Endogenous amino acid transport and translation of rat liver mRNA in Xenopus laevis oocytes

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Title:
Endogenous amino acid transport and translation of rat liver mRNA in Xenopus laevis oocytes
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Campa, Michael Joseph, 1955-
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Amino Acids   ( mesh )
Biological Transport   ( mesh )
Oocytes   ( mesh )
Xenopus Laevis   ( mesh )
RNA, Messenger   ( mesh )
Biochemistry and Molecular Biology thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 139-153.
Statement of Responsibility:
by Michael Joseph Campa.
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Typescript.
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Vita.

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University of Florida
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Full Text











ENDOGENOUS AMINO ACID TRANSPORT AND
TRANSLATION OF RAT LIVER mRNA
IN XENOPUS LAEVIS OOCYTES















by

MICHAEL JOSEPH CAMPA


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




UNIVERSITY OF FLORIDA


1989















To Pam and Darwin.














ACKNOWLEDGEMENTS

I would like to thank the members of my committee for

their invaluable guidance and support throughout the course

of my graduate education. Special thanks go to Dr. Carl

Feldherr for his helpful discussions concerning the

microinjection of Xenopus laeves oocytes and for access to

his extensive knowledge of the field. In addition, I would

like to express my appreciation to Dr. Rusty Mans for

sharing with me his pervasive understanding of the science

of biochemistry. I would also like to thank my fellow

graduate students, Ron Laine, Barrie Bode, Rohit Cariappa,

Neil Shay, and Jan Dugan for their friendship and helpful

discussions over the past five years. A special word of

thanks go to Mary Handlogten and Elizabeth Dudenhausen for

their indispensable technical assistance, and to John

Berceann for his special assistance and questionable humor.

In addition, I would like to acknowledge especially my

mentor, Dr. Michael Kilberg, for allowing me to work under

his direction and for his invaluable guidance and support

throughout the completion of the work presented in this

dissertation.

Finally, I am forever indebted to my parents, Fred J.

and Barbara S. Campa, for their unfaltering support and

love.


iii
















TABLE OF CONTENTS

page

ACKNOWLEDGEMENTS ..................................... iii

LIST OF TABLES ........................................

LIST OF FIGURES ..................................... vi

ABSTRACT ................................................. iii

CHAPTERS

1 INTRODUCTION ..................................... 1

2 CHARACTERIZATION OF NEUTRAL AND CATIONIC AMINO
ACID TRANSPORT IN XENOPUS LAEVIS OOCYTES ......... 9

Introduction .................................... 9
Materials and Methods ............................ 11
Results ........................................ 17
Discussion ....................................... 37

3 EFFECT OF MEIOTIC MATURATION ON THE TRANSPORT OF
AIB, THREONINE, AND LEUCINE IN XENOPUS LAEVIS
OOCYTES .......................................... 43

Introduction .................................... 43
Materials and Methods ............................ 49
Results ......................................... 51
Discussion .......................................... 58

4 TRANSLATION OF RAT LIVER mRNA IN XENOPUS LAEVIS
OOCYTES ................. ......................... 65

Introduction ..................................... 65
Materials and Methods ........................... 71
Results ............................................ 95
Discussion ......................................... 120

5 CONCLUSIONS AND FURTHER DIRECTIONS ............... 130

REFERENCES .............................................. 139

BIOGRAPHICAL SKETCH .................................. 154















LIST OF TABLES
Table page

2-1 Transport of neutral and cationic amino acids
into oocytes in the presence of Na+, Li+, or
choline ....................................... 19

2-2 MeAIB inhibition of Na+-dependent transport
for selected amino acids ...................... 22

2-3 Inhibition analysis of Na+-independent leucine
transport ................................... 29

3-1 Measurement of Na+-dependent amino acid
transport in oocytes following induction of
GVBD by progesterone or TPA ................... 56

3-2 Measurement of saturable, Na-independent amino
acid transport in oocytes following induction
of GVBD by progesterone or TPA ............... 57

4-1 Determination of the relative concentration of
PEPCK mRNA in total RNA extracted from the
livers of control, glucagon-treated, and
diabetic rats ................................. 101















LIST OF FIGURES
Figure page

2-1 Time-course for transport of MeAIB into
X. laevis oocytes ........................... 21

2-2 Arginine and serine inhibition of AIB
transport ................................... 24

2-3 Inhibition of Na+-dependent arginine transport
by basic, acidic, and neutral amino acids ..... 26

2-4 Arginine and serine inhibition of Na-dependent
threonine transport ........................... 28

2-5 Inhibition of saturable Nat-independent
arginine transport by basic, acidic, and
neutral amino acids .......................... 31

2-6 The pH dependence for histidine inhibition of
Na*-independent arginine transport ............ 32

2-7 Stereo-selectivity for inhibitors of
Na+-independent BCH or threonine transport .... 36

3-1 Effect of K+ concentration on progesterone-
induced GVBD in oocytes ..................... 53

3-2 Time course of TPA-induced GVBD in oocytes .... 54

3-3 Effect of solvent, in the presence or absence
of progesterone, on the saturable
Na-independent transport of leucine .......... 62

4-1 Ethidium bromide-stained agarose gels of rat
liver RNA isolated from control, glucagon-
treated, or diabetic animals .................. 99

4-2 Results of Northern analysis of rat liver RNA
extracted from control (C), glucagon-treated
(G), or diabetic animals (D) .................. 100

4-3 Immunoblot analysis of rat liver proteins using
PEPCK and RSA anti-sera ..................... 104

4-4 Rat liver mRNA-induced synthesis of PEPCK in
oocytes ..................... .................. 105









4-5 Rat liver mRNA-induced synthesis of RSA in
oocytes ....................................... 106

4-6 Synthesis of PEPCK and RSA in oocytes to 96
hours following the microinjection of rat
liver mRNA .................................... 109

4-7 Synthesis of PEPCK and RSA in oocytes to 96
hours following the microinjection of rat
liver mRNA: results of laser scanning
densitometry .................................. 110

4-8 Synthesis of PEPCK and RSA in oocytes to 48
hours following the microimjection of rat
liver mRNA: results of laser scanning
densitometry ................................. 112

4-9 Synthesis of PEPCK and RSA in oocytes following
the microinjection of rat liver mRNA: effect of
mRNA quantity ................................ 114

4-10 Northern and immunoblot analyses of rat
liver RNA ................. .................... 116

4-11 Degradation of mRNA for rat liver PEPCK and
RSA in oocytes ............................... 119


vii














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

ENDOGENOUS AMINO ACID TRANSPORT AND
TRANSLATION OF RAT LIVER mRNA
IN XENOPUS LAEVIS OOCYTES

by

MICHAEL JOSEPH CAMPA

December, 1989

Chairman: Dr. Michael S. Kilberg
Major Department: Biochemistry and Molecular Biology

Neutral and cationic amino acid transport was

studied in stage 6 Xenopus laevis oocytes and found to be

mediated by Na+-dependent and Na+-independent processes.

Using the criteria of cis- and trans-inhibition, pH

sensitivity, and stereoselectivity, several distinct

transport activities were identified. The characteristics

of some of these activities resembled those of transport

systems previously described in other tissues. Evidence is

presented which supports the presence of activities similar

in many respects to Systems ASC, asc, y*, L, and B0+.

Although a portion of the transport of 2-aminoisobutyric

acid (AIB) into oocytes was found to be Nat-dependent, the

data do not support mediation by System A.

The effect of meiotic maturation, also referred to as

germinal vesicle breakdown (GVBD), on the transport of AIB,


viii








threonine, and leucine in oocytes was also investigated.

The in vitro induction of GVBD was achieved using

progesterone or tetradecanoylphorbol acetate (TPA).

Following the induction of GVBD, the Na*-dependent and Na+-

independent transport rates of all substrates tested

declined markedly.

The results of experiments investigating the oocyte-

mediated synthesis of liver phosphoenolpyruvate

carboxykinase (PEPCK) and serum albumin (RSA) following

microinjection of rat liver mRNA are also reported. The

quantities of PEPCK and RSA were found to increase with

time up to about 48 hours after microinjection. Further

incubation resulted in a decline in the amount of the two

proteins. In addition, the microinjection of 20 ng or less

of mRNA per oocyte was found to be more efficient than the

microinjection of quantities greater than 20 ng.

Furthermore, the oocyte-mediated translation of mRNA

extracted from the livers of fed, glucagon-treated, or

diabetic rats was shown to reflect the relative quantities

of PEPCK and RSA mRNA present as revealed by Northern

analysis. Experiments involving the microinjection and re-

extraction of mRNA revealed that the transcripts coding for

the two proteins are quite stable in the oocyte. The mRNAs

coding for both PEPCK and RSA were estimated to have half-

lives of at least three days in the oocyte.














CHAPTER I

INTRODUCTION

The South African clawed frog, Xenopus laevis, was

first described in 1803 by F.M. Daudin. Originally

believed to belong to the genus Bufo, it was not classified

as a member of the family Pipidae until many years later

and its present Latin name not officially adopted until

1890 (Leslie, 1890). The skin of the animal is smooth and

the body flattened dorsoventrally. The fore and hind limbs

are splayed out to the sides and, while in an ideal posture

for swimming, do not effectively support terrestrial

locomotion. The claws of Xenopus laevis, located on the

hind feet, are used to help tear food into manageable

chunks. Breeding takes place during the rainy season at a

time when the ponds contain ample water to support the

growth of the larvae. The female is capable of ovulating

from 500 to 1,000 eggs in a single 24-hour spawning period.

Fertilization occurs in the water and metamorphosis takes

approximately 2 months to complete (Deuchar, 1975). The

animals do well in captivity and can remain healthy for

many years on a diet of vertebrate heart or liver. Unlike

Rana pipiens, the ovary of the captive Xenopus female

contains oocytes at all stages of maturation. The oocytes








2

are easily obtained from the anesthetized animal and can

survive many days in a comparatively simple buffered saline

solution supplemented with essential ions and antibiotics

(Wallace et al., 1973). The impressive size of the fully

matured oocyte, over 1 mm in diameter, facilitates

manipulation. For these reasons, the African clawed frog

has become an important tool in biochemical and biological

research.

The use of Xenopus laevis oocytes as a means of

carrying out the translation of exogenous mRNA began in

1971 in the laboratories of Gurdon and Marbaix (Gurdon et

al., 1971; Lane et al., 1971). In these series of

experiments, the co-injection of purified haemin and rabbit

reticulocyte 9S RNA was shown to bring about the synthesis

in oocytes of a hemoglobin-like moiety that was

chromatographically indistinguishable from the authentic

molecule isolated from rabbits. Soon after this landmark

work, further reports of the oocyte-mediated translation of

foreign mRNA began appearing in the literature. Examples

of the translation of mRNA isolated from vertebrates,

(Vassart et al., 1975; Lane and Knowland, 1975; Reynolds et

al., 1975), invertebrates (Lane et al., 1983), viruses

(DeRobertis et al., 1977), and plants (Lane et al., 1981;

Matthews et al., 1981) clearly demonstrated the flexibility

and fidelity of the Xenopus oocyte system.








3

Although the translation of heterologous mRNA in

Xenopus oocytes has been used effectively in diverse

spheres of research, it has been particularly beneficial to

the study of integral membrane proteins, such as hormone

receptors (Bahouth et al., 1988), ion channels (Leonard et

al., 1987), and nutrient transporters (Hediger et al.,

1987a,b). Unlike cytoplasmic enzymes, whose presence can

often be followed easily through a purification scheme by

means of appropriate activity assays, channels and

transporters generally must be situated at a hydrophobic

interface separating 2 hydrophilic compartments in order to

be detected. Although the reconstitution of solubilized

membrane proteins into artificial proteoliposomes

accomplishes this partitioning (Hirata, 1986; Maloney and

Ambudkar, 1989), many channels and transporters do not

possess the stability necessary to survive rigorous

solubilization procedures. Translation of foreign mRNA in

oocytes, however, results in the correct positioning of

functional integral membrane proteins into the oocyte

plasma membrane (Sumikawa et al., 1981; Barnard et al.,

1982; Parker et al., 1985). Further, purification

strategies can be designed which focus on the

identification of the mRNA, or corresponding cDNA, coding

for a particular protein rather than on the protein itself

(Hirono et al., 1985). The use of the Xenopus oocyte in

this role, that is, as a screening device for the








4

characterization of specific cloned cDNAs, is exemplified

by the work of Wright and his co-workers with the cloning

of the intestinal Na-dependent glucose transporter

(Hediger et al., 1987a,b). Lacking an antibody directed

against the protein, the authors identified a cDNA coding

for the glucose carrier by means of mRNA translation in

Xenopus oocytes. Initial experiments involving the

microinjection of rabbit intestinal poly(A)+ mRNA into

oocytes resulted in an 8-fold increase in the Na+-dependent

transport of methyl-a-D-glucopyranoside (MeGlc). Following

successive rounds of mRNA size fractionation via agarose

gel electrophoresis and microinjection, a class of mRNA

averaging 2.3 kb in length was identified that increased

the rate of Na'-dependent MeGlc transport into oocytes by

10-fold over that seen with adjacent size fractions

(Hediger et al., 1987b). The active fraction was used to

prepare a cDNA library using an in vitro expression vector

(Hediger et al., 1987a). Synthetic RNAs transcribed from

increasingly smaller pools of clones were microinjected

into oocytes which were then assayed for Na+-dependent

MeGlc transport. The single clone that was ultimately

identified boosted MeGlc transport by more than 1,000-fold

over controls.

This manuscript, in essence, describes endogenous amino

acid transport in Xenopus oocytes and the development of

methodology to study the oocyte-mediated synthesis of








5

hepatic proteins following the microinjection of rat liver

mRNA. Although the bulk of the experiments which are

described were conducted with the eventual expression of

the rat liver System A amino acid transporter in mind, the

approach should be applicable to all plasma membrane

transporters.

Prior to studies involving the investigation of

specific heterologous transport or channel activities in

Xenopus oocytes, it is necessary to first verify the level

of expression in the uninjected oocyte of the activity in

question. For example, before undertaking experiments

involving the microinjection of rabbit intestinal mRNA,

Hediger et al. (1987b) initially characterized the

endogenous glucose transport activities in the oocyte. The

results of these preliminary investigations revealed the

presence of a transport activity resembling the facilitated

glucose transporter by virtue of its sodium independence.

A Na/glucose co-transport activity, however, appeared to

be absent from the uninjected oocyte (Hediger et al.,

1987b). Given this low background level of endogenous

activity, an increase in the Na*-dependent transport of

glucose resulting from the microinjection of mRNA, should

be relatively easy to detect. As described above, such was

the case for this particular transporter. In a similar

manner, Houamed et ai. (1984) have described the expression

in oocytes of functional receptors for Y-aminobutyric acid,








6

glycine, and glutamic acid following the microinjection of

rat brain mRNA. Earlier experiments had failed to detect a

response in uninjected oocytes after the application of

these amino acids (Kusano et al., 1982; Houamed et al.,

1984). Likewise, investigations into the subunit

structures of fetal and adult acetylcholine receptors were

possible because of the absence of native receptors of this

type in the oocyte (Kusano et al., 1982; Mishina et al.,

1986). With these examples in mind, it is apparent that an

investigation involving the mRNA-induced expression in

oocytes of an amino acid transporter must begin with the

characterization of endogenous transport activities.

