-12 hr after release from HU),

were synthesized de novo during the G2-M-G1 transition or whether

the peptides present in the early G1 NE pre-existed in the cell

prior to mitosis.















IS

















-













Figure 8. SDS Gel Electrophoresis of NE Peptides
Isolated from Synchronized Cells

Nuclear envelopes were isolated from cells in
logarithmic growth and from cells at various
stages of the cell cycle. Nuclear envelope
fractions were solubilized in SDS and electro-
phoresis was performed as described in
Materials and Methods. Lane 1, log phase;
Lane 2, G2/M; Lane 3, M/G1; Lane 4, Gl/S.







Specifically CHO cells were grown for 5 generations in 0.2pCi/ml

[3H] leucine and then synchronized in the presence of 0.2pCi/ml [3H]
leucine as described in Materials and Methods. In late G2 (5 hr

after HU removal, Figure 7) the cell culture was harvested, washed

twice with PBS and then the 6 liters of cells divided into two equal

aliquots. From one aliquot NE were immediately isolated (described

in Table IV as the G2/M NE population). The remaining cells were

returned to fresh medium free of [ 3H] leucine but containing 13.2 mg/

liter cold leucine. Four hours later (after 80% of the cells had

completed M, Figure 7) these cells were harvested and NE immediately

isolated (described as the M/G1 NE population in Table IV). The

specific activity of these two NE fractions was then determined and

compared. As can be seen in Table IV, the average specific activity

of the M/G1 NE is approximately 25% less than the specific activity

of the NE's isolated from the G2/M cell population. These data

suggest that although some de novo synthesis of NE protein has

occurred over the 4 hr time span between the isolation of the G2/M

and M/G1 NE, the majority of the peptides present in the M/G1 NE's

pre-existed in the G2/M cells. The data presented in Table IV do

not, of course, take the precursor pool into account. Resolution

of this problem will be discussed below.

The reduction in specific activity broadly described in

Table IV was further investigated by isolating NE at a time point

(6.5 hr after release from HU) at which less than 5% of the cells

have completed M and every hour thereafter until greater than 80%

of the cells have completed M (Figure 9). Cells were grown and











Table IV

Dilution of [3H] Leucine Labeled NE Protein

Specific Activities of NE Protein (cpm/mg protein)

G2/M M/G1 Ratio (M/G1:G2/M)

5.3 x 105 3.9 x 105 0.74

9.3 x 105 6.7 x 105 0.72

6.3 x 105 4.2 x 105 0.67

2.7 x 105 2.1 x 105 0.78

1.4 x 105 1.1 x 105 0.79

x= 0.74

SD = + 0.05


Cells were grown and synchronized in media containing 0.2pCi/ml [3H] leucine and NE
isolated from half of the cell population immediately before synchronous division (G2/M NE).
Four hours later (when '80% of the cells had completed M) NE were isolated from the other
half of the cell population which had proceeded through M in the absence of [3H] leucine (M/G1 NE).
Specific activities were determined as described in Materials and Methods.








synchronized in the presence of [ H] leucine as described for the

experiments presented in Table IV. Six and one-half hours after

release from HU (Figure 9), cells were harvested and washed twice

in PBS. One aliquot of cells was used immediately for NE isolation

(G2/M NE), while the remaining cells were returned to culture in

complete medium lacking [3H] leucine. Each hour thereafter, for the

next 6 hr, an aliquot of cells was harvested, NE isolated and the

specific activity of NE protein was determined. The specific

activity of the NE's at each time point is displayed in Figure 9.

Clearly, NE's which are in the late G2 phase of the cycle (8.5 hr

after release from HU) have lost 10% of the label associated with

the envelopes suggesting that during very late G2 labeled components

of the NE are rapidly diluted with unlabeled components. As the

NE's enter M there appears to be a sharp decrease in the rate of

dilution and then as NE's enter G1 a resumption of label dilution

occurs (Figure 9). Dilution of labeled NE peptides is greatly

reduced during that period of time in which the cell population is

dividing when compared to the dilution observed in NE's isolated from

cells in the G2 and G, phases of the cell cycle.

In order to determine whether a particular peptide or group of

NE peptides were preferentially lost during the G2-M-G1 transition,

[3H] leucine-labeled NE, isolated during the experiments described in

Table IV were solubilized in SDS, labeled peptides and glycopeptides

separated on a discontinuous SDS-PAGE, and the profile examined by

fluorography. Figure 10 contains both the coomasie blue stained

profile of the SDS-PAGE and the fluorogram obtained from the labeled






















Figure 9. Dilution of [3H] Leucine Labeled NE Protein

A suspension culture was grown to stationary
phase in the presence of O.2pCi/ml [3H] leucine
and synchronized as described in Materials and
Methods in O.2pCi/ml [3H] leucine. Six and one-
half hours after release from HU NE were prepared
from an aliquot of cells. The remaining cells
were washed twice with PBS and returned to
culture in complete medium lacking [3H] leucine.
Nuclear envelopes were isolated each hour after
removal of [3H] leucine and specific activities
of each fraction were determined as described
in Materials and Methods. Fraction of cells divided
was determined from 4 replicate counting in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0-0; fraction divided D-0.





