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cztclrcmma-pn

TRANSCRIPTION ASSAY DEVELOPMENT WITH
NUCLEI ISOLATED FROM PORCINE SKELETAL MUSCLE
BY

Chun-hsiang Chang

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1990

ABSTRACT
TRANSCRIPTION ASSA! DEVELOPMENT HITS
NUCLII ISOLATED IRON PORCINS SNELETAL NUSCLE
BY

Chun-heiang Chang

A system for isolation of nuclei from porcine skeletal
muscle (SH) and for in_xi§zg RNA transcription was
established in order to investigate the regulatory mechanism
of developmental changes in SM protein. In addition, SM
alpha-actin and beta-tubulin mRNA abundance for pigs of
different ages were determined by Northern blot analysis.

SM nuclei were isolated from longissimus dorsi muscle (LD)
of 1- and 28-day old pigs by using a modification of a
method used to isolate nuclei from cardiac muscle (Surk et
al.,1974). Results from the tritiated UTP incorporation
assay indicate that these nuclei preparations have the
capacity to synthesize RNA and attain maximum incorporation
after 40-45 minutes. SM nuclei from l-day-old pigs
synthesized more RNA than SM nuclei from 28-day-old, but the
difference was not significant (P>0.25). The appropriate
condition of the transcription assay was determined to be
3x107 nuclei in a 400 ul assay volume. All nascent tRNA,

rRNA and mRNA in the nuclei were elongated since [3H1-UTP

incorporation was reduced after addition of 0.05 ug/ml
alpha-amanitin in the transcription mixture. Transcription
assay results indicated that more (P<0.02) SM alpha-actin
hnRNA was synthesized in the 28-day-old pig SM nuclei than
in the l-day-old pig SM nuclei. Northern blot analysis
indicated that there are developmental increases in alpha-
actin mRNA (P<0.03) and no changes in beta-tubulin mRNA
(P>0.1) from one- to twenty eight-day-old pigs. A
decreasing trend of beta tubulin was actually observed.
These results indicate that the relative increase in SM
alpha-actin mRNA observed was due, at least in part, to an
increase in the transcriptional activity of the SM alpha-

actin gene.

This is dedicated to my family.

iv

ACKNOWLEDGMENTS

I would like to thank the department of Food Science
and Human Nutrition and the College of Agriculture and
Natural Resources for their support during my degree
program. I wish to express my sincere gratitude to Dr. W.G.
Helferich for his support and guidance as major professor,
and for his patience, Understanding and friendship
throughout the research program.

Appreciation is extended to Dr. W.G. Bergen, Dr. R.A.
Merkel and Dr. G.M. Strasburg for serving on my graduate
committee as well as for their support and encouragement of
this research. I wish to thank Dr. A.L. Grant for his
assistance and technical advice with this research project.
I am thankful to have the opportunity to work with him. I
would like to thank Dr. J.L. Gill for his helpful
consultation of statistical analysis. Also, I would like to
thank a number of faculty, staff and graduate students that
have helped me during my degree program.

Finally, my deepest gratitude to my family and my wife,
Annie, for their continual love and support of this. I wish
to thank Annie's patience, understanding and consulting in

the statistical analysis.

TABLE OF CONTENTS

Page
LIST OF TABLESoooooooooooooooo0.0000000000000000.oeoooeoeVii
LIST OF FIGUREOOOOOOOOOOOOOOOOOOOOOOOOOOZOOOOOOOOOOOOOOOViii
LIST OFAPPENDICESOOCO00.00.0000...0....OOOOOOOOOOOOOOOOOOXi
LITERATURE REVIEW (Introduction)...........................1
General information on regulation of gene

transcription in eucaryotes.......... ..... ....1

General information on regulation of skeletal
muscle protein synthesis......................9
MTERIAE ANDMTHODSOOOOOOOOOCOOOOO00......0.00.00.00.00018

Background for methods utilized.......................18

Materials and methods............................ ..... 24
(1) Relative abundance of alpha-actin and
beta-tubulin mRNA in LD muscle...................24
Tissue sample collection and RNA extraction.....24
Northern blot analysis..........................26
Statistical analysis............................28
(2) Transcription assay..............................28
Tissue sample collection and nuclei isolation...28
Tritiated UTP Incorporation assay...............29
Statistical analysis............................3l
Transcription in vitro..........................34

RESULTS...................................................36
DISCUSSION................................................55
CONCLUSION................................................59
APPENDICES................................................60

LITERATURE CITEDOOOOOOOOOOOOOOOOOO0.00.0.0... ..... 00......74

vi

Table
Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES
Page
Characteristic of eucaryotic RNA polymerase.......3
Summary of RNA modification and processing. ....... 6

Composition of transcription mixture for
tritiated UTP incorporation assay....... ......... 32

Relative abundance of alpha-actin and

beta-tubulin mRNA among 1-day-old, 28-day-old

and adult pigs. Data was obtained by

densitometry scan of autoradiograms in

figure 4.........................................38

Relative abundance of alpha-actin mRNA and
beta-tubulin mRNA in LD muscle from 1-day-old

and 28-day-old pigs. Data were obtained by
densitometry scan of autoradiograms shown in

figure 5........................... ........ . ..... 40

Total RNA synthesized by isolated skeletal

muscle nuclei. Tritiated UTP incorporated

into RNA (tritium incorporation assay) was
conducted as described in Materials and