Chapters II and III of this manuscript describe such an

investigation. Chapter II concerns the transport

properties of 13 naturally-occurring and synthetic amino

acids in the prophase-arrested oocyte. Chapter III, on the

other hand, deals with alterations in amino acid transport

observed following the in vitro induction of meiotic

maturation. The investigation described in Chapter III is

limited to 4 distinct transport activities in the oocyte

which correspond in many ways to transport systems

previously characterized in other species.

Chapter IV of this manuscript focuses on events taking

place within the oocyte subsequent to the microinjection of

rat liver mRNA. Based on experiments involving the

microinjection of globin mRNA (Gurdon et al., 1971), it is








7

often assumed that foreign mRNA molecules are relatively

resistant to degradation once inside the oocyte. However,

globin mRNA, as well as the mRNAs coding for most other

proteins studied by means of the oocyte system (Berns et

al., 1972; Barnard et al., 1982), is particularly long-

lived in vivo. The fate of mRNAs with short half-lives,

however, such as that coding for the System A amino acid

transporter, has not been investigated in the oocyte.

Furthermore, although the majority of investigations

referenced in this manuscript utilize post-microinjection

incubations of 2 or 3 days, the optimum incubation time

could vary greatly depending on the particular protein

under investigation. In addition, the observation of

literally hundreds of oocytes has demonstrated that the

viability of the cell deteriorates after microinjection and

that this deterioration is dependent, in part, upon the

quantity and type of mRNA injected. Extension of the post-

microinjection incubation period beyond the point where

viability begins to decline would serve little purpose.

Due to the fact that the System A carrier has yet to be

isolated in pure form, neither an antibody nor a cDNA

corresponding to this transporter is available. Thus, the

direct monitoring of the System A protein or its mRNA in

the oocyte is not possible. For this reason, 2 other

proteins, phosphoenolpyruvate carboxykinase (PEPCK) and rat

serum albumin (RSA), were chosen to serve as models.








8

Following the microinjection of rat liver mRNA into

oocytes, the oocyte-mediated synthesis of PEPCK and RSA was

monitored via immunoblot analysis and the stability of the

corresponding mRNAs by means of Northern analysis.

Experiments are also described which examine differences in

the quantities of PEPCK and RSA synthesized in the oocyte

resulting from the microinjection of varying amounts of

mRNA. In addition, a novel utilization of the oocyte mRNA

translation system, that is, as a means of estimating

relative quantities of specific mRNAs, is also described in

this chapter. Following the microinjection of RNA

extracted from the livers of rats under 3 conditions (fed,

diabetic, or glucagon-injected), proteins are synthesized

in the oocyte in quantities reflecting, in general, the

abundance of their corresponding mRNAs in the liver donor.














CHAPTER II

CHARACTERIZATION OF NEUTRAL AND CATIONIC AMINO ACID TRANSPORT
IN XENOPUS LAEVIS OOCYTES

Introduction

Amphibian oocytes have been used extensively in the

study of numerous and diverse aspects of metabolism as well

as cell and developmental biology. Because of their large

size and relative ease of handling, oocytes from the African

clawed frog, Xenopus laevis, are used routinely for

investigations involving transcriptional and translational

regulation, protein processing and targeting, and

intracellular signalling (reviewed in Huez and Marbaix,

1986). Furthermore, isolated Xenopus oocytes have been

employed extensively as an in vivo translation system for

mRNA from other cell types (Colman, 1984; Huez and Marbaix,

1986). These studies have allowed the detailed investigation

of a number of membrane-bound receptors (Williams et al.,

1988; Kumar et al., 1988), ion-channels (Hirono et al., 1985;

Sigel, 1987), and the intestinal Na+-dependent transport

system for glucose (Hediger et al., 1987a,b). Recently,

Aoshima et al. (1988) have reported increased amino acid

uptake in oocytes following injection of mRNA isolated from

rat intestine. Prior to undertaking studies to test for

expression of specific amino acid transport systems in

9








10

oocytes following microinjection of exogenous mRNA, it is

helpful to have some knowledge of the characteristics of

amino acid transport in the "basal" state, i.e., the

prophase-arrested oocyte. For this reason, an investigation

of the endogenous amino acid carriers in Xenopus oocytes was

undertaken.

Transport of amino acids by oocytes has been reviewed

recently (Van Winkle, 1988). Previous reports on alanine

(Jung and Richter, 1983; Jung et al., 1984) and leucine

(Belle' et al., 1976) transport into Xenopus oocytes have

revealed that both amino acids are taken up from the

surrounding medium in a saturable, Na+-dependent fashion.

Jung et al. (1984) concluded that a transport system

analogous to the System A transporter exists in the oocyte

and mediates Na+-dependent alanine uptake. However, their

studies also demonstrated that the Na+-dependent alanine

transport was not inhibited by an excess of the System A-

specific amino acid analog 2-(methylamino)-isobutyric acid

(MeAIB). Bravo et al. (1976) have examined the transport of

several amino acids into oocytes. For example, alanine

transport was shown to be inhibitable by the neutral amino

acids glycine, valine, and leucine, but not by the charged

amino acids arginine and glutamic acid. These data suggest

the presence of a neutral amino acid carrier with broad

substrate specificity, perhaps similar to System ASC

(Kilberg, 1982). With regard to charged amino acids,








11

competition for transport between glutamic acid and aspartic

acid, and between lysine and arginine was observed (Bravo et

al., 1976). However, apart from proposing that oocytes may

have separate transporters for the various classes of amino

acids, no attempt was made by Bravo et al. (1976) to identify

further the specific agencies responsible for the observed

transport.

This chapter is an investigation of amino acid transport

in prophase-arrested oocytes from X. laevis with emphasis on

the identification and partial characterization of the

agencies responsible for the transport of neutral and

cationic substrates. Although the unambiguous verification

of individual transporters must await protein

identification, the utilization of substrate specificity,

trans-effects, and inhibition analysis has made possible the

preliminary identification of distinct carrier processes as

defined previously in other cell types.

Materials and Methods

Isolation of Oocytes

Mature female Xenopus laevis (Xenopus I, Ann Arbor,

Michigan) were housed in an aquarium containing synthetic

pond water (1.4 mM NaCl, 0.05 mM NaHCO3, 0.03 mM KC1, 0.003 mM

Na2HPO4) at 18 to 20'C. The animals were fed either bovine

liver or heart 3 times per week.

Ovarian tissue was removed from hypothermically-

anesthetized animals and immediately placed into amphibian









12

Ringer's solution (115 mM NaCl, 2 mM KC1, 1.8 mM CaCl2, 5 mM

HEPES, pH 7.6) at room temperature. The tissue was cut into

smaller pieces of approximately 20-50 oocytes each and rinsed

several times with fresh Ringer's solution followed by 2

rinses with modified Barth's medium (MBM) containing 88 mM

NaC1, 1 mM KC1, 2.4 mM NaHCO3, 0.3 mM Ca(NO3),, 0.41 mM CaC12,

0.82 mM MgSO4, 10 /g/ml penicillin, 10 gg/ml streptomycin

sulfate, 15 mM HEPES, pH 7.6 (Colman, 1984). Oocytes were

manually separated from surrounding membranes

(defolliculated) with Dumont #5 forceps and placed into fresh

MBM at 18 to 20*C. Prior to experimentation, defolliculated

oocytes were incubated overnight to facilitate recognition

and removal of those damaged during the denuding process.

Other studies have employed oocytes still surrounded by

follicle cells ("follicles") or oocytes defolliculated by

treatment with collagenase. We have determined that

enzymatic isolation of oocytes with collagenase can cause

loss of endogenous transport activity which is not regained

upon incubation in vitro. As a result, all the oocytes used

in the present work were isolated by hand defolliculation.

Amino Acid Transport

All transport assays described below were carried out

using uptake tubes made from 5 ml polycarbonate centrifuge

tubes. The conical end of the tube was cut off and a small

piece of nylon mesh (Tetko Inc. No. HC-3-75, 75 AM mesh) was

glued to the opposite end using plastic model cement. Excess








13

nylon mesh was trimmed from around the tube after the glue

had dried thoroughly. All incubations and washes, described

below, were carried out with the oocytes inside the uptake

tubes. Oocytes were transferred by means of glass pipettes

(1.5 mm inside diameter).

Prior to measurement of amino acid uptake, 10-12 oocytes

were incubated twice for 10 min each in 15 ml of Na*-free

MBM. The Na+-free MBM was made by substituting choline

chloride and choline bicarbonate for the corresponding

sodium salts. After the second incubation, the oocytes were

transferred to 0.5 ml of uptake buffer, consisting of either

Na*-containing or Na*-free MBM with 50 AM of 3H-labelled amino

acid. Following a 15 to 60 min incubation at room

temperature, the oocytes were washed free of external isotope

with 10 rinses of 4 ml each of ice-cold amphibian Ringer's

and single cells were placed into individual scintillation

mini-vials. Solubilization was achieved by incubating each

cell in 200 Al of 0.2 N NaOH containing 0.2% sodium dodecyl

sulfate. The amount of time required for complete

solubilization varied depending on whether or not the vials

were agitated throughout the incubation period. Vials

vigorously agitated on an orbital shaker (American Rotator V,

approximately 170 rpm) required only 3 hours while unagitated

vials were normally incubated overnight. Following

solubilization, the trapped radioactivity was quantitated

via liquid scintillation spectrophotometry. Neutralization








14

of the solubilization buffer was not required with the

scintillation cocktail utilized (3a70B, Research Products

International Corp.).

The rate of Na+-dependent transport was calculated by

subtracting the rate observed in the absence of sodium from

that observed in the presence of sodium. For cis-inhibition

experiments, 5 mM of unlabeled amino acid were included in

the uptake mixture. Unlabeled amino acids that are

transported by the same carrier as the radioactively-labeled

substrate will, in effect, cause a decrease in the net uptake

of the substrate under scrutiny. Unless specified otherwise,

all amino acids and analogs were the L-isomer. Where

necessary, uptake mixtures were balanced with regard to

osmolarity using choline chloride.

Given the long duration of the uptake assay, the

question of metabolism of the substrate molecule by the

oocyte was addressed. The degree to which radioactive

substrate was incorporated into TCA-precipitable material

was determined by first homogenizing oocytes after transport

in buffer containing 100 mM NaCl, 1 mM phenylmethyl-

sulfonylfluoride, 1% (v/v) Triton X-100, and 20 mM Tris, pH

7.6 (oocyte homogenization buffer, OHB). A 5 to 10 Al

aliquot of the homogenate supernatant fraction was then added

to 1 ml of 1 N NaOH, 1.5% (v/v) H202 in a plastic 15 ml conical

tube and vortexed briefly. The tube was capped tightly and

placed into a 37C water bath. This incubation in alkali








15

serves to degrade any tRNA that may be charged with

radioactive amino acid substrate. After 10 minutes, the tube

was removed from the bath and 4 ml ice-cold 24% (w/v) TCA

containing 2% (w/v) casein hydrolysate added and the mixture

vortexed briefly. The tube was then cooled on ice for at

least 30 minutes prior to filtration, with suction, through a

glass fiber filter disk (Whatman GF/C). The tube was rinsed

with 5 ml ice-cold 8% (w/v) TCA and the rinse poured over the

filter. The filter was further washed with an additional 5

ml of 8% (w/v) TCA followed by 10 ml acetone. The filter was

left under vacuum until dry and the radioactivity adhering to

it quantitated via liquid scintillation spectrophotometry.

The incorporation of histidine, alanine, threonine, serine,

or cysteine into precipitable material was monitored for 60

min and shown to be less than 3.5% of the total amino acid

accumulated. Even after a 24 h incubation the incorporation

of the amino acids tested was less than 10%. For AIB and

MeAIB there was no detectable incorporation into protein.

Metabolism of substrate molecules was further examined

by means of paper chromatography. Following transport,

oocytes were homogenized in OHB as described previously and

100 gl of the supernatant fraction added to 300 p1 of

absolute ethanol in a 1.5 ml Eppendorf microcentrifuge tube.

The tube was placed at -20*C for at least 1 hour after which

time it was centrifuged at 12,000 x g at 4C for 30 minutes.

The supernatant fraction was transferred to a clean tube and









16

centrifuged as above but only for 15 minutes. The

supernatant fraction was again transferred to a clean tube

and evaporated to dryness under vacuum. The residue was

solubilized in 20 Al distilled, deionized water (ddH2O) and 5

gl was used to determine total radioactivity via liquid

scintillation spectrophotometry. Depending on the quantity

of radioactivity present, 5 to 10 Al of the extract (i.e. the

resolubilized residue) was spotted onto Whatman 3MM

chromatography paper. Adjacent lanes contained unlabelled

amino acid as well as pure radioactively-labelled amino acid.

The chromatography paper was rolled into a tube shape,

fastened with a staple, and placed upright in a glass

chromatography tank containing approximately 150 ml N-

butanol, acetic acid, and ddH2O in the ratio of 12:3:5. The

separation was allowed to proceed until the solvent front had

migrated approximately 12 cm from the origin. The

chromatography paper was removed from the tank, laid flat,

and the solvent front marked with a pencil. The paper was

allowed to air dry in a fume hood and individual lanes were

then cut from the paper. Lanes containing unlabelled amino

acid were sprayed with a 3% (w/v) solution of ninhydrin in

95% (v/v) ethanol, allowed to dry, and then heated in a

drying oven at 100C for 10 to 15 minutes or until color

appeared. Heating the paper with a hand-held hair drier was

also an effective means of catalyzing the ninhydrin reaction.

Lanes containing radioactively-labelled amino acid or oocyte








17

extract were cut transversely into 1 cm strips and the

radioactivity quantitated via liquid scintillation

spectrophotometry. The calculation of R values was carried

out by dividing the distance from the origin that the amino

acid had migrated, as evidenced by the ninhydrin-positive

spot or radioactivity peak, by that of the solvent front.

Results

In the animal cells studied to date, the Na+-dependent

transport of neutral amino acids is mediated principally by

two distinct carriers, Systems ASC (Christensen et al., 1965)

and A (Oxender and Christensen, 1963). The substrate

specificity for System ASC is broad with some affinity for

all neutral amino acids, but preference is shown for those

with a sulfhydryl or hydroxyl group as typified by serine,

threonine, or cysteine. In contrast, System A is

characterized by a high sensitivity to H+, trans-inhibition,

and tolerance for some, but not all, N-monomethylated

substrates (Christensen et al., 1965; Christensen, 1984;

Kilberg et al., 1985). The latter characteristic is

generally considered to be the most useful defining property

to distinguish System A mediation from that of System ASC

(Oxender and Christensen, 1963; Christensen, 1984).

Historically, the non-metabolizable alanine analogs AIB (2-

aminoisobutyric acid) and MeAIB have been the substrates of

choice because their Na+-dependent transport is nearly

restricted to System A in a wide variety of cell types.








18

However, a portion of Na+-dependent AIB uptake is mediated by

System ASC in some cells (Kilberg et al., 1981).

The results of initial experiments investigating the

transport into oocytes of several neutral and cationic amino

acids are shown in Table 2-1. The highest rates of substrate

transport in the presence of sodium were seen with cysteine

(98.6 7.4 pmol-oocyte1-h'), alanine (73.4 4.4

pmol.oocyte' h1) and leucine (62.5 4.1 pmol.oocyte' h').

As described in the Materials and Methods section, the

portion of the total substrate transport that is dependent

upon the presence of alkali ions in the medium can be

estimated by performing the transport assay in the presence

and absence of sodium or lithium, and then calculating the

difference between the observed velocities. The amino acids

for which uptake exhibited, proportionally, the greatest

amount of sodium dependence were glycine and threonine. For

both of these substrates, approximately 80% of the total

transport was eliminated when sodium was replaced by choline

in the uptake buffer. Conversely, approximately 93% of the

total oocyte uptake of tryptophan occurred in the absence of

sodium. Although most of the amino acids tested exhibited

very little or no Li co-transport, 3 amino acids, glycine,

serine, and leucine, were shown to have Li+-dependent

transport velocities which were at least 30% of the Na+-

dependent value (Table 2-1).