50
















4 1.0

-.9

-.8

-.7
S3-

a_ -.5 z





o2- 2
2..3


0 3
ax -




S1 ,00


6 7 8 9 10 11 12 13
TIME AFTER RELEASE
FROM HU (HRS)






















Figure 10. SDS Gel Electrophoresis of NE Peptides Isolated
After Removal of [3H] Leucine at the G2/M
Boundary

A cell culture was grown for 5 generations in
0.2pCi/ml [3H] leucine and synchronized in
0.2pCi/ml [3H] leucine as described in Materials
and Methods. Nuclear envelopes were isolated
before label removal (G2/M interface, 7 hr after
removal of HU) and after division in the absence
of label (M/G1 interface, 11 hr after removal of
HU). The isolated NE were solubilized in SDS,
electrophoresed and fluorographed as described
in Materials and Methods. (A) Fluroograph of
NE peptides isolated at G2/M (lane 1) and M/G1
(lane 2) interfaces. Equal numbers of counts
(31,000 cpm) were applied to the gel. (B)
Coomasie blue stained profiles corresponding
to the fluorogram in A. The G2/M (lane 1)
fraction contained 75pg of protein while the
M/G1 (lane 2) fraction contained 50pg protein.



















I~ K


S..


CM


V-


S- om


1~1








components. From the fluorogram presented in Figure 10 it is apparent

that, within the detection limit of the fluorographic process and

one dimensional gel electrophoresis, no individual NE peptide or

glycopeptide is preferentially lost during the G2-M-G1 transition.

Incorporation of the [3H] Leucine into NE Protein

One likely, if not the most likely, explanation for the reduced

specific activity found in M/G1 NE's relative to G2/M NE's (Table IV)

is dilution of the pre-existing (i.e. G2/M) label with unlabeled NE

components synthesized and inserted into the NE during either G2, M,

or G1 phase of the cell cycle after removal of [3H] leucine from the

culture medium. The data presented in Figure 9 suggests that some

dilution of label occurs in G2 immediately after removal of [3H]

leucine from the culture medium as well as in G1. In order to

determine whether this dilution results from phase-specific biosyn-

thesis of NE peptides, cells were synchronized as described above and

then 5.25 hr after release from HU an aliquot of cells was taken and

pulsed for 1 hr in medium containing O.21Ci/ml [3H] leucine but only

25% of the standard F-10 leucine concentration. After 1 hr in labeled

precursor an aliquot was taken from the homogenate and the NE was

isolated. Cells were pulsed with O.2pCi/ml [3H] leucine and NE iso-

lated every hour from 5.25 11.5 hr after release of cells from an

HU blockade (Figure 11).

The specific activity of each NE and homogenate fraction was

determined and the specific activities plotted as a function of time

after release from HU block. As can be seen, the specific activity

of the NE fractions remain relatively constant (t 5%) from 6 9 hr




















Incorporation of [3H] Leucine into NE Protein


A suspension
phase and syn
and Methods.
blockade into


culture was grown to stationary
chronized as described in Materials
The culture was released from HU
Media containing 25% of the normal


F-10 leucine concentration. One hour before
synchronous division (i.e. 5.25 hr after removal
from HU), an aliguot of cells was removed, pulsed
with 0.2pCi/ml [ H] leucine for one hour and
harvested for NE isolation. Each hour thereafter,
an additional aliquot of cells was pulsed for 1 hr
with 0.2pCi/ml [3H] leucine, NE isolated, and the
specific activity determined on each NE and
homogenate fraction. Fraction of cells divided
was determined from 4 replicate counting in a
Levy-Hausser counting chamber. Nuclear envelope
specific activity 0 0; homogenate specific
activity A-A ; fraction divided[-[].


Figure 11.































-.9


-.8


.7


-.6


-.5 z
0
-.4 i


-.3 L


-.2


TIME AFTER RELEASE
FROM HU (HRS.)








after release from HU (by which time approximately 40% of the cells

in the population have divided). However between 9 and 10 hr after

release from HU when the majority of the cell population is in the

early portion of the G1 phase of the cell cycle (Figure 11), incor-

poration of [ H] leucine into the NE's increased 1.25 times that

seen in M and by 11 hr after release, incorporation of [3H] leucine

into the NE is 1.56 that seen in M. Taken together, the data in

Figure 9 and Figure 11 suggest that the majority of NE synthesis (and

consequently dilution of pre-existing label) occurs in late G2 and

early to mid Gl, with NE specific synthesis in M being low relative

to either of these time periods.