Methods. Data are expressed as CPM X 103

for each time point..............................44

ANOVA of tritiated UTP incorporation assay.
The experimental design is a split-split-plot
with repeated measurements design................46

Effects of nuclei concentration and

sampling time on total RNA synthesized

in vitro. Data are expressed as CPM X 10

for each sampling time point.....................48

Relative abundance of alpha-actin hnRNA
synthesized by l-day-old and 28-day-old porcine

SM nuclei. Data are expressed as densitometry
unit from the autoradiograms shown in

figure ll............................... ......... 54

vii

Figure

Figure

Figure

Figure

1.

2.

LIST OF FIGURES

Page

Schematic representation of the structure

of skeletal muscle fibers. This figure

was taken from "Muscle as Food” 1986

(Edited by Dr. P.J. Bechtel). The location

of skeletal muscle nuclei is beneath the
sarcolemma......................................11

A general scheme outlining cellular processes
involved in muscle growth. Muscle protein
accretion is a function of intracellular protein
synthesis and protein degradation, and myogenic
cell proliferation encompasses processes involved
in the replication of muscle precursor cells in
prenatal and postnatal muscle (satellite cells)
(From Allen, Merkel, and Young, Michigan State
University, J. Anim. Sci. 49, 116)..............12

Schematic picture of nuclei separated from

other cellular organelles after
ultracentrifugation at 105,500 g for 80 minutes
with 2.4 M sucrose buffer.......................30

Autoradiogram of alpha-actin (A) and
beta-tubulin (B) mRNA abundance in LD muscle

from 1-day-old, 28-day-old and adult pig.

RNA was isolated and analyzed via a Northern
blot. The mouse MBS tubulin cDNA hybridized

to mRNA species of 1800 and 2800 nucleotide (B),
then the blot was stripped with 0.01x SSC and
0.1% SDS for 2 hours at 70°C and

re-hybridized with a human alpha-actin cDNA.

A single band of 1650 nucleotide was observed (A).
Northern blot of beta-tubulin (B was washed
with 0.2x SSC and 0.1% SDS at 55 C and

exposed for 24 hours. Northern blot of
alpha-actin was washed with 0.1x SSC and 0.1% SDS
at 65°C and exposed for 30 minutes.

Lane 1: 10 ug 28-day-old pig LD RNA, lane 2:

20 ug 28-day—old pig LD RNA: lane 3: 10 ug
1-day-old pig LD RNA, lane 4: 20 ug 1-day-old
pig LD RNA: lane 5: 10 ug adult pig LD RNA,

lane 6: 20 ug adult pig LD RNA. These data

viii

are preliminary data............................37

Figure 5. Autoradiograms of alpha-actin and beta-tubulin
mRNA abundance in LD muscle from four 28-day-old
and four 1-day-old pigs. Each lane contains
10 ug of RNA isolated from LD muscle from
each animals. Animals are number 1 to 4 for
each age group evaluated. The human alpha-actin
cDNA hybridized to mRNA species of 1650
nucleotide (A). The Northern blot of alpha-actin
was washed with 0.1x SSC and 0.1% SDS at 65° C
then exposed for 30 minutes. The mouse M35
tubulin cDNA hybridized to mRNA species of
1800 and 2800 nucleotide (B), Northern blot of
beta-tubulin (B) was washed with 0. 2X SSC and
0.1% SDS at 55° C then exposed for 24

hourSIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.039

Figure 6. Northern blots of alpha-actin and beta-tubulin
stained with methylene blue to visualize the
18S and 288 ribosomal bands.....................42

Figure 7. Photograph of nuclei (400x magnification) using a
phase-contrast microscope. Nuclei were isolated by
differential centrifugation as described in
Materials and Methods...........................43

Figure 8. Effects of age and nuclei concentration on
transcription in vitro (Tritiated UTP
incorporated into RNA). Incorporation assay
was conducted as described in Materials and
Methods. Skeletal muscle nuclei isolated from
l-day-old and 28-day-old pig LD muscle and
3 different nuclei concentrations total
nuclei number equal to 3X10 2X10 and 1X107
nuclei per 400 ul assay volume)
for each age group were evaluated...............45

Figure 9. Effects of nuclei concentration and sampling time
on total RNA synthesized in vitro. Polynomial
curves were calculated by Stepwise-regression.
Equation of 1X10 nuc ei is
Y=0.748+0.444X-0 005x and R Square=0.976.
Equation of 2X10 nuc ei is

=2.27O+0.69lX-07007X and R Square=0.909.
Equation of 3X10 nuc ei is
Y=l.873+0.855X-0.008X and R Square=0.973.
Tritium incorporation is represented by Y
and sampling time is represented by X.
The maximum of Y for equation 1 and 2 is
45 minutes and the maximum of Y for
equation 3 is 40 minutes........................49

Figure 10. Effect of alpha-amanitin inhibition on

ix

Figure 11.

transcription in vitro was evaluated as
described in Materials and Methods.