Table 2-1. Transport of neutral and cationic amino acids into
oocytes in the presence of Na*, Li', or choline.


Substrate Na+ Li choline



Ala 73.4 4.4 34.2 3.2 38.9 3.2

Gly 8.9 0.6 6.4 0.7 1.8 0.3

Gln 31.5 3.6 13.8 1.8 13.6 1.8

Ser 46.0 5.1 39.1 3.6 30.2 3.3

Thr 48.6 2.9 11.8 1.8 9.9 2.4

CysH 98.6 7.4 52.5 6.0 44.3 4.2

Arg 44.7 3.5 32.0 3.0 29.4 5.4

His 39.2 3.2 9.3 1.9 23.4 1.2

Leu 62.5 4.1 38.7 3.1 27.8 3.2

Phe 43.0 2.8 18.9 3.0 21.5 2.2

Trp 18.3 0.9 11.4 0.6 17.1 1.1

AIB 10.4 1.6 5.2 0.5 4.1 0.2




The oocyte transport of the amino acids listed above was
assayed at 20"C for 15 or 60 minutes as described in the
Materials and Methods section. The concentration of the
substrate amino acids was 50 pM in all cases. Uptake mixes
were made using MBM (Na+ condition) or MBM with the
corresponding lithium (Li+ condition) or choline (choline
condition) salts replacing sodium chloride and sodium
bicarbonate. The data are expressed as pmol*oocyte'*lh'1 and
are the averages S.D. of at least 10 oocytes.
Abbreviations: Ala, alanine; Gly, glycine; Gln, glutamine;
Ser, serine; Thr, threonine; CysH, cysteine; Arg, arginine;
His, histidine, Leu, leucine; Phe, phenylalanine; Trp,
tryptophan; AIB, 2-aminoisobutyric acid.








20

Experiments involving the Na -dependent transport of 200

MM MeAIB (Fig. 2-1) and AIB (data not shown) by isolated

oocytes showed a slow uptake that was linear for at least 2

hours. Although 90% of 50 pM [3H]AIB uptake was prevented by

the addition of 5 mM unlabelled AIB, MeAIB at the same

inhibitor concentration inhibited only 26% of the Na+-

dependent AIB transport (Table 2-2). Similar results were

obtained when the Na+-dependent transport of characteristic

System ASC substrates such as alanine, serine, threonine, and

cysteine was challenged with excess MeAIB (Table 2-2). To

test for MeAIB-dependent trans-inhibition, another defining

property of System A (Handlogten et al., 1981; Kilberg et

al., 1985), the intracellular concentration of MeAIB was

brought to 1.3 mM by incubating oocytes overnight in MBM

containing 10 mM MeAIB. There was no detectable decrease in

Na+-dependent AIB transport by the MeAIB-loaded cells (data

not shown). Although it is possible that a higher intra-

oocyte MeAIB concentration might have caused trans-

inhibition of AIB uptake, this is unlikely given the

inability of excess MeAIB to cis-inhibit AIB transport (Table

2-2). Two other systems could possibly account for the Na+-

dependent AIB uptake by the oocyte, System B0'* or System ASC.

These two activities can be easily distinguished because of

the acceptance of cationic amino acids by the B0'+ carrier

(Van Winkle et al., 1985). The Na+-dependent uptake of AIB

was measured in the presence of increasing concentrations of























3 0 Choline
u A Na+-dependent
0 A
0 2




1 I
E
T O

0
0 30 60 90 120
Minutes













Figure 2-1. Time-course for transport of MeAIB into X. laevis
oocytes. The Na+-dependent and Na+-independent transport of
200 pM [14C]MeAIB was measured at 20'C as described in the
Materials and Methods section. The data are expressed as
pmol-oocyte" and each point represents the average S.D. of at
least 8 individual oocytes. Where not shown, the standard
deviation bars are contained within the symbol.

















Table 2-2. MeAIB inhibition
selected amino acids.


of Na-dependent transport for


Substrate MeAIB Velocity Percent of
Control


AIB 4.9 1.2
+ 3.6 1.1 74

Ala 20.9 3.6
+ 28.7 4.8 137

Ser 23.0 5.4
+ 20.1 1.7 87

Thr 30.6 4.7
+ 24.5 5.1 80

CysH 43.8 9.3
+ 54.0 10.3 123


The Na'-dependent transport of 50 pM [3H]AIB, [H]alanine,
[3H]serine, or [3H]threonine was measured for 60 minutes at
200C in the presence or absence of 5 mM MeAIB as described in
the Materials and Methods section. The [3H]cysteine was
handled in an equivalent manner except that the concentration
was 100 PM and 1 mM dithiothreitol was included in the uptake
mixture. The Na-dependent uptake rates are reported as
pmol.oocyte'1*h' and are the averages S.D. of at least 10
individual oocytes. The addition of unlabelled AIB (5 mM)
decreased Na-dependent [3H]AIB uptake from 4.9 1.2 to 0.45
0.2 pmol-oocyte"'*h1, a 91% reduction.








23

arginine with or without serine (Fig. 2-2). Arginine

concentrations up to 10 mM did not significantly affect Na+-

dependent AIB uptake, but the addition of as little as 1 mM

serine, a System ASC substrate, nearly eliminated all alkali

ion dependent AIB uptake. Furthermore, the Na+-dependent

transport of 50 AM AIB (4.9 1.2 pmol*oocyte'*h'1) was

decreased to 0.5 0.2, 0.4 0.2, and 0.9 0.4 pmol*oocyte"

1*h' by the presence of 5 mM threonine, leucine, or histidine,

respectively. These results are indicative of a carrier with

broad substrate specificity, such as System ASC (Kilberg et

al., 1981; Christensen, 1984). Interestingly, the

concentration-dependent decrease of Na-independent AIB

transport by arginine suggests the presence of the recently

defined System bo,+ (Van Winkle et al., 1988), a Na+-

independent transport system with the ability to accept

neutral as well as cationic amino acid substrates. The

saturability of Na+-independent AIB uptake was verified by

the strong inhibitory activity of 5 mM unlabelled AIB on the

Na+-independent transport of 50 MM [3H]AIB. In this

experiment, only 15% of the total [3H]AIB entry into oocytes

in the absence of sodium could be attributed to non-saturable

uptake (i.e., non-carrier mediated).

In contrast to several mammalian cell types (White,

1985), the transport of positively-charged (basic) amino

acids into oocytes was found to occur by Na+-dependent as

well as Na+-independent processes. When measured in the


















'.. A ,-- } Na+ Media

2 I O-- } Choline Media
A--AL

o 0 T

m 1

0-----0
E A

0
0 1 5 10 10 10 Arg
00 0 1 5 10 Ser
Inhibitor, mM










Figure 2-2. Arginine and serine inhibition of AIB transport.
The transport of 50 AM [3H]AIB by oocytes was measured in Na*-
containing (filled symbols) or Na+-free (open symbols) media
for 60 minutes at 20*C as described in the Materials and
Methods section. Inhibitory amino acids (circles = arginine,
triangles = serine + arginine) were included in the uptake
mixtures at 1, 5, or 10 mM. Choline chloride was used to
maintain the osmolarity of all uptake mixtures at a constant
level. Data are expressed as pmol'oocyte'.h'- and each point
represents the average S.D. of at least 10 oocytes. Where not
shown, the S.D. bars are contained within the symbol.








25

presence of other amino acids, Na'-dependent arginine

transport was inhibited by the presence of not only other

cationic amino acids such as lysine, homoarginine,

ornithine, and 2-amino-3-guanidinopropionic acid (AGPA), but

by zwitterionic amino acids such as alanine, threonine, and

leucine as well (Fig. 2-3). Cysteic acid, which is

negatively-charged at physiological pH, had virtually no

effect on Na-dependent [3H]arginine transport. Consistent

with the lack of arginine inhibition of Na+-dependent AIB

uptake (Fig. 2-2), AIB was a relatively poor inhibitor of

Na+-dependent arginine uptake. These results, which suggest

the existence in oocytes of a transport system capable of

interacting with both neutral and positively-charged amino

acids, are similar to those reported by Van Winkle et al.

(1985) for a Na+-dependent carrier identified in pre-

implantation mouse blastocysts. The murine system, which was

named System B0', displayed mutual competition among alanine,

lysine, and 3-aminoendobicyclo-(3,2,1)-octane-3-carboxylic

acid (BCO). The lack of inhibition by cysteic acid is in

agreement with the observation that Na+-dependent alanine

transport in the blastocyst was insensitive to the presence

of cysteine sulfinate, a negatively-charged amino acid (Van

Winkle et al., 1985).

To delineate further the System B0' activity, mutual

inhibition of threonine and arginine was assayed. The

transport of Na-dependent arginine was shown to be inhibited



















o 120

a 100 **
C
S 80
CL
0 <
60 -4
r 60
oQ
40
0 40 -
a-
20 "-
+ T 1 *
20
Z 0
O W C7 C <" 0 < mn
< -J < 0 -
: <

Inhibitor










Figure 2-3. Inhibition of Na+-dependent arginine transport by
basic, acidic, and neutral amino acids. The Na+-dependent
transport of 50 iM [3H]arginine in oocytes was assayed for 60
minutes at 200C as described in the Materials and Methods
section. The concentration of the indicated inhibitor was 5 mM
in all cases. The data were compiled from two separate
experiments and are expressed as the percentage of the
transport rate in the absence of inhibition (7.7 4.2 and 7.8
2.5 pmol-oocyte"' .hh for the two experiments). Each bar
represents the average Na+-dependent transport rate for at
least 10 oocytes. Both the Na+-containing and the Na+-free
buffers contained the inhibitory amino acid. The statistical
significance is indicated with asterisks (*p<0.005, and **
p<0.025).









27

almost entirely by 5 mM threonine (Fig. 2-3). Conversely,

when the Na*-dependent transport of threonine was challenged

with excess arginine, only 50 to 60% was inhibited (Fig. 2-

4). The remaining Na+-dependent threonine transport was

eliminated by the inclusion of 1-10 mM serine in the uptake

mixture. These data demonstrate the presence of at least two

Na+-dependent carriers in the oocyte plasma membrane. One

carrier is able to interact with both positively-charged and

neutral amino acids (Na-dependent arginine transport

inhibited by threonine, Table 2-3), analogous to System B0',

and the other with zwitterionic amino acids only (serine-

sensitive portion of Nat-dependent threonine transport, Fig.

2-4). The substrate specificity and the lack of interaction

with MeAIB (Table 2-2) of the latter system argues for

analogy to System ASC.

The Na+-independent transport of positively-charged

amino acids by a number of eukaryotic cell types has been

shown to be mediated by a process termed System y that is

relatively pH insensitive and stereo-selective (White,

1985). Data obtained with a broad spectrum of cell types

suggest that System y+ is distinct from those routes

characteristically utilized by neutral amino acids, although

it has been demonstrated that a neutral amino acid and a Na

can competitively inhibit the carrier (Christensen and

Handlogten, 1969; White, 1985). The Na+-independent

transport of arginine by oocytes was inhibited by positively



















0120
-- Arginine alone
S100 \ 0- Arginine and Serine
C
c_
o 80
C -
60 -
'- C
0 (D \ O
-0
O Q)
O c 40 -

0 20 0 *
+
z 0
0 1 5 10 10 10 Arg
0 0 0 1 5 10 Ser
Inhibitor, mM









Figure 2-4. Arginine and serine inhibition of Na+-dependent
threonine transport. The Na+-dependent transport of 50 pM
['H]threonine by oocytes was measured for 60 minutes at 20C as
described in the Materials and Methods section. As indicated,
inhibitory amino acids were included in the uptake mixtures at
1, 5, or 10 mM. Choline chloride was used to maintain the
osmolarity of all uptake mixtures at a constant level. The
data are expressed as the percentage of the Na+-dependent
transport rate in the absence of inhibition. The uninhibited
threonine transport rates in Na*-containing and Na-free
buffers were 48.3 9.2 and 28.6 5.3 pmol-oocyte'*h" ,
respectively. An asterisk indicates that the value is
statistically (p<0.05) different from control.






















Table 2-3. Inhibition analysis of Na-independent leucine
transport.


Inhibitor Velocity Percent of
Saturable


none 19.9 6.6 100
Leu 0 0
Phe 0 0
His 2.7 14
Thr 4.3 22
BCH 8.5 43


The Na*-independent transport of 50 AM [3H]leucine in oocytes
was measured in the presence or absence of 5 mM competitor
amino acid as described in the Materials and Methods section.
Transport was carried out for 60 minutes at 20*C. The
saturable leucine transport rate was calculated by subtracting
the rate observed in the presence of excess unlabelled test
amino acid from that observed in the absence of inhibitor.
The data are expressed as pmol*oocyte'*.h" or as the percent
of the saturable transport rate in the absence of inhibition.
The results represent the average of at least 10 oocytes.








30

charged amino acids, but neutral and negatively-charged

amino acids were significantly less effective (Fig. 2-5).

Over 80% of the saturable Na+-independent transport of 50 AM

arginine could be eliminated by 5 mM lysine, homoarginine, or

ornithine. System y' exhibits increasing affinity for

substrate molecules as the length of the side-chain

increases. For example, the K values for inhibition of

arginine uptake in HTC cells (White and Christensen, 1982)

and human fibroblasts (White et al., 1982) are the lowest for

homo-arginine, with four methylene groups in the side-chain,

and the highest for 2-amino-3-guanidinopropionic acid

(AGPA), for which the side-chain contains only one methylene

group. Consistent with the data obtained for fibroblasts

(White et al., 1982), 5 mM AGPA decreased arginine uptake by

only 20% or less. Essentially equivalent results were

obtained with lysine as the test substrate (data not shown).

In an effort to investigate the effect of charge alone

on an amino acid's ability to inhibit System y', arginine

transport was challenged with an excess of histidine at two

different pH values (Fig. 2-6). At pH 6.0, approximately

half of the histidine molecules possess a net positive charge

and half are zwitterionic in form. At pH 8.0, most of the

histidine molecules are zwitterionic. Thus, it is possible

to separate the inhibitory effect of charge from that

imparted by the size, shape, and hydrophilicity of the amino

acid's side-chain. The data of Figure 2-6 illustrate that


















O
100

Q)
e 80
C

4 < 60
V) 0





z 0
o c 40
-0

C 20
T

z 0


- n-
2:i


3 < 0
0 C)


Inhibitor












Figure 2-5. Inhibition of saturable Na+-independent arginine
transport by basic, acidic, and neutral amino acids. The Na*-
independent transport by oocytes of 50 MM [3H]arginine was
measured for 60 minutes at 20*C as described in the Materials
and Methods section. The concentration of the indicated
inhibitor was 5 mM in all cases. The data are expressed as the
percentage of the saturable transport rate in the absence of
inhibition (13.1 1.5 pmol.oocyte'1-h'1). The total saturable
transport rate was calculated by subtracting the uptake rates
measured in the presence or absence of 5 mM unlabelled
arginine. Each bar represents the average transport rate
S.D. of at least 10 individual oocytes.




