Determination of acid soluble [3H] leucine counts (cpm/mg protein)

in the whole cell homogenate at each time point clearly demonstrated

that the increase in specific activity of NE and homogenate protein

seen in Gl was not due to increased transport of labeled precursor

but rather was due to enhanced de novo biosynthesis.

To determine whether specific peptides of the NE might be

preferentially synthesized during the G2-M-G1 segment of the cell cycle

NE fractions, obtained from the time points taken in Figure 11, were

solubilized in SDS and the peptide and glycopeptide composition dis-

played by coomasie blue staining and fluorography on a 7.5 12.5%

discontinuous SDS-PAGE. The fluorogram presented in Figure 12A

indicates that, at the limits of detection by one dimensional SDS gel

electrophoresis and fluorography, all NE peptides are synthesized at

some point in the G2-M-G1 transition. Comparison of the coomasie

blue stained profile (Figure 12B) with the intensities of the individ-

ual bands in the exposed fluorogram (Figure 12A) suggests that the





















Figure 12. SDS Gel Electrophoresis of [3H] Leucine Pulse Labeled
NE Peptides

Nuclear envelopes isolated during the experiment
described in Figure 11 were solubilized and subjected
to electrophoresis and fluorography as described in
Materials and Methods. (A) Fluorogram depicting
[ H] leucine labeled NE isolated at the six time
points indicated in Figure 11. Lane 1, 5.75 hr
after HU removal; Lane 2, 6.75 hr after HU removal;
Lane 3, 7.75 hr after HU removal; Lane 4, 8.75 hr
after HU removal; Lane 5, 9.75 after HU removal;
Lane 6, 11 hr after HU removal. Equal numbers of
counts (11,000 cpm) were applied to each lane.
(B) Coomasie blue stained profile corresponding
to the fluorogram in (A). Lane 1, 72pg protein;
Lane 2, 74pg protein; Lane 3, 76pg protein; Lane 4,
71ig protein; Lane 5, 56ug protein; Lane 6, 42pg
protein.










I -II- (








I




S- (0


C


c
L








NE peptides were labeled approximately in proportion to their relative

staining with coomasie blue.

Dilution of [3H] Choline Labeled NE

Using [3HI choline as a precursor of phosphatidyl choline, I

have examined the relative reutilization of NE phospholipids during

the G2-M-G1 transition. As in the work with [ H] leucine labeled NE,

I have initially followed the dilution of label at two time points

within the cell cycle, the so-called G2/M stage and the M/G1 stage of

the cell cycle (Table V).

Chinese hamster ovary cell cultures were grown for 5 generations

in medium supplemented with O.lpCi/ml [3H] choline and then syn-

chronized in complete medium containing O.liCi/ml [3H] choline. Five

hours after release from HU (i.e. at the G2/M transition) the labeled

culture was harvested, washed twice with PBS, and divided into equal

aliquots. G2/M NE were prepared from one aliquot while the other

aliquot was returned to culture in complete F-10 medium without

[3H] choline but containing 0.7 mg/liter cold choline. Following
division (i.e. 9 hr after release from HU, see Figure 7), M/G1 NE

were prepared and chloroform-methanol extracts (Rouser and Fleischer,

1967) were prepared from both preparations. The specific radio-

activity of the G2/M NE and the M/G1 NE phospholipid (cpm/pg lipid

phosphorus) was determined and the specific activities compared

(Table V).

The simplest interpretation of the data in Table V would suggest

that approximately 65% of the M/G1 NE phospholipid was present in the

cell prior to mitosis while 35% of the M/G1 phospholipid was










Table V

Dilution of Labeled NE Phospholipids


NE Phospholipid Specific Activity (cpm/pg lipid phosphorus)

Radiolabel G2/M M/G1 Ratio (M/G1:G2/M)

[3H] choline 2.0 x 104 1.4 x 104 0.70

[32P] orthophosphatet 0.8 x 104 0.5 x 104 0.63


x= 0.67


Cells were grown and synchronized in media containing O.l~Ci/ml [3H] choline or 0.4pCi/ml [32p]
orthophosphate and NE isolated from half of the cell population immediately before synchronous
division (G2/M NE). Four hours later (whenm80% of the cells had completed M) NE were isolated
from the other half of the cell population which had proceeded through M in the absence of
label (M/G1 NE). Specific activities were determined as described in Materials and Methods.
$ [32p] data were calculated from experiment shown in Figure 13.








synthesized during the G2-M-G1 transition. Reutilization of label

is not considered in these data but will be discussed below.