Q)

- 100

0Q)
.- 80
C

S60
C

40
0-
-a

+
20

z 0


I -1 pH 6.0
f55 pH 8.0



_E
-N





ST


10


Histidine, mM















Figure 2-6. The pH dependence for histidine inhibition of Na'-
independent arginine transport. The Na+-independent transport
by oocytes of 50 pM [3H]arginine was measured for 60 minutes at
20*C in the presence or absence of 5 mM unlabelled histidine at
pH 6.0 or 8.0. The data are expressed as the percentage of the
transport rate in the absence of inhibition measured at the
same pH (pH 6 = 30.4 1.9, pH 8 = 22.3 1.2 pmol.oocyte'*h-1)
and represent the averages S.D. of at least 10 separate
cells.








33

the charged form of histidine was a more effective inhibitor

of Na-independent arginine transport than the neutral form.

At pH 6.0, 10 mM histidine inhibited approximately 70% of

arginine transport, while at pH 8.0, only 30% was blocked. As

seen for System y+ in other cell types (White et al., 1982;

White and Christensen, 1982), Na+-independent arginine

transport was found to be insensitive to inhibition by H+

over a wide range, the rate at pH 6.0 being equal to, or

slightly higher than, that at pH 8.5 (Fig. 2-6). In

agreement with previous results regarding the stereo-

selectivity of Na+-independent arginine transport (White et

al., 1982; White and Christensen, 1982), L-arginine uptake

(50 AM) was inhibited approximately 95% by 5 mM unlabelled L-

arginine (20.8 2.9 versus 0.98 0.2 pmol.oocyte',.hr'),

whereas the same concentration of D-arginine was much less

effective, decreasing arginine transport by about 45% (20.8

2.9 versus 11.7 2.5 pmol.oocyte'1 hr1).

In a variety of eukaryotic cells, the transport of amino

acids possessing aromatic or branched side-chains is

mediated largely by the System L amino acid carrier (Shotwell

et al., 1983). System L is characterized by Na+-

independence, pH and N-ethylmaleimide (NEM) insensitivity,

trans-stimulation, and affinity for the non-metabolizable

analog, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid

(BCH) (Vadgama and Christensen, 1985a,b). In erythrocytes,

there exists a Na-independent neutral amino acid carrier








34

distinct from System L (Vadgama and Christensen, 1985a,b;

Fincham et al., 1985). This carrier has been named System

asc; the letters chosen to reflect the substrate similarity

to the Na+-dependent System ASC (i.e. alanine, serine,

cysteine, and threonine) and the use of lower case to

indicate Na+-independence (Bannai et al., 1984). Although

the subset of amino acids characteristically transported by

Systems L and asc do overlap, the two carriers can be

functionally distinguished (Vadgama and Christensen,

1985a,b). Preliminary evidence in oocytes indicated the

presence of multiple Na+-independent neutral amino acid

carriers. As a result, the transport of threonine, BCH, and

leucine was monitored to determine whether these activities

appeared similar to those already described (i,e, Systems L

and asc). Historically, the Na+-independent uptake of

leucine and BCH has been considered relatively specific for

System L, and recent evidence suggests that threonine is a

good test substrate for System asc (Vadgama and Christensen,

1985a,b).

When Na+-independent leucine transport was assayed,

phenylalanine, histidine, and threonine eliminated greater

than 70% of the saturable leucine uptake (Table 2-3). The

branched-chain amino acid analog BCH was a relatively poor

inhibitor when compared to leucine itself, lowering the

transport of leucine by about 60%. To test for trans-

stimulation, oocytes were incubated for approximately 20








35

hours in MBM containing 10 mM unlabelled leucine. This pre-

loading with leucine resulted in a greater than 2-fold

stimulation of saturable Na+-independent leucine transport

(12.0 0.9 versus 28.2 4.8 pmol*oocyte" *h'1, p<0.01). An

analogous experiment utilizing oocytes preloaded with

alanine, a presumed substrate for System asc, if present,

showed no change in saturable Na+-independent alanine

transport.

The Na+-independent uptake of BCH was inhibited most

effectively by an excess of phenylalanine, leucine, or BCH

itself, but was resistant to inhibition by threonine,

alanine, or serine (Fig. 2-7). AIB, cysteic acid, and

arginine were also totally ineffective as inhibitors (data

not shown). As can be seen in Figure 2-7, a portion of the

saturable Na+-independent threonine transport in oocytes can

be inhibited by leucine and BCH even though, as noted above,

threonine was a relatively poor inhibitor of BCH transport.

In two separate experiments, 10 mM threonine was able to

inhibit only 31% (not shown) and 35% (Fig. 2-7) of saturable

Na+-independent BCH transport. This is in agreement with the

inability of threonine, probably the inhibitor/substrate of

choice to monitor System asc, to inhibit BCH uptake via

System L in human erythrocytes (Vadgama and Christensen,

1985b).

Differences in stereo-selectivity between threonine and

BCH transport into oocytes were also apparent (Fig. 2-7).



















100 E3 Threonine Transport

80 -

B 60

40
% 4 0 0 .-1





-I I I I I I I in I I
Y-J















Figure 2-7. Stereo-selectivity for inhibitors of Na-
independent BCH or threonine transport. The Na+-independent
transport of 200 gM ['H]BCH or [3H]threonine by oocytes was
measured for 60 minutes at 20*C in the presence or absence of
the indicated inhibitory amino acid at a concentration of 10
mM. Each bar represents the average of at least 10 oocytes and
the standard deviations, omitted for clarity, were typically
20% or less. An asterisk indicates that the difference in
effectiveness between the D- and L-isomers is statistically
significant (p<0.005).








37

Na+-independent threonine transport was relatively

stereospecific, approximately 75-80% of its saturable

transport was inhibited by the L-forms of alanine, serine,

and leucine. However, the D-forms of these amino acids

inhibited only 20% or less of threonine uptake. In contrast,

the agency responsible for the Na+-independent, saturable

transport of BCH is less stereo-specific, as both D-leucine

and D-phenylalanine inhibited greater than 50% of the

saturable uptake (Fig. 2-7).

Discussion

The data shown in Table 2-1 demonstrate that amino acid

uptake in Xenopus laevis oocytes occurs by Na+-dependent as

well as Nat-independent means. Greater than 50% of the total

transport of 6 of the substrates tested (glycine, threonine,

AIB, glutamine, leucine, and cysteine) was shown to be

dependent upon the presence of sodium. Conversely serine,

arginine, histidine, phenylalanine, and tryptophan were

transported primarily by Na+-independent means. Although

variations in transport rates for different batches of

oocytes were often observed for individual amino acids,

leucine transport was found to be particularly variable.

While in most experiments the great majority of leucine

transport was Na-independent, occasionally a sizeable

portion was Na+-dependent. For alanine, transport was

mediated equally by Na+-dependent and independent processes.

Such would be the case if transport systems analogous to








38

Systems ASC and asc, to be described more fully below, exist

in the oocyte plasma membrane.

The low rates of Na+-dependent MeAIB or AIB transport in

oocytes make their use for routine assays of endogenous

System A, if present at all, difficult, but this low activity

should prove helpful in experiments designed to detect

elevated levels of carrier following micro-injection of

foreign mRNA coding for that particular transport protein.

Although Na+-dependent uptake of the System A-specific

substrate MeAIB was detected, when tested for either trans-

or cis-inhibition, Na+-dependent uptake of AIB was

insensitive to the presence of intra- or extra-cellular

MeAIB. In freshly isolated hepatocytes in suspension, the

Na+-dependent entry of MeAIB is almost totally restricted to

System A, whereas that of AIB is slightly less restricted,

approximately 10% of its transport is the result of System

ASC mediation (Kilberg et al., 1981). AIB entry into CHO-K1

cells has been shown to be mediated by a combination of Na+-

dependent and Na+-independent agencies (Bass et al., 1981;

Shotwell et al., 1981; Moffett et al., 1983). The Na+-

dependent portion has been shown to be due to System A (60%)

and System ASC (10%), whereas System L (30%) has been shown

to be responsible for the majority of the Na-independent

entry. Similarly, in oocytes the transport of AIB appears to

be the result of a combination of multiple distinct transport

activities. Approximately 75% of the Na+-independent








39

transport of AIB in oocytes can be eliminated by arginine,

suggesting an activity analogous to System b0' (Van Winkle et

al., 1988), and the remaining 25% by the addition of an

excess of serine (Fig. 2-2). We find that threonine and

serine, while mutually inhibitory, are not effective

inhibitors of the System L substrate BCH (e.g., Fig. 2-7).

Thus, the serine-inhibitable Na'-independent AIB transport

may be indicative of System asc activity (Vadgama and

Christensen, 1985a,b). System b0' is a Na'-independent

transporter that accepts both neutral and cationic amino

acids in a manner similar to the Na+-dependent System B0'.

System b0'+ has a more limited substrate specificity than

System BO'+ and has been shown to exhibit decreased reactivity

with amino acids having large, branched side-chains such as

BCH (Van Winkle et al., 1988). The Na+-dependent portion of

AIB transport is insensitive to inhibition by arginine, but

easily inhibitable by an excess of serine. Therefore, it

appears that the majority of Na+-dependent AIB transport in

oocytes is mediated by a transporter with characteristics

similar to System ASC (Van Winkle et al., 1988).

With regard to Na+-dependent transport of naturally-

occurring neutral amino acids, the evidence presented points

to mediation by activities similar to Systems ASC and B0'+.

The assignment of Na+-dependent alanine transport in oocytes

to System A by Jung et al. (1984) was based on pH sensitivity

and stereo-selectivity, even though alanine uptake was not








40

inhibited by the System A-specific probe, MeAIB.

Furthermore, the precise pH at which alanine transport

declined cannot be gauged as the actual data were not

published. We believe that affinity for amino acids with N-

mono-methylation such as MeAIB, to be a hallmark of System A.

Using this criterion, the results presented indicate that the

Na+-dependent uptake of many neutral amino acids by Xenopus

oocytes is mediated by MeAIB-insensitive carrierss. Using

threonine as an example, we find that only about 50% of its

Na'-dependent uptake is inhibitable by an excess of arginine

(System B0'+), whereas the remainder is strongly inhibited by

serine and is probably mediated by an activity similar in

scope and properties to the ASC system.

The Na+-independent transporters were found to represent

the major pathways for accumulation of neutral amino acids by

Xenopus oocytes. The processes that appear to be responsible

are, in general terms, analogous to Systems L, asc, and b0'*

described for other cell types (Vadgama and Christensen,

1985a,b; Van Winkle et al., 1988). The distinguishing

feature of System b0' is the ability to accept cationic as

well as neutral amino acids (Van Winkle et al., 1988). In

the oocyte, the Na+-independent uptake of threonine is

mediated by processes that appear similar in many respects to

Systems b0'+ and asc as evidenced by the partial inhibition of

its transport by the cationic amino acid arginine (data not

shown). Increasing the concentration of unlabelled arginine








41

from 5 to 10 mM caused no further inhibition of Na+-

independent threonine transport, demonstrating the ability

of arginine to distinguish between two distinct activities

rather than merely exhibiting partial inhibition of a single

process. The remainder of the threonine Na+-independent

transport was saturable by serine. This result is indicative

of System asc given the relatively poor inhibitory action of

serine and threonine on System L-mediated BCH transport. A

carrier analogous to System y+ appears to be responsible for

most of the Na -independent arginine transport. The cationic

forms of lysine and histidine were effective in blocking

arginine uptake. For the latter amino acid, the importance

of the net positive charge was demonstrated directly by

altering the pH of the assay medium. The previously reported

insensitivity to pH changes between 6 and 8 of the carrier

itself (White, 1985) was confirmed for the oocyte.

With regard to active accumulation of amino acids by the

oocyte; i.e., Na'-dependent transport, an activity that

appears to be analogous to System Bo0' may predominate. Our

observations support a similar proposal by Van Winkle (1988)

in a recent review. However, given the resistance to

arginine inhibition of a portion of Na+-dependent threonine

and AIB transport, the presence of a carrier preferring

zwitterionic amino acids only, such as System ASC, is also

indicated. When expressed on a per milligram non-yolk

protein basis, the rates of amino acid transport measured in








42

oocytes are, on average, considerably slower than that

observed in other cell types. For example, cationic amino

acids, such as arginine, homoarginine, and lysine, are

transported into human fibroblasts at a rate of approximately

1 nmol'mg1proteinmin"' (White et al., 1982). The same amino

acids are transported into Xenopus oocytes at a rate which is

only 3% of the fibroblast rate. Similarly, the neutral amino

acids alanine, serine, and threonine are transported into

oocytes at rates that are only one-fiftieth of that seen in

rat hepatocytes. These slow rates of amino acid transport in

oocytes may be a reflection of the relatively quiessent

metabolic state characteristic of fully-grown germ cells. In

summary, it is clear that amino acids enter defolliculated

Xenopus oocytes by means of several distinct transport

activities which correspond, in general characteristics, to

systems observed in other animal cells.














CHAPTER III

EFFECT OF MEIOTIC MATURATION ON THE TRANSPORT OF AIB,
THREONINE, AND LEUCINE IN XENOPUS LAEVIS OOCYTES

Introduction

Fully-grown, stage 6 (Dumont, 1972) Xenopus laevis

oocytes, which are arrested in prophase of the first meiotic

division, can be induced to mature in vitro by the action of

specific hormones or growth factors. It is only the fully-

matured oocytes, now called eggs, that are fertilizable. In

vivo, meiotic maturation is triggered by gonadotropins

released from the pituitary gland (Wasserman, 1986). The

hormones interact with the follicle cells surrounding the

oocytes, stimulating them to synthesize and secrete

progesterone. The maturation process commences following

the interaction of progesterone with the oocytes. During the

course of this maturation process, which culminates in a

meiotic blockade at metaphase of the second division, the

oocyte nuclear envelope breaks down. The disappearance of

the nucleus, also called the germinal vesicle, signals the

completion of maturation and can be easily verified in vitro

by the presence of a non-pigmented spot at the brown-colored

animal pole. Germinal vesicle breakdown (GVBD) can also be

gauged by fixing the oocyte in trichloroacetic acid and

confirming the absence of the nucleus following dissection.

43








44

In this report, the terms "GVBD" and "maturation" are used

interchangeably.

The most widely studied hormones used to induce

maturation in vitro are progesterone and insulin. Much work

has been carried out in recent years in attempts to elucidate

the precise mechanism involved in progesterone- or insulin-

induced GVBD (Stith and Maller, 1984; Maller, 1987).

Although there is still debate with regard to the nature and

order of events taking place, there is general acceptance

that the induction of GVBD by either of these hormones is

accompanied by the following: 1) a decrease in the intra-

oocyte concentration of 3',5'-cyclic adenosine monophosphate

(cAMP); 2) an increase in overall protein synthesis; 3) an

increase in total protein phosphorylation; and 4) the

activation of a specific cytoplasmic factor, named the

maturation promoting factor (MPF), which is responsible for

mediation of the late meiogenic events of the maturation

process (Taylor and Smith, 1987; Maller, 1987).

Recognition of the existence of MPF arose from

experiments which demonstrated the meiotic maturation of

oocytes after they had been injected with a portion of the

cytoplasm obtained from a fully-matured (i.e. post-GVBD) egg

(Masui and Markert, 1971; Smith and Ecker, 1971). In

contrast to the hormone-induced event, which takes from 6 to

12 hours, MPF-induced maturation is relatively rapid,

occurring in less than 2 hours. Further, this factor appears








45
to function post-translationally as its effect is unaltered

by the presence of the protein synthesis inhibitor,

cycloheximide (Taylor and Smith, 1987). The MPF from Xenopus

eggs has been partially purified (Wu and Gerhart, 1980) and

shown to possess a protein kinase activity. In agreement

with this finding, earlier work (Maller et al., 1977) had

demonstrated an increase in total protein phosphorylation

following the transfer of mature oocyte cytoplasm into

prophase-arrested oocytes. This increase in protein

phosphorylation is also observed following incubation of

oocytes in medium containing progesterone or insulin. In

addition, the action of either of these hormones results in a

rapid decline in the cytoplasmic level of cAMP (Stith and

Maller, 1985; Maller, 1987); progesterone acting by

inhibition of adenylate cyclase and insulin by activation of

phosphodiesterase (Maller, 1987). Interestingly, insulin,

as well as insulin-like growth factor-1 (IGF-1), has been

shown, in vitro, to bring about a decrease in the activity of

adenylate cyclase (Sadler and Maller, 1987). Conversely,

activators of adenylate cyclase, such as cholera toxin, and

inhibitors of phosphodiesterase, such as theophylline and

isobutylmethylxanthine, have been shown to inhibit hormone-

induced GVBD in oocytes (Stith and Maller, 1984,1985; Sadler

and Maller, 1987).

Although the mechanisms by which progesterone and

insulin bring about meiotic maturation in oocytes appear to









46

be very similar, both operating by causing a decrease in the

cytoplasmic concentration of cAMP, evidence has demonstrated

that the two pathways are actually quite distinct. For

example, following treatment with either progesterone or

insulin, Xenopus oocytes have been shown to have elevated

intracellular pH (pH,) (Lee and Steinhardt, 1981) as well as

increased phosphorylation of ribosomal protein S6 (Hanocq-

Quertier and Baltus, 1981). Inclusion of cholera toxin in

the incubation medium, while able to block both progesterone-

and insulin-induced GVBD, inhibits only the progesterone-

mediated increase in pH, and S6 phosphorylation while having

no effect on the increases caused by insulin (Stith and

Mailer, 1984). In addition, incubation of oocytes in Na+-

free medium containing amiloride, a Na/H exchange blocker,

prevents the decline in pH, following treatment with either

hormone. However, in the presence of progesterone, the

percentage of oocytes undergoing meiotic maturation is

unchanged from that in amiloride-free Na+-containing medium

while it is reduced by 60% when insulin is used (Stith and

Maller, 1985). This result also indicates that the decrease

in pH, accompanying GVBD is not necessary for maturation to

occur.

In addition to insulin's effects on cAMP levels via

phosphodiesterase activation, the tyrosine kinase domain of

the oocyte insulin receptor has been shown to be intimately

involved in catalyzing GVBD. In 1986, Morgan et al. showed








47
that insulin- (but not progesterone-) induced GVBD could be

prevented by the injection of antibodies directed against the

kinase domain of the human insulin receptor. Furthermore,

injection of antibodies directed against a highly conserved

region of the ras gene product (p21) was shown to block

insulin-mediated GVBD while having no effect on

progesterone-mediated GVBD (Korn et al., 1987). The ras

protein is a membrane-bound, guanine nucleotide binding

protein that is believed to function in the response of cells

to insulin (Kamata and Feramisco, 1984; Heyworth et al.,

1985). Among the actions attributed to both insulin (Saltiel

et al., 1986) and p21 (Fleischman et al., 1986) is activation

of a specific phospholipase C. Activation of phospholipase C

results in a transient increase in the intracellular

concentrations of diacylglycerol and inositol phosphates as

a result of the enzyme's action upon phosphatidylinositol

4,5-bisphosphate (Houslay et al., 1987). The inositol

phosphates are involved in the release of Ca++ from non-

mitochondrial stores (Berridge, 1983). Evidence has also

been reported which implies a role for inositol 1,3,4,5-

tetrakisphosphate in increasing the permeability of sea

urchin egg plasma membrane to Ca++ (Irvine and Moor, 1986).

Diacylglycerol, on the other hand, interacts with protein

kinase C, causing its activation. The tumor promoting agent,

TPA (12-O-tetradecanoylphorbol-13-acetate), contains a

diacylglycerol-like moiety and has been shown to bring about








48
the activation of protein kinase C (Nishizuka, 1984),

presumably by mimicking the action of diacylglycerol itself.

TPA has also been shown to be an effective inducer of GVBD in

Xenopus oocytes (Stith and Maller, 1987).

In addition to the changes mentioned above, meiotic

maturation of Xenopus laevis oocytes also results in a

generalized decline in plasma membrane transport activity to

near zero levels (Richter et al., 1984). Following induction

of GVBD via progesterone, Richter et al., (1984) noted a

dramatic decrease in the uptake of L-alanine, thymidine,

chloride, phosphate, and alkali ions. Likewise, the Na+-

dependent transport of L-leucine was shown in earlier work to

decrease markedly following progesterone-induced GVBD

(Belle' et al., 1976). The protein synthesis inhibitor,

cycloheximide, was shown to prevent not only GVBD but the

associated decrease in Na+-dependent leucine transport as

well. Because the matured oocytes are normally ovulated into

pond water, it has been hypothesized that this decrease in

transport activity may serve to minimize the loss of

intracellular solute molecules, such as amino acids, through

carrier-mediated efflux (Van Winkle, 1988).

This chapter describes an investigation into the effects

of progesterone- or TPA-induced GVBD on the transport of

leucine, threonine, and AIB. Studies described in Chapter II

involving cis-inhibition and trans-stimulation of leucine

transport indicate that this amino acid enters oocytes by








49
means of a carrier similar in many respects to System L

(Shotwell et al., 1983). For this reason the saturable, Na+-

independent transport of leucine subsequent to in vitro

induced meiotic maturation was investigated. Additional

experiments described in Chapter II (Figs. 2-4 and 2-7)

indicate that threonine enters oocytes both by Na+-dependent

and Na+-independent means. Data from this investigation

implies that threonine transport is mediated by two

previously studied Na+-dependent transporters: Systems ASC

(Kilberg et al., 1981) and BO'+ (Van Winkle et al., 1988) and

one Na-independent transporter, System asc (Vadgama and

Christensen, 1985a,b). The effect of GVBD on the relative

activities of these carriers was examined by monitoring the

Na+-dependent (System ASC and B0') and saturable, Na+-

independent (System asc) transport of threonine.

Maturation-induced effects on the Na+-dependent oocyte

transport of AIB were also conducted. Although the Na+-

dependent transport of AIB in rat hepatocytes is mediated

almost entirely (greater than 90%) by System A, it appears to

be transported primarily by a System ASC-like carrier in

Xenopus oocytes (see Chapter I).

Materials and Methods
Isolation of Oocvtes and Amino Acid Transport

Methodology for the isolation of oocytes and amino acid

transport is included in the Materials and Methods section in

Chapter II.












Induction of Meiotic Maturation in Oocvtes

Manually defolliculated oocytes were incubated in

plastic Petri dishes at 20C in MBM containing either

progesterone or 12-O-tetradecanoylphorbol-13-acetate (TPA)

for the specified period of time. Progesterone and TPA stock

solutions were stored in 0.2 ml aliquots at -20C. The stock

solutions were made by dissolving progesterone in 95% (v/v)

ethanol at a concentration of 2 mg/ml (6.4 mM) or TPA at a

concentration of 1 mg/ml (1.62 mM) in dimethylsulfoxide

(DMSO). Control oocytes for progesterone- or TPA-induced

GVBD were incubated in MBM containing ethanol or DMSO

respectively, at concentrations equivalent to those in the

experimental conditions. Bovine serum albumin (BSA), at a

concentration of 0.2% (w/v), was included in the TPA-

containing medium to minimize nonspecific adsorption of the

phorbol ester to the plastic dish. Meiotic maturation (i.e.

GVBD) was verified by placing oocytes in 10% (w/v)

trichloroacetic acid for approximately 10 minutes at room

temperature followed by dissection using Dumont #5 forceps.

Absence of the germinal vesicle was taken as evidence of

meiotic maturation. For transport experiments, only those

oocytes possessing a white spot at the animal pole were used.

Fixation and dissection of such oocytes revealed a perfect

correlation between the presence of the spot and the absence

of the germinal vesicle.








51

Results

Initial attempts to induce GVBD in stage 5 and 6

(Dumont, 1972) oocytes with progesterone were without

success. After 24 hours of incubation in MBM or O-R2 (82.5

mM NaC1, 2.5 mM KC1, 1.0 mM CaC12, 1.0 mM MgCl2, 1.0 mM

Na2HP04, 5.0 mM HEPES, pH 7.8) (Wallace et al., 1973)

containing 10 AM progesterone, all oocytes tested were found

to have intact germinal vesicles following trichloroacetic

acid fixation and dissection. In 1976, Vitto and Wallace

investigated the effects of ouabain, an inhibitor of Na/K

ATPase, on progesterone-induced GVBD in oocytes. Their

results demonstrated that ouabain, while unable to induce

GVBD on its own, was able to facilitate maturation when

included in the incubation medium in conjunction with

progesterone. Further, it was also shown that the omission

of K+ from the incubation buffer had the same effect as the

inclusion of ouabain (Vitto and Wallace, 1976). These

studies were expanded by Wasserman et al. (1986) with the

demonstration of the induction of GVBD in stage 4 oocytes

that had been incubated in progesterone-containing, K-free

O-R2 medium. This same buffer containing 2.5 mM K+ and

progesterone brought about GVBD in stage 5 and 6 oocytes only

(Wasserman et al., 1986).

The oocyte incubation medium used in our laboratory,

MBM, contains 1 mM K. However, in order to avoid

interference in determinations of Na+-dependent amino acid








52

transport rates, the pH of the MBM was routinely adjusted

with KOH, resulting in a final K' concentration of 10 to 20

mM. In order to ascertain the effect of this unnaturally

high K level on the induction of GVBD via progesterone

action, buffers of varying K+ concentration were tested (Fig.

3-1). In this experiment, 100, 87, and 32% of the oocytes

incubated for 12 hours in progesterone-containing MBM with no

K+, 1 mM K%, or 15 mM K+, respectively, underwent GVBD.

Interestingly, approximately 1 in 10 oocytes incubated in K'-

free MBM without progesterone underwent GVBD as well. Based

on this data, all subsequent experiments described in this

chapter were carried out using MBM containing a final

potassium concentration of 1 mM.

The inclusion of the tumor promoting compound, TPA, in

oocyte incubation medium results in the induction of GVBD in

stage 5 and 6 oocytes (Stith and Maller, 1987). The

concentration of the phorbol ester required to bring about

maturation in 50% of the oocytes tested was found to be 150

nM. In agreement with the data of Stith and Maller (1987),

TPA at a concentration of 600 nM, in MBM containing 1 mM K+,

was indeed found to be an effective inducer of GVBD (Fig. 3-

2). Although 5% of the oocytes tested were shown to have

undergone GVBD at 2 and 4 hours, a consistent increase in

those maturing was not seen until 6 hours after the addition

of TPA. After 10 hours, 90% of the oocytes had undergone

GVBD as evidenced by the absence of a germinal vesicle

















120

100

80

> 60




20

0
















Control) or in the following solutions containing 10 pM
progesterone: K+-free MBM (K+ Free); MBM containing 1 mM K+ (Low
K+); or MBM containing 15 mM K+ (High K) After 12 hours of
incubation at 20'C, the oocytes were fixed in 10% (w/v)
trichloroacetic acid and dissected as described in the
Materials and Methods section. Absence of the germinal vesicle
was taken as evidence of GVBD. The results are expressed as
the percentage of oocytes undergoing GVBD.


















100

80
C3
m 60
C 40 /



0


0 2 4 6 8 10 12

Incubation time (hours)











Figure 3-2. Time course of TPA-induced GVBD in oocytes.
Oocytes were incubated at 20C in MBM alone; MBM containing
0.2% (w/v) BSA and 0.04% (v/v) DMSO; or MBM containing 0.2%
(w/v) BSA, 0.04% (v/v) DMSO, and 0.6 AM TPA. After varying
periods of time, 20 to 40 oocytes were fixed in 10% (w/v)
trichloroacetic acid and dissected as described in the
Materials and Methods section. Absence of the germinal
vesicle was taken as evidence of GVBD. The results are
expressed as the percentage of oocytes undergoing GVBD. None
of the oocytes incubated in the absence of TPA were found to
have undergone GVBD.








55

following trichloroacetic acid fixation and dissection. The

time course of progesterone-induced GVBD was essentially the

same as that just described for TPA (data not shown).

Following progesterone- or TPA-induced maturation, the

Na+-dependent transport of threonine and AIB was measured in

oocytes (Table 3-1). At a substrate concentration of 50 MM,

the Na-dependent transport rate of threonine declined 93.1

and 99.6% compared to control following progesterone- or TPA-

induced GVBD, respectively. As described in Chapter II, Na+-

dependent threonine transport in oocytes appears to be the

result of transport activities analogous to Systems B0'+ and

ASC. Although no attempt was made to distinguish between

mediation by one or the other carrier, the almost complete

elimination of Na+-dependent threonine transport following

GVBD indicates that both activities declined equally.

Although a decline in the Na+-dependent AIB transport rate

was also observed, the difference was found to be non-

significant at the P<0.05 level. The saturable, Na+-

independent transport rates of 50 pM threonine and leucine

were also shown to decline following induction of GVBD (Table

3-2). In the absence of sodium, the saturable rate of

threonine entry declined 78.3% following TPA-induced GVBD.

However, the decline following progesterone-induced

maturation was only 16% of the control value. The decrease

in the rate of leucine entry into oocytes seen following

progesterone-induced maturation (63.8%) was also of lesser





















TABLE 3-1. Measurement of Na-dependent amino acid transport
in oocytes following induction of GVBD by progesterone or
.TPA.

Velocity
Substrate Control GVBD % Decrease P<

Progesterone:

AIB 0.9 0.4 0.3 0.3 69.4 N.S.
Thr 38.6 21.2 2.7 2.7 93.1 0.025
---------------------------------------------------------
TPA:

AIB 3.8 1.6 3.0 2.8 21.7 N.S.
Thr 34.2 7.8 0.1 3.2 99.6 0.005


Germinal vesicle breakdown (GVBD) was induced in oocytes by
the inclusion of progesterone (3.2 MM) or TPA (0.6 pM) in the
surrounding medium and incubating for 10 to 12 hours as
described in the Materials and Methods section. The Na+-
dependent transport of 50 AM [H]AIB or [3H]threonine was then
measured for 60 minutes at 20C in those oocytes having
undergone GVBD. GVBD was verified in oocytes incubated in
parallel by fixation in 10% (w/v) trichloroacetic acid and
dissection. Transport velocities are expressed as pmol*
oocyte'*h"1. (N.S. = not statistically significant)




















TABLE 3-2. Measurement of saturable, Na+-independent amino
acid transport in oocytes following induction of GVBD by
progesterone or TPA.

Velocity
Substrate Control GVBD % Decrease P<

Progesterone:

Thr 12.5 0.7 10.5 1.2 16.0 0.025
Leu 17.4 4.5 6.3 1.1 63.8 0.005
--------------------------------------------------------
TPA:

Thr 14.4 4.4 3.1 2.9 78.3 0.005
Leu 13.8 3.9 1.8 1.8 91.5 0.005


Germinal vesicle breakdown (GVBD) was induced in oocytes by
the inclusion of progesterone (3.2 MM) or TPA (0.6 IM) in the
surrounding medium and incubating for 10 to 12 hours as
described in the Materials and Methods section. The Na'-
independent transport of 50 AM [(H]threonine or [3H]leucine
was then measured for 60 minutes at 200C in those oocytes
having undergone GVBD. Saturable transport velocities were
calculated by subtracting the velocities observed in the
presence of 5 mM unlabeled substrate from those in the absence
of excess substrate. GVBD was verified in oocytes incubated
in parallel by fixation in 10% (w/v) trichloroacetic acid and
dissection. Transport velocities are expressed as pmol-
oocyte1 h1.