Dilution of [32P] Orthophosphate Labeled NE Phospholipid

Since choline labels a specific phospholipid which could con-

ceivably behave differently than the majority of the NE phospholipid,

I felt it important to determine the dilution of total NE phospho-

lipid. To do this I chose to label cells to constant specific

activity with [32P] orthophosphate. In these experiments CHO cell

cultures were maintained and synchronized in complete F-10 medium

supplemented with 0.4pCi/ml [32P] orthophosphate. As in the previous

radiolabel dilution experiments (Tables IV and V, Figure 9), the

culture, labeled to constant specific activity with [32 P, was

harvested approximately 6.5 hr after release from HU (at the G2/M

transition) and washed twice with PBS. One aliquot of the washed cells

was immediately used for preparation of NE while the remaining cells

were returned to complete medium lacking [32P] and allowed to proceed

through M. Each hour after the cells were returned to unlabeled media,

a percentage of the cells was harvested, homogenate prepared, and NE

isolated. After the last isolation was completed (11.5 hr after

release from HU, Figure 13), phospholipid was extracted into chloro-

form methanol, phosphorous anlaysis was performed on each fraction,

and the NE phospholipid specific activity (cpm/ug lipid phosphorus)

determined. Figure 13 describes the time course of the change in

phospholipid specific activity in cell homogenate and the NE fraction.

Clearly the specific activity of both the NE and homogenate remains

relatively constant while the majority of the cells are in late G2























Figure 13. Dilution of [32P] Labeled NE Phospholipid

A suspension culture was grown to stationary phase
in the presence of O.4iCi/ml [32p] orthophosphate
and synchronized as described in Materials and
Methods in 0.4uCi/ml [32p] orthophosphate. Six
and one-half hours after release from HU NE were
prepared from an aliquot of cells. The remain-
ing cells were washed twice with PBS and returned
to culture in complete medium lacking [32p].
Nuclear envelope and homogenate were isolated each
hour after removal of [32P] and phospholipid
specific activities were determined on each frac-
tion as described in Materials and Methods.
Fraction of cells divided was determined from 4
replicate counting in a Levy-Hausser counting
chamber. Nuclear envelope specific activity
0-0; homogenate specific activity A-A; fraction
divided -D .





63




















1.0

-.9




I -.7

I0 /
0

0 t
--.

.4

oA _.30
x-
.2u
0 5
N .1


6 7 8 9 10 11 12
TIME AFTER RELEASE
FROM HU(HRS.)








and M and then begins to drop as the cells enter G1. After 80% of

the cells have completed division (Figure 13, 11.5 hr after release

from HU), the specific activity of the NE is approximately 65% of

the NE phospholipid specific activity before M (Figure 13 and Table V).

The simplest explanation for these data is that 65% of the phospho-

lipid present in NE 12 hr after release from HU (i.e. early G1, see

Figure 13) was present in the cell prior to M.

Interestingly the specific activity of the NE phospholipid

begins to decrease after 20-30% of the cells have entered the G1 phase

of the cell cycle whereas the specific activity of the whole cell

phospholipid drops only after %80% of the cells have completed M

(i.e. the majority of the cells are in early to mid G1). The data

in Figure 13 clearly suggest that synthesis of phospholipid to be

incorporated into NE and other cellular phospholipid increases as the

cells enter G1.
32
Incorporation of [ 32] Orthophosphate into NE Phospholipid

In order to confirm that the dilution of NE phospholipid specific

activity, seen in Table V and Figure 13, is due to synthesis of new

phospholipid, which is, in turn incorporated into NE, cell cultures

were grown to stationary phase and then synchronized as described

previously in complete medium containing no label. Seven hours after

release from HU (i.e. at the G2/M transition, Figure 14), an aliquot

of cells was removed from the stock culture and pulsed for 1 hr with

0.8pCi/ml [32P] orthophosphate. After one hour in [32 P, the labeled

culture was harvested and both NE and homogenate prepared. Similarly

treated aliquots of cells were pulsed for 1 hr with [32P] over the








next 5 hr. The specific activity of phospholipid was then deter-

mined by chloroform-methanol extraction and phosphorus analysis on

each homogenate and NE fraction. The time course of [32P] incorpora-

tion into NE and homogenate phospholipid can be seen in Figure 14.

As would have been predicted from Figure 13, after 30-40% of the

cells have completed division, the homogenate and NE phospholipid

specific activity begins to rise. This increase in specific activity

of the two fractions remains linear over the remainder of the time

course studied. These data (Figure 14) clearly suggest that a major

fraction of the phospholipids which are destined to become part of the

NE are synthesized and incorporated into the organelle during early G1.

Rate of Precursor Pool Equilibration

To more rigorously interpret the label dilution data described

throughout the previous experiments (Tables IV and V and Figures 9

and 13), I examined the kinetics of precursor pool equilibration in

synchronized cultures which were manipulated in a fashion identical

to that already reported in Tables IV and V and Figures 9 and 13.

Such data is, of course, essential if I am to make more precise

estimates of the amount of NE peptide and phospholipid synthesis (and

subsequently dilution of pre-existing label) which occurs during the

G2-M-G1 transition in our synchronized cultures.