58

magnitude than the decrease seen following TPA-induced GVBD

(91.5%).

Discussion

Previous studies investigating the effects of GVBD on

the transport of small solute molecules (Belle' et al., 1976;

Richter et al., 1984) have focused primarily on the net

changes in transport rather than on maturation-induced

changes of specific carriers. This chapter describes an

investigation into the effects of progesterone- or TPA-

induced GVBD on four distinct transport activities. The

activities studied were shown in Chapter I to be similar in

many respects to Systems ASC, B0'+, L, and asc. Maturation-

induced alterations in the rates of substrate transport

attributable to these carriers were investigated by

measuring the Na+-dependent AIB and threonine transport

(Systems B0+ and ASC), and the Na+-independent, saturable

transport of threonine (System asc) and leucine (System L).

In agreement with other laboratories (Belle' et al.,

1976; Richter et al., 1984), the experiments just described

demonstrate a marked decrease in oocyte plasma membrane amino

acid transport activity following GVBD. Although the

physiological reason for this decrease in transport activity

is as yet unknown, it has been theorized that this

generalized decline serves to protect the oocyte from

unbridled loss of soluble cytoplasmic constituents via

carrier-mediated efflux (Van Winkle, 1988). Inherent in this








59

notion is the assumption that oocytes do, in fact, lose

solute molecules to the surrounding medium through plasma

membrane transporters. However, data reported in 1976 by

Bravo et al. indicate that efflux may not be a problem for

defolliculated oocytes. In this investigation, manually-

defolliculated oocytes were assayed for soluble amino acid

content before and after a 24-hour incubation in amino acid

free medium. The results show a net decrease in the intra-

oocyte concentration of only 4 (lysine, glutamic acid,

valine, and leucine) of the 17 amino acids tested (Bravo et

al., 1976). Therefore, the decline in transport activity

seen following meiotic maturation may not serve to curtail

efflux but may simply be a reflection of the fact that

oocytes have no need for plasma membrane transporters while

in the nutrient-poor pond water. Normal, or accelerated,

turnover of existing transporters, subsequent to cessation

of the translation of new carriers, could account for the

observed decrease in transport activity. A report in 1981 by

Kado et al. has shown that the total plasma membrane surface

area of the oocyte decreases following progesterone-induced

GVBD. Although the reason for this reduction in surface area

is not known, it is plausible that the decrease is a

reflection of enhanced internalization of existing plasma

membrane proteins. Besides eliminating transporters that

are no longer of use, the metabolites resulting from their

proteolytic degradation could serve to supplement the








60

oocyte's yolk stores as a source of energy and amino acids to

be used following fertilization. Further support for the

endocytotic removal of plasma membrane transporters could be

gathered by investigating the changes in hormone binding site

number brought about by the addition of progesterone or TPA

to the incubation medium. For example, estimation of the

size of the oocyte insulin receptor population could be

obtained by Scatchard analysis of [1251]insulin binding as

described by Shimizu et al. (1980). Unfortunately, the

relative affinities of the substrate amino acids used in the

preceding study for their respective carriers are not

sufficiently high to allow for direct estimation of

transporter number via binding assays.

Whatever the mechanism by which the oocyte brings about

the reduction in the uptake of amino acids following GVBD, it

is evident that both progesterone and TPA are effective in

inducing the change. As outlined in the introduction to this

chapter, incubation of stage 5 or 6 oocytes in progesterone-

containing medium leads to a rapid decline in the cytoplasmic

level of cAMP. This is followed by intra-oocyte

alkalinization, increased protein phosphorylation, and,

lastly, GVBD (Wasserman et al., 1986). However, the initial

event in TPA-induced maturation is activation of protein

kinase C (Stith and Mailer, 1987), presumably due to the

similarity in structure between diacylglycerol, a product of

phosphoinositide breakdown, and the phorbol ester









61

(Nishizuka, 1984). Further, insulin, but not progesterone,

has been shown to lead to phosphoinositide breakdown (Stith

and Maller, 1987) and, hence, diacylglycerol production in

oocytes. Given this dissimilarity in the initial event of

progesterone- and TPA- (or insulin-)induced meiotic

maturation, there exists the possibility of parallel

differences in the response of amino acid transport activity

to maturation. The data in Table 3-2 indicate that this may,

in fact, be the case. Progesterone-induced GVBD caused a

reduction of 16 and 63.8% in the saturable, Nat-independent

transport of threonine and leucine, respectively. The

decrease seen for threonine and leucine following TPA-

induced GVBD were 78.3% for the former and 91.5% for the

latter. In all of these experiments, the non-saturable, or

diffusion-mediated, Na+-independent component of threonine

and leucine transport was also seen to decline following GVBD

(data not shown). The decrease in the non-saturable

transport was observed whether TPA or progesterone was used

although the decrease was of lesser magnitude when TPA was

used. Non-saturable threonine and leucine transport

declined 67 and 75% in the presence of progesterone but only

45 and 25%, respectively, in the presence of TPA. Although

it is possible that these changes in membrane permeability

are caused by the solvents used in the progesterone and TPA

stock solutions, it is unlikely given the data in Fig. 3-3.

For this experiment, progesterone was dissolved in ethanol or


















40 3 Total transport
I-I Non-saturable transport
-C

I
S30


o 20
0

E 10


0
EtOH DMSO EtOH DMSO
Control GVBD









Figure 3-3. Effect of solvent, in the presence or absence of
progesterone, on the saturable, Na+-independent transport of
leucine. Progesterone was dissolved in 95% (v/v) ethanol or
DMSO at a concentration of 2 mg/ml. Oocytes were then
incubated in MBM containing ethanol alone (0.05% (v/v)), DMSO
alone (0.05% (v/v)), ethanol (0.05% (v/v)) plus progesterone
(3.2 AM), or DMSO (0.05% (v/v)) plus progesterone (3.2 AM).
After 10 hours of incubation at 20C, those oocytes
exhibiting a white spot at the animal pole were assayed for
Na+-independent leucine transport. Leucine transport was
measured at 50 pM substrate concentration in the presence
(Non-saturable transport) or absence (Total transport) of 5
mM unlabeled leucine as described in the Materials and
Methods section.








63

DMSO prior to dilution into the incubation media. Following

verification of GVBD, the saturable, Na-independent

transport of leucine was determined as described in the

Materials and Methods section. No difference between the

effects of ethanol and DMSO, either alone or in conjunction

with progesterone, was detectable (Fig. 3-3). Therefore, it

is possible that the alterations in membrane permeability, as

evidenced by changes in the rate of diffusion-mediated

leucine uptake, are due to dissimilar effects of TPA and

progesterone on the oocyte plasma membrane.

In contrast to the changes seen in transport rates

measured in the absence of sodium, decreases in the Na+-

dependent transport of threonine caused by progesterone or

TPA were essentially equal (Table 3-1). Although a decline

in Na+-dependent AIB transport was also observed, the change

was found to be non-significant at the P<0.05 level. The

lack of statistical significance is more likely a reflection

of the extremely low AIB transport velocities, in combination

with comparatively large standard deviation values, rather

than a refractoriness of the oocyte AIB transporter to

undergo a decline in activity in response to meiotic

maturation.

In summary, the Na+-dependent transport of threonine,

and the saturable, Na+-independent transport of threonine and

leucine were shown to decrease markedly following induction

of GVBD by progesterone or TPA. Differences in the magnitude









64

of the reduction in the saturable, Na-independent, and non-

saturable components of threonine and leucine transport

caused by progesterone and TPA were noted. No such

differences between the effects of progesterone- and TPA-

mediated GVBD on Na+-dependent threonine transport were seen.














CHAPTER IV

TRANSLATION OF RAT LIVER mRNA
IN XENOPUS LAEVIS OOCYTES

Introduction

Since its first mention in the literature (Lane et al.,

1971; Gurdon et al., 1971), the use of frog oocytes as a

means to study the metabolism and translation of exogenously-

derived mRNA has steadily increased. The basic techniques

involved, including the in vitro handling, incubation, and

microinjection of the oocytes, have remained essentially

unchanged since the early 1970's. Pioneering experiments

employing the Xenopus laevis oocyte as an "in vitro"

translation system often utilized relatively abundant or

easily-isolated mRNA species. Examples include the mRNA's

coding for rabbit globin (Lane et al., 1971), duck globin

(Lane et al., 1973), and calf lens aA2 crystallin (Berns et

al., 1972). Further, the translation of viral polypeptides

in Xenopus laevis oocytes was demonstrated in 1972 by Laskey

et al. using encephalomyocarditis RNA. Proper post-

translational modification of foreign proteins has been

demonstrated repeatedly. Glycosylation (Colman et al.,

1981), signal sequence removal (Lane et al., 1981),

phosphorylation (Gedamu et al., 1978), and multi-subunit

protein assembly (Sumikawa et al., 1981) are just a few of

65








66
the cases in which the Xenopus oocyte has faithfully

translated and processed foreign proteins.

Accurate post-translational targeting of foreign

proteins by the frog oocyte is further evidenced by the

correct membrane insertion of functional ion channels

(Sigel, 1987) and plasma membrane hormone receptors

(Williams et al., 1988) following the microinjection of mRNA

extracted from chick leg muscle or AR42J pancreatic cells in

culture, respectively. Utilization of the oocyte system as

an assay for a specific mRNA was demonstrated by Hirono et

al. (1985) with the identification of a specific size

fraction of mRNA capable of supporting the synthesis of rat

brain Na+-channels in Xenopus oocytes. More recently, it has

been demonstrated that the oocyte system can be used as a

functional screen in the molecular cloning of the cDNA for a

specific protein. A case in point is the cloning of the

intestinal Na+/glucose co-transporter (Hediger et al.,

1987a). Messenger RNA extracted from rabbit small intestine

was size-fractionated via agarose gel electrophoresis and

individual fractions microinjected into Xenopus laevis

oocytes. After a 3-day incubation, the injected oocytes were

assayed for the presence of the transporter. The size

fraction of mRNA resulting in the most significant increase

in oocyte Na+-dependent glucose transport was then used to

create a cDNA library. Messenger RNA transcribed in vitro

from the library was then injected into oocytes which were








67
subsequently assayed for Na+-dependent glucose transport.

Identification of the cDNA for the transporter was achieved

by injecting mRNA transcribed from increasingly smaller

pools of cDNA.

Research in our laboratory has, for the past several

years, focused on the System A amino acid transporter

(Kilberg et al., 1986; Chiles et al., 1988; Fafournoux et

al., 1989). As is the case with most other amino acid

transporters, the isolation and purification of the System A

carrier has proven to be an exceedingly difficult task. Our

laboratory is currently pursuing several diverse routes in

attempts to isolate the carrier. One of these, which has

been the major focus of my thesis, involves the use of the

Xenopus laevis oocyte as a functional screen to identify the

mRNA coding for the System A carrier. The method is modeled

after that utilized in the cloning of the intestinal Na+-

dependent glucose transporter described above (Hediger et

al., 1987a).

Characterization of the endogenous oocyte amino acid

transport activities, a necessary prerequisite to studies

involving the identification of an exogenous carrier in the

oocyte following microinjection of foreign mRNA, has been

described in Chapter II. Although the capability of amino

acid uptake assays in oocytes to distinguish among several

different amino acid transport activities was illustrated in

these initial experiments, it was not known if the assays








68

were of sufficient sensitivity to detect subtle changes in

amino acid uptake rates subsequent to mRNA injections. As

described in Chapter II, the principle means by which the

presence of the System A carrier is documented in any cell or

tissue, including oocytes, is via measurement of the Na+-

dependent transport of MeAIB or AIB. Direct monitoring of

the carrier protein, or the mRNA coding for the protein, is

not possible due to the fact that neither cDNA clones nor

antibodies directed against the carrier exist. Therefore,

the question of the stability of the System A mRNA or protein

in the oocyte could not be answered. For these reasons, two

additional proteins, for which both cDNA clones and antisera

are available, were chosen to be monitored directly. The

proteins selected were rat serum albumin (RSA) and

phosphoenolpyruvate carboxykinase (PEPCK). An investigation

of the expression of these two proteins will allow our

laboratory to optimize the oocyte system for expression of

rat liver mRNA.

Rat serum albumin, like the System A protein, is

translated by membrane-bound ribosomes. Experiments

described by Teraoka et al. (1982) have demonstrated that a

related protein, mouse serum albumin, is synthesized in

oocytes following the microinjection of mouse liver mRNA.

Further, the protein is secreted into the surrounding medium

by the oocyte and appears to be of identical molecular weight

to authentic mouse serum albumin (Teraoka et al., 1982). A








69
report in 1981 by Richter and Smith, however, has indicated

that the oocyte may, in fact, have a relatively limited

capacity to handle mRNAs normally translated by membrane-

bound ribosomes, such as that coding for serum albumin or the

System A carrier. By monitoring the synthesis of RSA in the

oocyte following the injection of rat liver mRNA, it should

be possible to gauge the efficiency of the oocyte protein-

translating machinery which would also be responsible for the

translation of the System A protein.

Phosphoenolpyruvate carboxykinase, on the other hand,

was chosen to be monitored in the oocyte following the

microinjection of mRNA because of the similarities in

regulation between it and System A (Kilberg et al., 1985;

Meisner et al., 1983). The injection of glucagon or N,02-

dibutyryl cyclic AMP (dibutyryl cAMP) into rats has been

shown to result in an 8-fold stimulation of liver PEPCK

enzyme activity and mRNA level in less than 2 hours (Cimbala

et al., 1981; lynedjian and Hanson, 1977). In an analogous

manner, System A activity in primary cultures of rat

hepatocytes has been shown to increase approximately 3-fold

when measured 2 hours after exposure to glucagon (Barber et

al., 1983). Under these conditions, System A activity

continues to increase for an additional 4 hours to a level 5

times that of control (Barber et al., 1983). However, as

mentioned above, direct measurements of the level of hepatic

System A mRNA are not possible. Indirect evidence, based on








70
experiments utilizing RNA polymerase inhibitors has,

however, demonstrated that essentially all of the increase in

System A activity can be attributed to elevated mRNA

production (Christensen and Kilberg, 1987).

In addition to similarities in the regulation of PEPCK

and System A, the mRNAs coding for these proteins appear to

have very short half-lives in vivo. The half-life of PEPCK

mRNA has been estimated to be 40 minutes (Cimbala et al.,

1982; Nelson et al., 1980). Similarly, the half-time for the

decay of System A activity in hepatocytes isolated from a

glucagon-injected rat is approximately 1.5 hours (Handlogten

and Kilberg, 1984). Because the System A value was

calculated from transport data and not mRNA levels, it is not

known whether the decay reflects rapid mRNA turnover as well

as protein degradation. However, because the glucagon-

induced elevation in System A activity involves, presumably,

mRNA synthesis, it can be argued that the rapid decay in

activity would be likely to include mRNA turnover as well.

This chapter describes the synthesis in oocytes of 3

distinct proteins, RSA, PEPCK, and the System A carrier,

subsequent to the microinjection of rat liver mRNA. Oocyte

production of two of the proteins, RSA and PEPCK, has been

monitored via immunoblotting techniques. Synthesis of the

rat liver System A carrier, on the other hand, has been

followed by measuring changes in the rate of oocyte Na+-

dependent AIB uptake. The degradation in the oocyte of the








71

mRNAs coding for RSA and PEPCK has been monitored by Northern

blot analysis. Experiments involving variations in the

amount and type (i.e. extracted from fed, glucagon-treated,

or diabetic rat liver) of mRNA injected, as well as the

length of the post-injection incubation period are

described.