In the experiments presented in Figure 15, CHO cells were

grown for 5 generations in medium supplemented with 0.2pCi/ml [3H]

leucine (i.e. until the peptides in the cell had reached a constant

specific activity) and then synchronized as described in Materials

and Methods in the presence of 0.2pCi/ml [3H] leucine. Five hours























Figure 14. Incorporation of [32P] into NE Phospholipid

A suspension culture was grown to stationary
phase and synchronized as described in Materials
and Methods. Seven hours after removal of HU an
aliquot of cells was removed, pulsed with O.8pCi/ml
[32p] orthophosphate for one hour and NE isolated.
Each hour thereafter (for 6 hr) another aliquot of
cells was pulsed with 0.8uCi/ml [32p] for 1 hr and
NE isolated. Phospholipid specific activity was
determined on NE and homogenate isolated after
each pulse. Fraction of cells divided was deter-
mined from 4 replicate counting in a Levy-Hausser
counting chamber. Nuclear envelope specific
activity 0-0; homogenate specific activity A-A ;
fraction divided I-0.I


















































TIME AFTER RELEASE
FROM HU(HRS.)








after release from HU an aliquot of cells was removed from the culture,

collected on a glass fiber filter, quickly washed with 10 volumes of

ice cold PBS and solubilized in 0.2 N NaOH. The remaining cells were

harvested, washed twice with PBS (as was done in all my previously

presented dilution data). Immediately after the PBS washes (6 hr

after HU removal) another aliquot of cells was collected on a glass

fiber filter, washed and solubilized. The remaining cells were

returned to complete media without [ 3H] leucine. Each hour after

returning the cells to unlabeled media an aliquot of cells was

collected, washed, and placed in 0.2 N NaOH. After all the samples

had been collected (10 hr after removal from HU, 90% of the cells

having divided, Figure 15), the filters were heated at 700C for 5 min,

cooled on ice, and precipitated overnight in 10% TCA (40C). The

specific activity of the TCA soluble [ 3H] leucine counts was then

determined and plotted as a function of time (Figure 15). As can

be seen in Figure 15, approximately 55% of the [3H] leucine, which

was in the cytoplasmic precursor pool before the cells were harvested,

was removed during the initial two PBS washes. Subsequent culturing

of the washed cells in [3H] leucine-free media reduced the soluble

[3H] leucine pool to a specific activity with was 30% of the initial
specific activity of the initial time point (Figure 13).

By first setting the initial, pre-PBS wash, soluble [3H] leucine

pool at 100% (initial time point, Figure 15) and then averaging the

fractional value of this initial pool which was measured during the

G2-M-G1 transition in the absence of label, I have calculated a mean

soluble [3H] leucine pool specific activity during the G2-M-G This
























Figure 15. [3H] Leucine Pool Dilution

A suspension culture was grown for 5 generations
in O.2uCi/ml [3H] leucine and synchronized in
O.2pCi/ml [3H] leucine as described in Materials
and Methods. Five and one-half hours after HU
removal a sample was taken and acid soluble [3H]
leucine determined as described in Materials and
Methods. The remaining cells were washed 2 times
with PBS and another sample taken for detemina-
tion of TCA soluble [3H] leucine counts. The
washed cells were then returned to unlabeled
media and samples taken each hour for determination
of TCA soluble [3H] leucine counts. Fraction of
cells divided was determined from 4 replicate
counting in a Levy-Hausser counting chamber.
Acid soluble [3H] cpm/mg total protein 0-0;
fraction dividedD-D .





70

















r 1.0

-.9

-.8

3H-LEUCINE -.7
removed
I .6

8.-
3. .52
x7-

u z
15- 0

33- i,,


1- .
.,. ....... 0
5 6 8 10
TIME AFTER RELEASE
FROM HU (HRS)








calculation results in a mean value equal to 36% (average of two

experiments) of the [3H] leucine found in the soluble pool of cells

prior to removal of label. This mean value represents the percentage

of [3H] leucine in the soluble pool of cells dividing in the absence

of label relative to those cells (before PBS washes) whose cytoplasm

had reached equilibrium with [3H] leucine in the medium. Since the

leucine pool size has been shown to remain constant in HeLa cells

during this phase of the cell cycle (Robbins and Scharff, 1966), I

have concluded that the protein synthesized following removal of

[3H] leucine from the medium, is synthesized with a specific activity
that is, on the average, 36% of the specific activity of proteins

synthesized when [3H] leucine was maintained in the culture.

Using the formula below (which is derived in the appendix), I

have estimated the amount of early G1 NE protein synthesized during

the G2-M-G1 transition. I have based these calculations on the ratio

of specific activities of G2/M NE to M/G1 NE presented in Table I.

SAG1 SAG2
SA' SAG2

where: p = proportion of G1 NE synthesized during G2-M-G1,

SAG1 = specific activity of M/G1-NE

SAG2 = specific activity of G2/M-NE
SA' = relative specific activity of pool after label removal

Setting SAG2 equal to unity gives SAG1 a value of 0.74 (Table IV).