Materials and Methods

Isolation of Oocytes

Methodology for the isolation of oocytes is included in

the Materials and Methods section in Chapter II.

Hepatoma Cell Culture

Rat hepatoma cells, from the Fao cell line, were grown

in 75 cm2 culture flasks (Falcon 3023) at 37*C in a humidified

atmosphere of 5% (v/v) CO2 and 95% (v/v) air. The cells were

maintained in minimal essential medium (MEM), pH 7.4,

supplemented with 25 mM sodium bicarbonate, 2.5 mM L-

glutamine, 10 gg/ml penicillin, 5 Ag/ml streptomycin, 28.5

Ag/ml gentamicin, 0.2% (w/v) bovine serum albumin (BSA), and

5% (v/v) fetal bovine serum (FBS).

The rat Fao cell line was derived from rat H4 hepatoma

cells after exposure to 8-azaguanine (Deschatrette and

Weiss, 1974). The rat H4 cell line was derived from the

Reuber H35 hepatoma (Reuber, 1961) which was in turn obtained

by feeding rats a diet supplemented with N-2-fluorenyl-

diacetamide, resulting in a bile-secreting, transplantable

hepatocellular carcinoma. The Fao cell line is a well-









72

differentiated cloned cell line expressing a number of liver-

specific proteins. Hormonal induction of tyrosine and

alanine aminotransferases is also observed (Deschatrette and

Weiss, 1974).

RNA Extraction from Cultured Cells

Total RNA was extracted from Fao cells using the method

of Chirgwin et al. (1979) as modified by Maniatis et al.

(1982). Cells were allowed to grow to confluency and were

then rinsed twice with phosphate-buffered saline (PBS: 10 mM

sodium phosphate, pH 7.4, 154 mM NaCl). The cells were

removed from the surface of the flasks by trypsinization.

This was done by the addition of 1 ml of a solution

containing 0.05% (w/v) trypsin and 0.6 mM

ethylenediaminetetraacetic acid (EDTA) in PBS to the flasks

which were then incubated at 370C for a few minutes.

Detached cells were rinsed from the flasks with 5 to 10 ml

PBS. Cells from 5 to 10 flasks were washed by centrifugation

(500 x g for 5 minutes at 40C in a swinging bucket clinical

centrifuge) and resuspension in PBS, and lysed by vortexing

the cell pellet in 5 ml guanidinium thiocyanate (GuSCN)

solution containing 4 M GuSCN, 0.5% (w/v) sodium lauroyl

sarcosine, 25 mM sodium citrate, 100 mM 2-mercaptoethanol,

and 0.1% (v/v) Antifoam A (Sigma). The solution was

transferred to a glass mortor and homogenized using a motor-

driven Teflon pestle. The homogenate was then transferred to

a 15 ml plastic centrifuge tube. The tube was centrifuged at








73

10,000 x g at 4C for 10 minutes in a Sorvall SM-24 rotor.

The supernatant fraction was layered over 1.25 ml of a 5.7 M

CsCl cushion containing 25 mM sodium acetate, pH 5, and

centrifuged at 36,000 x g at 20*C for 19 to 21 hours in an

Beckman SW50.1 swinging bucket rotor. Before use, the CsCl

solution was treated overnight with 0.2% (v/v)

diethylpyrocarbonate (DEPC) and then heated at 65'C for at

least 1 hour to eliminate residual DEPC. Following

centrifugation, the resulting RNA pellet was dissolved in 0.4

ml of solubilization buffer containing 10 mM Tris, pH 7.5, 1

mM EDTA, 5% (w/v) sodium lauroyl sarcosine, and 5% (v/v)

phenol (added just before use) and transferred to a clean

polypropylene 50 ml Oak Ridge tube. The volume was brought

to 5 ml with solubilization buffer, vortexed hard, and 4 M

NaCl added to a final concentration of 0.1 M. Ten

milliliters of a mixture of phenol, chloroform, and isoamyl

alcohol (in the ratio of 50:49:1) were added, the tube

shaken, and then centrifuged at 12,000 x g at 40C for 10

minutes in a Sorvall SS-34 rotor. The aqueous layer was

transferred to a clean tube and 2 ml solubilization buffer,

minus phenol but including 0.1 M NaC1, was added to the

organic phase. After shaking and centrifuging as above, the

aqueous phases from the two extractions were combined and the

RNA contained therein precipitated by the addition of one-

tenth volume 2 M sodium acetate, pH 5.5 and three volumes

absolute ethanol followed by incubation overnight at -20C .








74

The RNA was sedimented by centrifugation and the pellet

washed two times by resuspension in 75% (v/v) ethanol. The

pellet was broken up and suspended in 1 ml of 0.4 M sodium

acetate and precipitated with 2.5 volumes absolute ethanol.

After sedimentation, the pellet was again washed with 75%

(v/v) ethanol, dried under vacuum, and solubilized in DEPC-

treated distilled, deionized water (ddH2O). Following

centrifugation at 20,000 x g at 10C for 10 minutes in a

Sorvall SS-34 rotor, the supernatant fraction was made 0.2 M

in sodium acetate and precipitated with 2.5 volumes absolute

ethanol. The RNA was sedimented, washed with 75% (v/v)

ethanol, dried, and brought up in DEPC-treated ddH2O. The

numerous washes and precipitations serve to remove residual

CsCl from the extracted RNA.

Total RNA extracted from CHO-K1, alar4-H2.1, and ala'4-

H3.9 cells was the generous gift of Dr. Ellis Englesberg.

Poly(A)* mRNA was isolated from the total RNA by oligo(dT)

cellulose column chromatography as described below. The

parent cell line, CHO-Kl, is a proline auxotroph whose growth

can be inhibited by amino acids that prevent the uptake of

proline (Moffett et al., 1983; Moffett et al., 1988). By

selecting cells for growth in media containing increasingly

higher concentrations of alanine, an amino acid known to

competitively inhibit proline transport, mutant cells were

developed which exhibit elevated proline transport. Two of

these mutants, ala'4-H2.1 and ala'4-H3.9, display increased








75

rates of proline transport through the System A carrier, a

transport system whose substrate specificity includes both

proline and alanine. The H2.1 and H3.9 mutants were found to

have System A-mediated proline uptake 18- and 29-fold higher,

respectively, than the parent cells (Moffett et al., 1988).

RNA Extraction from Rat Liver

Total RNA was extracted from the livers of rats that had

been treated as follows: "Control" rats were fed ad libitum

and then given glucose by gastric gavage (5 g/kg body weight)

2.5 hours before killing. "Glucagon" rats were fasted for 24

hours, injected intraperitoneally with 1 mg glucagon, and

killed 2.5 hours later. The glucagon was delivered in a

50:50 mix of PBS and ethanol (0.3 ml total volume). Diabetes

was induced in rats by the intraperitoneal injection of 10 mg

streptozotocin/100 g body weight. The streptozotocin was

dissolved in 50 mM sodium citrate, pH 4.3. Urine glucose was

monitored daily with Tes-Tape (Lilly) and blood glucose

levels were measured in samples taken at the time of

sacrifice (Sigma Procedure No. 635), generally 2 to 3 days

following the injection of streptozotocin.

Rat liver RNA was extracted using the method of

Chomczynski and Sacchi (1987) from male Sprague-Dawley rats

(approximately 200 g body weight) that had been maintained on

standard laboratory rat chow. After anesthesia, the liver

was perfused free of blood with ice-cold PBS according to the

method of Kilberg (1989). The liver was removed, weighed,








76

and then minced prior to homogenization at room temperature

in solution D (4 M guanidinium thiocyanate, 0.5% (w/v) sodium

lauroylsarcosine, 100 mM 2-mercaptoethanol, 25 mM sodium

citrate, pH 7) using a motor-driven Teflon pestle. Solution

D was used in the ratio of 8 to 10 ml per gram liver. The

homogenate was then made 0.2 M in sodium acetate, pH 4, and

extracted with an equal volume of water-saturated phenol and

one-fifth volume of a mixture of chloroform and isoamyl

alcohol (49:1). The mixture was placed on ice for

approximately 15 minutes, after which time the aqueous and

organic phases were separated by centrifugation at 12,000 x g

for 20 minutes at 4C in a Sorvall SS-34 rotor. Total RNA

was precipitated from the aqueous phase by the addition of an

equal volume of ice-cold isopropanol and chilling at -20C

for at least one hour. Sedimentation of the RNA was achieved

by centrifugation at 12,000 x g for 20 minutes at 4C. The

resulting pellet was then solubilized in solution D and 2

volumes of ice cold absolute ethanol were added. The RNA was

allowed to precipitate overnight at -20*C and was then

sedimented as above. The pellet was washed twice with ice-

cold 75% (v/v) ethanol, dried under vacuum, and solubilized

in ddHO2 that had been treated with 0.1% (v/v)

diethylpyrocarbonate (DEPC) and autoclaved.

RNA Extraction from Oocytes

In experiments designed to gauge the relative half-

lives of various rat liver mRNA's in oocytes following









77

microinjection (described in a separate section), total RNA

was extracted from oocytes using a modified version of the

RNA extraction protocol described above. Typically, 10

oocytes were homogenized in 0.6 ml solution D and the

homogenate centrifuged at 12,000 x g for 10 minutes at 4'C.

The supernatant fraction was transferred to a clean tube and

extracted with phenol and chloroform as described

previously. After separation of the aqueous and organic

phases by centrifugation, the aqueous phase was extracted a

second time with one-half the original volumes of phenol and

chloroform. The RNA present in the aqueous phase was

precipitated with an equal volume of isopropanol and the

resulting pellet solubilized in 0.3 ml solution D. The

remainder of the extraction is identical to that described

for rat liver.

DNA Assay

Because determinations of relative quantities of PEPCK

and RSA mRNA in total rat liver RNA depend, in part, on the

precise quantity of RNA analyzed, precautions were taken to

verify the absence of DNA, a potential source of interference

in the measurement of RNA concentration. To test for the

presence of DNA in the RNA preparations extracted from rat

liver by the GuSCN procedure, the diphenylamine (DPA)

reaction, described by Burton (1956), was used. Samples of

total rat liver RNA were dissolved in 0.5 ml DEPC-treated

ddH2O and 0.5 ml perchloric acid added to each sample. The








78
samples were then heated in 4 ml polycarbonate centrifuge

tubes at 70*C for 15 minutes, cooled, 2 ml DPA reagent added,

and the tubes incubated overnight at room temperature. The

DPA reagent was made by adding 0.1 ml of a 16 mg/ml solution

of acetaldehyde to 20 ml of a DPA stock solution. The stock

solution was made by dissolving 1.5 g DPA in 100 ml glacial

acetic acid followed by the addition of 1.5 ml concentrated

sulfuric acid. The stock solution was stored at room

temperature in a brown glass bottle. After the overnight

incubation, the absorbance at 600 nm of each sample was

compared to that of DNA standards treated in an identical

manner. The standards were prepared from salmon testes DNA.

The results of this assay indicate that no more than 0.006 mg

DNA/mg RNA was present in the RNA extracted from rat liver by

the method described.

Determination of In Vivo System A Activity

Because the System A amino acid transporter and

phosphoenolpyruvate carboxykinase (PEPCK) respond in

parallel to changes in the metabolic state of the animal, the

in vivo monitoring of the rat liver System A carrier allows

one to estimate the status of PEPCK as well. The in vivo

determination of rat liver System A activity in fed,

glucagon-treated, and diabetic rats to be used as RNA donors

was carried out according to the method of Kilberg and

Neuhaus (1975) as follows: Approximately 1 hour prior to

anesthesia, 2 LCi of [3H]AIB (8 to 10 Ci/mmol) was injected








79
into the tail vein of the rat. Samples of blood (2 to 3 ml)

and perfused liver (0.5 to 1.0 g) were taken and stored on

ice while total RNA was extracted from the remainder of the

liver as described above. The liver sample was then weighed,

homogenized in 2 ml normal saline (154 mM NaC1), and the

proteins precipitated from the homogenate with the addition

of an equal volume of 10% trichloroacetic acid. After

centrifugation at 12,000 x g for 15 minutes at 4"C, the

radioactivity contained in a portion, typically 0.2 ml, was

determined via liquid scintillation spectrophotometry. The

blood sample was allowed to clot and the resulting serum was

treated in manner equivalent to that for liver. The quantity

of radioactivity present in 1 g liver divided by that present

in 1 ml serum is referred to as the distribution ratio (DR).

When compared among the fed, diabetic, and glucagon-treated

conditions, the DR can be used to gauge the relative degree

of glucagon-dependent induction of System A and, presumably,

PEPCK in the rat liver. Following the RNA extraction,

Northern analysis is used to verify the augmentation in PEPCK

mRNA level.

Oliqo(dT) Cellulose Column Chromatographv

Poly(A)+ mRNA was purified by oligo(dT) cellulose

(Sigma) column chromatography. Oligo(dT) cellulose

chromatography columns were made by first washing 0.25 g

oligo dT cellulose in a sterile plastic 12 ml tube using

DEPC-treated ddH20. The tube was gently rotated end-over-








80

end by hand and the cellulose allowed to sediment under unit

gravity. The supernatant fraction and fines were poured off

and the wash repeated. The washed cellulose was then

resuspended in fresh DEPC-treated ddH2O and the slurry poured

into a disposable plastic chromatography column (BioRad no.

731-1550) that had previously been rinsed with DEPC-treated

ddH2O. The ddH2O was allowed to flow through and the column

equilibrated with at least 10 column volumes of DEPC-treated

RNA loading buffer (LB) containing 0.5 M NaCl, 10 mM Tris, pH

7.4. The Tris was added after the buffer had been treated

with DEPC and autoclaved due to the incompatability of DEPC

and this compound. An equal volume of RNA diluting buffer

(DB), containing 1 M NaCl, 20 mM Tris, pH 7.4, was added to

the RNA solution consisting of total RNA in DEPC-treated

ddH2O. Best results were obtained with final RNA

concentrations of not more than 1 mg/ml as higher

concentrations sometimes resulted in very slow flow rates

through the column. The solution was mixed and heated at

65'C for 3 minutes and immediately cooled on ice. The

solution was then poured over the column, allowed to flow

through, and non-binding (i.e. poly(A)') RNA washed through

with 5 to 10 column volumes of LB. The poly(A)+ mRNA was

eluted with DEPC-treated ddHO2. Fractions enriched in mRNA

were pooled, divided into aliquots of approximately 30 jg

nucleic acid each, made 0.3 M in sodium acetate, and

precipitated overnight with ethanol. Following








81

sedimentation, the pellets were washed twice by resuspension

in ice-cold 75% ethanol and stored at -70C under 75% (v/v)

ethanol.

Microiniection

Messenger RNA to be microinjected into oocytes was first

dissolved in DEPC-treated ddH2O and then centrifuged at

12,000 x g at 4"C for a few seconds immediately prior to use.

This centrifugation step was included in order to ensure that

the mRNA solution was free of particulate matter that could

clog the microinjection needle. Microinjections were

performed using a Hamilton Micro Lab P microprocessor-

controlled pipettor programmed to deliver 40 nl/injection

(Hitchcock and Friedman 1980; Hitchcock et al. 1987).

Microinjection needles were pulled from 2 pl Drummond

"MICROCAPS" using a Narishige PN-3 micro-pipette puller.