Substituting these values into the equation we have:


S(0.74) 1.0 0.41
p = (0.36) 1.0 41







Thus when one takes into account the fact that the soluble pool

is not completely depleted of [ H] leucine after removal of [3H]

leucine from the media, one calculates that%40% of the M/G -NE

protein was synthesized during the G2-M-G1 transition. By simple sub-

traction then 160% of the M/G1 NE protein must have pre-existed M.

I have used the same experimental design and logic to examine

the dilution of the [32P] labeled phosphatidic acid pool in order to

more precisely estimate NE phospholipid biosynthesis during the

G2-M-G1 transition. Phosphatidic acid was chosen since it is a

precursor of phospholipids and is not shunted into other pathways

(Howard and Howard, 1974; Spector, 1972).

In these experiments (Figure 16) CHO cells were grown for 5

generations in 0.4 pCi/ml [32P] and then synchronized as described

in Materials and Methods in 0.4 pCi/ml [32P] orthophosphate. Six

hours after release from HU (Figure 16) an aliquot of cells was

harvested and immediately extracted with chloroform-methanol to give

me a base value for phosphatidic acid specific activity. The

remaining cells were also harvested, washed twice in PBS and another

aliquot of cell population extracted with chloroform-methanol

(Figure 16, 6.5 hr after HU removal). The remaining cells were then

returned to media containing 60 mg/liter cold phosphate but free of

[32P]. As shown in Figure 16, aliquots were harvested from the cell
population each hour through the G2-M-G1 transition and extracted

with chloroform-methanol. After all samples had been extracted, each

was Folch backwashed (Rouser and Fleischer, 1967), filtered, and

concentrated for application to thin layer chromatography plates.

























Figure 16. [32P] Phosphatidic Acid Pool Dilution

A suspension culture was grown for 5 generations
in O.4pCi/ml [32p] and synchronized in 0.4iCi/ml
[32p] as described in Materials and Methods.
Five hours after HU removal an aliquot of cells
was harvested by centrifugation and phosphatidic
acid specific activity was determined as
described in Materials and Methods. The remain-
ing cells were washed twice with PBS, another
aliquot removed for phosphatidic acid specific
activity determination and the bulk of the
cells returned to unlabeled media. Aliquots
were removed for phosphatidic acid specific
activity determination each hour thereafter
until 90% of the cells had completed division.
Fraction of cells divided was determined from
4 replicate counting in a Levy-Hausser counting
chamber. Phosphatidic acid (PA) specific
activity 0-0; fraction divided 0-D.































In
0

15-


'0



x 5
0.
a
U
('I


TIME AFTER RELEASE
FROM HU (HRS.)








Thin layer chromatography was run in solvent system I of Skipski and

Barclay (1969) for acidic phospholipids. After development, the

phosphatidic acid spot was visualized by spraying with distilled

water, scraped from the plate, eluted from the silica gel with

chloroform-methanol, and rechromatographed in the same developing

solvents. The phosphatidic acid spot was visualized in an iodine

chamber, scraped, and eluted as before. The specific activities of

phosphatidic acid at each time were determined on the eluted material

as described in Materials and Methods.

The rate of decrease in the specific activity of [32P] labeled

phosphatidic acid is presented in Figure 16. The arithmetic mean

intracellular specific activity during the G2-M-G1 transition was

calculated to be 38% (average of two experiments) of that found in

cells prior to removal of [32P] from the media. Calculating from

the observed dilution of [32P] labeled NE phospholipid (Table V) I

find the proportion of M/G1-NE phospholipid synthesized during the

G2-M-G1 transition to be 0.53.

0.67 1.0
P = 0.38 1.0 0.53

Again by subtraction, I estimate that 50% of the M/G1-NE pre-existed

M.

Changes in Nuclear Surface Area

Since a change in total nuclear surface area might reflect

synthesis of NE and at the same time affect the interpretation of

my data, I have determined mean nuclear surface areas in a population

of cells before and after the G2-M-G1 transition (i.e. at 5 hr and








8 hr in Figure 7). Cells were synchronized as described in Materials

and Methods and 5 hr after release from HU, an aliquot of cells was

removed, and prepared for sectioning as described in Materials and

Methods. After 75% of the cells had completed division (8 hr after

HU removal) another aliquot of cells was removed and prepared for

sectioning. Thick sections were taken from both cell samples,

stained, and photographed as described in Materials and Methods.

Measurements of the surface area of individual nuclei were made by

planimetry (Weibel, 1969). The nuclei in both cell populations were

found to have a mean nuclear surface area of"lO02. If we assume

that the mean nuclear surface area after the cells have completed

mitosis represents the mean surface area of "daughter" nuclei only,

then the magnitude of the G1 (8 hr after release from HU) nuclear

surface area relative to G2 nuclear surface area will reflect what

increase, if any, has occurred during the G2-M-G1 transition. If no

increase in NE occurred during the G2-M-G1 transition, this would be

reflected by a mean nuclear surface area in G1 which is exactly one-

half of the G2 mean nuclear surface area. My measurements suggest

that the total nuclear surface area has doubled during the G2-M-G1

transition (G1 and G2 mean nuclear surface areas are approximately

equal). If such a doubling in nuclear surface area was due entirely

to NE synthesis (i.e. no stretching or expansion of available NE),

I would expect to see a 50% dilution of NE protein during the G2-M-G1

transition. My label dilution experiments have indicated that the Gl

NE peptides have a specific activity which is 60% of the G2 NE while

the G1 NE phospholipids have a specific activity which is 50% that

of the G2 NE. In my opinion both my labeling data and my surface

area data are, within the limits of resolution, complementary.













CHAPTER IV
DISCUSSION

In Chapter II of this dissertation I have described a procedure

for the isolation of highly enriched nuclear and NE fractions from

CHO cells. The nuclei and NE fractions obtained by use of this

procedure have been characterized by light and electron microscopy;

chemical and enzymatic assay; as well as SDS gel electrophoresis.

These characterizations have demonstrated that the nuclei and NE

fractions isolated are distinct from each other and that both frac-

tions are distinct from the plasma membrane and whole cell homogen-

ate.

Although electron microscopy and marker enzyme assays indicate

that contamination of the nuclei and NE fraction with other organ-

elles is very low, the true enrichment of NE in the final pellet as

compared to the homogenate is impossible to determine since no

dependable enzymatic marker has been unequivocally localized to this

organelle in CHO cells. However, it must be again stressed that

the final NE fraction obtained by the procedures outlined in this

dissertation is morphologically similar to the intact NE found in

the cell in that the isolated NE's contain a double membrane bilayer

as well as the pore-lamina complex, thus strongly suggesting that

the isolated procedure described produces a highly enriched NE

fraction.








The NE fraction isolated from CHO cells is composed primarily

of protein and phospholipid having 200pg phospholipid/mg protein.

This phospholipid/protein ratio suggests that, in comparison to other

isolated cellular membranes (Fleischer and Kervina, 1974), the isolated

NE fraction is relatively protein rich. This finding may derive

from the fact that the isolation procedure described, isolates both

the nuclear membrane and the so-called proteinaceous lamina. The

inclusion of the proteinaceous lamina in the NE fraction would be

expected to increase the protein to lipid ratio.

Examination of the peptide composition of the NE fraction by

SDS gel electrophoresis demonstrates that the major coomasie blue

staining peptides of the NE fraction chromatograph with molecular

weights between 55,000 and 75,000 daltons. These peptides are

undoubtedly related to the pore-lamina and nuclear matrix proteins

identified in rat liver (Dwyer and Blobel, 1976) and HeLa cell

(Hodge et al., 1977) NE's. It is of interest to note that the

majority of the remaining NE peptides chromatograph with a molecular

weight greater than 75,000 daltons. Relatively few coomasie blue

staining bands are found to run at molecular weights below 50,000

daltons and no peptides appear to co-migrate with the histones seen

in the nuclear fraction.

It can not be emphasized too much that although the isolation

technique described required careful handling of material during the

preparation of the nuclei and NE, it does produce material in

sufficient quantity and of sufficient purity to permit biochemical

studies directed at elucidating the molecular basis of both NE

structure and biosynthesis.








In Chapter III of this dissertation the NE isolation procedure

(described in Chapter II) has been employed to examine the question

of whether or not cellular proteins and phospholipids which pre-existed

the mitotic phase of the CHO cell cycle are used by the CHO cell in

the reformation of the NE which occurs during telophase.

Sodium dodecyl sulfate gel electrophoresis of NE isolated at

various stages of the CHO cell cycle showed no consistent differences

in peptide or glycopeptide composition which could be assigned to

any phase of the cell cycle. It is of interest to note that I did not

see any cell cycle dependent changes in the peptides migrating between

55,000 and 75,000 MW, as have been reported by Hodge et al. (1977) to

occur in the HeLa cell nuclear matrix. In this regard, the data in

this dissertation agree with those of Sieber-Blum and Burger (1977)

who also did not see cycle specific changes in CHO cell NE's.

In Chapter III of this dissertation,evidence, obtained primarily

from what I have dubbed "label dilution" studies, has been presented

which strongly suggest that at least 60% of the early G1 NE protein

and at least 50% of the early G1 NE phospholipid existed in the cell

prior to mitotic breakdown and reformation (i.e. in late G2). These

data further suggest that the remaining 40% of early G1-NE protein

and the remaining 50% of early G1-NE phospholipid come from de novo

synthesis of NE components. Pulse label experiments suggest that

relatively little NE protein or phospholipid synthesis occurs in M

but rather that the majority of this NE synthesis occurs in late G2

and early G1. Unfortunately the degree of synchrony obtainable with

such a large number of cells does not allow me to determine whether








some NE components are specifically synthesized in the G2, M, or G1

phases of the cell cycle. However, my data do indicate that at some

point in the G2-M-G1 transition all of the NE peptides separable on

a one-dimensional SDS-PAGE are labeled with [3H] leucine suggesting

that no specific components) is carried through the transition while

another components) is degraded and totally resynthesized during

the G2-M-G1 transition.