Following microinjection, all incubations were carried out

in MBM at 18-200C with daily changes of medium.

Oocvte Homogenization. SDS-polvacrylamide Gel

Electrophoresis, and Immunoblotting

Oocytes were homogenized in ice-cold buffer (20 to 40 Al

buffer per oocyte) containing 100 mM NaCl, 1 mM

phenylmethylsulfonylfluoride, 5 mM benzamidine, 1% (v/v)

Triton X-100, and 20 mM Tris, pH 7.6 (Colman, 1984) using a

motor-driven teflon pestle designed to fit in a standard 1.5

ml Eppendorf microcentrifuge tube. The homogenate was spun

in a microcentrifuge at 12,000 x g for 10 minutes at 4C and








82

the infranatant fraction, between the yolk platelet pellet

and the lipid pellicle, was removed using a 25 gauge needle

and stored at -70C.

In experiments designed to investigate proteins secreted

from the oocyte, incubation medium (i.e. MBM) was collected

at specified times after microinjection. Immediately after

collection, all media were made 1 mM in PMSF and 5 mM in

benzamidine prior to storage at -700C. Oocytes were

typically incubated in 30 pl MBM per oocyte in order to

maximize the concentration of secreted proteins in the

medium. Utilization of smaller amounts of MBM per oocyte

resulted in reduced oocyte viability.

Oocyte proteins contained in homogenate or medium

samples were separated via sodium dodecyl sulfate (SDS)

polyacrylamide gel electrophoresis according to the method

of Laemmli (1970). Briefly, oocyte proteins were solubilized

and denatured by boiling for 2 to 5 minutes in sample

dilution buffer (SDB) containing 1% (w/v) SDS, 30 gg/ml

bromophenol blue, 12% (v/v) glycerol, 720 mM 2-

mercaptoethanol, and 125 mM Tris, pH 6.8. The ratio of

homogenate or medium to SDB could be as high as 1:1 without

obvious deleterious effects. In some cases, however, the

volume of protein sample (i.e. homogenate or medium) was too

large to be loaded into the wells of the polyacrylamide gel.

In these instances, proteins were first precipitated from

homogenate or medium samples by the addition of 24% (w/v) TCA








83

to a final concentration of 10% (w/v) and incubation on ice

for 30 to 60 minutes. The samples were then centrifuged at

12,000 x g for 15 minutes at 4C and the pellet washed twice,

with resuspension, in 0.75 ml ethyl ether to remove the TCA.

Complete removal of TCA was crucial at this time in order to

ensure solubility in SDB. The final pellet was allowed to

air dry, SDB was added, and the solution vortexed thoroughly.

After boiling for 2 to 5 minutes, the samples were allowed to

cool to room temperature and were then centrifuged at 12,000

x g for 1 minute at 4*C before being loaded into the wells of

a 7.5% (w/v) polyacrylamide separating gel equipped with a

4.5% (w/v) polyacrylamide stacking gel. Both the separating

and stacking gels contained 0.1% (w/v) SDS. Electrophoresis

was carried out in buffer containing 192 mM glycine, 25 mM

Tris, pH 8.3, and 0.1% (w/v) SDS at 20 to 30 mA, constant

current, until the bromophenol dye front was within 1 cm of

the bottom of the gel.

For electroblotting, the gel was removed from the

electrophoresis apparatus, the stacking gel was cut away and

discarded, and the separating gel immediately placed into

vacuum-degassed transfer buffer containing 192 mM glycine,

25 mM Tris-base, pH 8.3, and 20% (v/v) methanol (Towbin et

al., 1979). After 15 minutes, a piece of Whatman 3MM

chromatography paper, cut slightly larger than the gel, was

maneuvered under the gel. The chromatography paper and the

gel were then lifted together and placed on a square of








84

Scotch-Brite pad that had been soaked for at least 15 minutes

in transfer buffer. Bubbles were removed from between the

gel and the chromatography paper using a glass stir rod. The

gel was then covered with a piece of nitrocellulose paper,

cut to the size of the gel, that had been equilibrated for at

least 15 minutes in transfer buffer. Bubbles were removed as

above. A second piece of wet chromatography paper was then

laid over the nitrocellulose paper, bubbles were removed, and

a second wet Scotch-Brite pad was laid on top. The

"sandwich", consisting of pad, chromatography paper, gel,

nitrocellulose, chromatography paper, and pad, was then

secured in a perforated plastic holding device and submerged

vertically in a tank of transfer buffer. (The Scotch-Brite

pads, plastic holding device, and tank were from BioRad.)

Proteins contained in the gel were electrophoretically

driven into the nitrocellulose paper by the application of 30

V, constant voltage, for 14 to 16 hours followed by 40 V for

an additional 2 hours. The plastic holding device was then

removed from the tank and disassembled. The nitrocellulose

paper was removed and proteins adhering to it were visualized

by staining with a 0.01% (w/v) amido black solution

containing 50% (v/v) methanol and 10% (v/v) acetic acid. The

blot was destined in 50% (v/v) methanol, 10% (v/v) acetic

acid.

After destaining, blots that were not to be used

immediately were placed, stain side up, on a clean glass








85

plate, covered with plastic wrap, and stored at -20*C.

Otherwise, blots were prepared for immunoblotting by first

rinsing twice, for about 30 seconds each rinse, in PBS

containing 0.02% (w/v) sodium azide (PBS-azide). Blocking of

exposed nitrocellulose was achieved by incubation in PBS-

azide containing 5% (w/v) Carnation non-fat dry milk

(blocking buffer) for 4 to 16 hours using 70 to 80 ml

blocking buffer per blot. This and all subsequent

immunoblotting incubations were carried out at room

temperature with constant agitation. The blocking buffer was

poured off and 50 to 60 ml fresh blocking buffer containing

primary antibody (i.e. either immune or non-immune serum) was

added and the incubation continued for an additional 2 hours.

Antiserum against PEPCK was provided by Daryl K. Granner and

that for RSA was purchased commercially. The exact quantity

of serum to be used was determined empirically and is stated

in the figure legends. Following this incubation, the

blocking buffer containing serum was poured into a plastic 50

ml centrifuge tube and stored for further use at -20C. The

blot was rinsed 4 times for a few seconds each time with PBS-

azide and then washed for 25 minutes in 70 to 80 ml PBS-

azide containing 0.3% (v/v) polyoxyethylenesorbitan

monolaurate (Tween 20). This was followed by 4 additional

rinses with PBS-azide as above. After rinsing, the blot was

incubated for 1 hour in 50 to 60 ml blocking buffer

containing secondary antibody. Detection of primary








86
antibody was accomplished by one of two methods:

colorimetric, using alkaline phosphatase-conjugated

secondary antibody; or autoradiographic, using 25I-coupled

secondary antibody. Secondary antibodies, purchased from

Sigma, were raised in donkeys using either sheep (for anti-

PEPCK primary antibody) or goat (for anti-RSA primary

antibody) IgG as antigen. Alkaline phosphatase-conjugated

secondary antibody was used at a 1:1,000 dilution and 25I-

coupled secondary antibody was used at 106 cpm/ml blocking

buffer. The procedure for coupling 125I to IgG is described

in a separate section below. Following incubation in

secondary antibody, blocking buffer containing alkaline

phosphatase-conjugated secondary antibody was discarded

while that containing 'I-coupled secondary antibody was

stored at 40C behind lead for further use. The blot was

washed with PBS-azide, PBS-azide-Tween 20, and PBS-azide as

described above. For immunoblots utilizing 12I-coupled

secondary antibody, the blot was placed on a dry paper towel

and allowed to dry in air at room temperature. The dried

blot was then placed between sheets of plastic wrap and

allowed to expose X-ray film (Kodak X-OMAT AR) at room

temperature for varying periods of time depending on the

strength of the signal. For alkaline phosphatase-conjugated

secondary antibody, the blot was rinsed 2 additional times

for a few seconds each time with 100 mM Tris, ph 8.8

containing 1 mM MgC1, (Tris-MgCl2). The colorimetric








87

substrate buffer was prepared by adding 5-bromo-4-chloro-3-

indolyl phosphate (BCIP) from a 100 mg/ml stock solution in

dimethylsulfoxide (DMSO) to Tris-MgCl2 containing 0.1 mg/ml

nitro blue tetrazolium (NBT). The final concentration of

BCIP was 0.125 mg/ml. Immediately after the addition of

BCIP, the colorimetric substrate buffer was poured over the

blot and the colorizing reaction was allowed to continue in

the absence of motion until specific bands had reached the

desired intensity; often in as little as 2 to 5 minutes but

in some cases as long as 2 hours. The reaction was

terminated by pouring off the colorimetric substrate buffer

and rinsing the blot 4 or 5 times with deionized water

(dH2O). The blot was then air dried on a paper towel, covered

with plastic wrap and photographed using Polaroid Type 55

Land film. The negative was treated for 5 to 10 minutes in

18% (w/v) sodium sulfate and then rinsed with several changes

of dH2O for at least 30 minutes.

Radioiodination of Secondary Antibodies

Radioiodination of secondary antibodies was performed

using the chloramine T method described by Greenwood et al.

(1963). Briefly, 0.2 to 0.3 mg of either anti-sheep or anti-

goat IgG (both from Sigma) were dissolved in 0.5 ml PBS-

azide in a screw-cap 1.5 ml Eppendorf microcentrifuge tube.

One millicurie of 25I, in the form of sodium ['25I]iodide (13

to 14 mCi/pg iodine), was added to the tube followed by the

addition of 10 Al of a chloramine T stock solution containing








88

2.5 mg chloramine T/ml PBS-azide. The tube was tightly

capped and rotated end-over-end for 30 seconds. The reaction

was stopped by the addition of 120 yg sodium metabisulphite

from a 6 mg/ml stock solution in PBS-azide. Unincorporated

radioactivity was removed using either a small, 5 to 8 ml bed

volume, Sephadex G-100 column or a 1 ml Sephadex G-50 (fine)

"spin column" as described below. Both the G-100 and G-50

were swollen in PBS-azide for at least 48 hours before use.

The G-100 column was prepared in a thick-walled glass Pasteur

pipette that had been plugged with a small quantity of glass

wool. Approximately 2 to 3 bed volumes of PBS-azide were

allowed to flow through the column which was then totally

submerged in PBS-azide, to prevent drying, until use.

Following the addition of the sodium metabisulphite, the

reaction mixture was applied to the top of the column bed,

allowed to flow into the bed, and then PBS-azide carefully

applied to the column. Fractions (10 to 15) of 0.5 ml each

were collected and the radioactivity contained in 10 Al

aliquots of each fraction monitored using a Searle Model 1185

gamma counter. The fractions in the void volume containing

the greatest amount of radioactivity were combined and stored

in a lead container at 4C. The spin column was prepared by

filling a 1 ml disposable syringe, plugged with glass wool,

with a slurry of Sephadex G-50 (fine) in PBS-azide and

centrifuging at approximately 100 x g for 45 seconds in a

table-top centrifuge equipped with a swinging-bucket rotor.








89
One-half milliliter PBS-azide was added to the packed bed

which was then centrifuged again and the flow-through

fraction discarded. The radioiodination reaction mixture

was then added and the column centrifuged as before. The

total flow-through fraction was collected and stored in a

lead container at 4C.

Aaarose Gel Electrophoresis and Northern Analysis

RNA samples to be investigated via Northern analysis

were first subjected to electrophoresis in a denaturing 1%

(w/v) agarose gel containing formaldehyde. The gel was

prepared by dissolving 1 g high-gelling-temperature agarose

in 80 ml ddH2O in a glass Erlenmeyer flask using a microwave

oven. Dissolution of the agarose was facilitated by first

stirring the agarose solution for 3 to 5 minutes using a

magnetic stir plate. The flask containing the melted agarose

was then placed in a 65C water bath to facilitate cooling to

this temperature. Formaldehyde (12 ml of a 37% (v/v)

solution) and 10x 3-[N-morpholino]propanesulfonic acid

(MOPS) buffer (10ml) containing 200 mM MOPS, pH between 5.5

and 7.0, 50 mM sodium acetate, and 10 mM EDTA were then added

and the mixture swirled gently. The agarose solution was

then poured into a 100 ml graduated cylinder and the volume

made to 100 ml, if necessary, with ddHO2. The solution was

returned to the flask, swirled gently again, and poured into

the slab gel mold and allowed to cool and solidify for at

least 30 minutes. The well-forming comb was carefully








90

removed from the cooled gel which was then placed into the

electrophoresis unit. Sufficient tank buffer, consisting of

Ix MOPS buffer, was then poured into the unit until the top

of the gel was approximately 0.5 to 1 cm under the surface.

RNA samples stored as pellets under 75% (v/v) ethanol were

dried under vacuum and dissolved, with heating at 65*C, in

DEPC-treated ddH2O. Ribonucleic acid was quantitated by

measuring the absorbance of light at 260 nm wavelength (1

absorbance unit at 260 nm = 40 pg RNA/ml). Measured aliquots

(5 to 30 gl) of the RNA solutions were then added to RNA

loading buffer such that the final total volume was 60 pl per

sample. Loading buffer was prepared by combining 0.72 ml

deionized formamide, 0.16 ml 10x MOPS buffer, 0.26 ml 37%

(v/v) formaldehyde, 0.18 ml ddHO2, 0.1 ml 80% (v/v) glycerol,

and 0.08 ml of a saturated solution of bromophenol blue in

ddHO (Davis et al., 1986). Formamide was deionized by

stirring for 30 minutes at 40C with AG 501-X8 mixed-bed resin

(Bio-Rad) using 5 g resin for 50 ml formamide. The solution

was filtered twice through Whatman No. 1 filter paper,

aliquoted, and stored at -700C. The RNA in the loading bufer

was denatured by heating at 65C for 15 to 20 minutes and

immediately cooled on ice. Following the addition of 1 pl of

a 1 mg/ml solution of ethidium bromide (EtBr) in ddH20 to each

sample, the samples were mixed gently and loaded into the

wells of the agarose gel. Electrophoresis was carried out at








91

a constant voltage of 30 V until the bromophenol blue had

migrated three-quarters of the length of the gel.

The gel was removed from the electrophoresis unit and

photographed under ultraviolet illumination using Kodak Type

55 or 57 film. Negatives (Type 55) were treated as

previously described. For transfer of the RNA to

hybridization membrane (i.e. Northern blot, based on the

Dupont/NEN protocol), the gel was first cleared of

formaldehyde by washing for 5 minutes in dH2O with changes of

water at 1 minute intervals. The gel was then slowly

agitated for 20 minutes in 50 mM NaOH, rinsed twice with

dH2O, and incubated for 30 minutes in 100 mM Tris, pH 7.0.

The gel was then laid, face down, on a fully wetted piece of

Whatman 3MM chromatography paper, the ends of which were

submerged in 10x SSPE (20x SSPE contains 3 M NaCl, 20 mM

EDTA, and 200 mM sodium phosphate, pH 7.4). The gel was

overlaid with GeneScreen (NEN) nylon hybridization membrane

that had previously been hydrated in dHO2 for 1 minute and

then soaked in 10x SSPE for 15 minutes. Bubbles were removed

from between the gel and the GeneScreen by rolling a glass

stir rod back and forth over the membrane. The GeneScreen

was overlaid with two lOx SSPE-saturated sheets of 3MM paper

followed by 2 dry sheets. A 10 to 15 cm stack of paper towels

was laid on top and compressed slightly with a mass of

approximately 400 g on a sheet of glass. Transfer of RNA to

the hybridization membrane was verified the next day, usually