Measurement of nuclear surface area in synchronized populations

of cells in G2 and G1 indicated that there was no significant differ-

ence in the average surface area of the two nuclear populations.

These data suggest that the total nuclear surface has increased

%2 fold between G2 and early G1. If one assumes that increases in

surface area are directly correlated to NE biosynthesis, these data

predict that "label dilution" experiments would detect an%50%

dilution of NE protein during the time period examined. This is in

good agreement with our calculated results of 40% dilution and 60%

reutilization of pre-existing peptides.

The 60% of the early G1-NE protein which the data suggest

pre-existed M, when taken together with the data suggesting a burst

of early G1 NE synthesis does strongly imply that a majority of

G1 NE proteins come from pre-existing cellular components. Thus,

in my opinion, these data unequivocably rule out "complete" de novo

synthesis of NE protein as being responsible for mitotic reassembly.

Unfortunately I cannot state unequivocally that the early G1 NE

protein which pre-existed M resided solely in the G2 NE. The

possibility clearly exists that G1 NE components are made prior to








M and then stored in some cellular compartment for use in restruc-

turing the NE after division. The most likely organelle for such a

"storage function" would be the ER. Testing such a possibility must

await the availability of NE specific probes which can be applied

to the cells at various phases of the cell cycle to determine whether

"NE specific" components exist in other cellular organelles prior

to or during M.

The same problems and interpretation just discussed with regard

to NE peptides apply to those experiments which indicate that 50% of

the early G1 NE phospholipid pre-existed M. This apparently higher

rate of NE phospholipid biosynthesis relative to NE protein bio-

synthesis over the same G2-M-G1 transition may reflect a high turn-

over has been reported in other tissue culture cells (Cunningham,

1972).

I feel that my data can be interpreted to rule out complete

de novo synthesis of the NE during mitotic breakdown and reformation.

I also feel that the majority of the very early G1 NE components

pre-exist mitosis. A question which should now be asked is what is

the exact fate of NE peptides during mitosis. This can be ascer-

tained only by applying probes (e.g. antibodies) specific for NE

peptides to small populations of mitotic cells. Such probes should

allow identification of the precise location of NE peptides during

M and thus determine the "storage" and reutilization of such com-

ponents.













APPENDIX

DERIVATION OF THE EQUATION FOR CALCULATING THE PROPORTION
OF M/G1 NE SYNTHESIZED DE NOVO DURING THE G2-M-G] TRANSITION


p = proportion of M/G1 NE synthesized during the G2-M-G1
transition
SAG1 = specific activity of M/G1 NE

SAG2 = specific activity of G2/M NE
SA' = mean intracellular leucine pool specific activity during

the G2-M-G1 transition assuming an initial pool specific
activity of 1.0

The NE specific activity at M/G1 (SAG1) is equal to some pro-
portion (p) which was synthesized at a diluted pool specific activity

(SA') plus some proportion (l-p) synthesized before label removal at
the G2/M NE specific activity (SAG2). This can be expressed in the
following equation:
SAG1 = p(SA') + (l-p) SAG2 (1)
Rearranging equation 1 gives:
SAG1 = p(SA') + SAG2 p(SAG2) (2)

SAG1 SAG2 = p(SA') p(SAG2) (3)
SAG1 SAG2 = p(SA' SAG2) (4)

SAG1- SAG2
SA' SAG2 p (5)














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BIOGRAPHICAL SKETCH

Gregory E. Conner was born on December 21, 1950 in Jacksonville,

Florida, where he received his primary and secondary education in

parochial schools. In June of 1972 he received a Bachelor of Arts

degree from Vanderbilt University in Nashville, Tennessee. In

September of 1973 the author began graduate studies in the Department

of Biochemistry and Molecular Biology at the University of Florida.

Upon completion of these studies he will assume a post-doctoral

position at the Rockefeller University, New York, New York.

The author is married to the former Daphne Ann Wales of

Jacksonville, Florida.









I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Kennet D Nonan, Chairman
Assistant Professor of
Biochemistry and Molecular Biology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Charles M. Allen, Jr.
Associate Professor of
Biochemistry and Molecular Biology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Carl M. Feldherr
Associate Professor of
Anatomy









I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Edward M. Hoffman /
Associate Professor o
Microbiology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Thomas W. O'Brien
Associate Professor of
Biochemistry and Molecular Biology



This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

August 1978




Dean, College of Medicine




D anV-waduate School





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CG73r



























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