Experiments were conducted using nuclei

isolated from 28-day-old pig LD muscle at

nuclei concentration equal to 3X10

in a 400 ul assay volume.......................51

Autoradiograms of alpha-actin hnRNA abundance
and beta-tubulin hnRNA abundance synthesized

by l-day-old and 28-day-old pig SM nuclei.

RNA was synthesized and isolated via a
transcription assay as described in

Materials and Methods. Plasmid pBR 322

was included as a negative control. Panel A,

B, and C each represent analysis of

1- and 28-day-old pigs.........................53

Appendix

Appendix
Appendix
Appendix

Appendix

LIST OF APPENDICES
Page
Acid guanidine phenol chloroform RNA
extraction method.............................60
Northern blot analysis........................63
Skeletal muscle nuclei isolation protocol.....66

Tritiated UTP incorporation assay protocol....69

Transcription assay in yit;g..................70

xi

LITERATURE REVIEW
In this study, we are focusing on the transcriptional
regulation of muscle protein synthesis using Northern blot
analysis and a transcription assay in 21:22. Therefore, the
following concepts are described in this introduction:
1. General information on transcription in eucaryotes.
2. General information on regulation of skeletal muscle

protein synthesis.

1. EEEEBAL IEIQBHAIIQ! Q! BEQELAIIQE 92 Q!!! TBANEQBIEIIQ!
IN_EEQAB¥QIE§

DNA is the hereditary material which replicates and is
transferred to progeny with high fidelity. In the nucleus
of all eurcaryotic cells, DNA serves as a template to be
transcribed to primary RNAs by RNA polymerases. These
primary RNAs undergo post-transcriptional processing and
modification by various nucleases, ligases and other
modifying enzymes. Following the post-transcriptional
changes, these transcripts become mature messenger RNA
(mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). The
mature transcripts are then transported out of the nucleus
to the cytoplasm, and ribosomal complexes composed of mRNA,
tRNA, rRNA and ribosomal protein are assembled. These

complexes function to translate codons into their

2
corresponding polypeptides. These polypeptides are modified
by post-translational processing and fold to become
functional proteins.

The transcriptional process includes initiation,
elongation and termination. The major difference in
transcription between eucaryotes and procaryotes is that
transcription of mRNA, tRNA and rRNA in procaryotes is
performed by a single RNA polymerase, but in eucaryotes
there are multiple forms of RNA polymerase, each responsible
for the transcription of a different RNA species. General
characteristics of eucaryotic RNA polymerases are summarized
in Table 1. RNA polymerase II has at least four subunits to
form a pentameric holoenzyme which can carry out mRNA
syntheses, and unlike procaryotic RNA polymerase, it does
not recognize the promoter on the DNA sequence by itself.
There are different transcription factors (TFs) required for
promoter selection to begin the initiation process of
transcription. These TFs are involved in initiation and
regulation of transcription. TFs can be divided into two
classes based on their functions, one composed of general
factors that are required for the transcription of all genes
transcribed by polymerase II, and the other class of
sequence specific factors that are required for optimal or
enhanced transcription of only one gene or subset of genes
(Manley et al., 1980; Berk et al., 1990: Tjian et al.,
1990).

In order to achieve basal level transcription from

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4
polymerase II in 2119, promoters require at least four

distinct general TFs for activity. These general TFs
include TFIIA, TFIID, TFIID, and TFIIE/F (Roeder et al.,
1988a). The first step in the assembly of the transcription
complex at the promoter is when TFIID binds to the TATA box
promoter element. This complex then regulates the
expression of most eucaryotic genes transcribed by
polymerase II (Sharp et al., 1989). DNA binding studies
suggest that several transcription factors might contact
TFIID (Roeder et al., 1985, 1988a, 1988b, 1988c). After
TFIID binds to the promoter TFIIA then binds to the
promoter, followed by the incorporation of TFIIB, RNA
polymerase II and TFIIE/F into the initiation complex. This
complex then functions to direct the basal level of
transcription (Greenblatt et al., 1990: Roeder et al., 1990:
Tjian et al., 1989, 1990). TFIID is also thought to be the
binding site for several upstream transcription factors
(enhancers) such as USF, ATF,and GAL4 (Roeder et al., 1988b,
1988c). However, the relationship between upstream .
regulators and TFIID remains unclear. Recently, Ptashne
and Gann (1990) indicated that in addition to TFIID which is
now defined by the cloned gene (Pei et al. 1990), the
chromatographic fraction of TFIID contains some component or
components, all of which are necessary for a response from
any activating factors. Ptashne and Gann (1990) also
concluded that all activators (upstream regulators) exert

their effects by interacting with TFIID. Acidic activators

5

work directly and hence universally, whereas other
activators such as 0ctl, SP1, Ela, perform activation via
intermediaries bearing the acidic activating regions. Walsh
and Schimmel (1987) conducted DNA-binding assays to indicate
that two nuclear factors compete for the chicken skeletal
muscle actin gene. Their conclusion was that one complex is
ubiquitous and the second appears to be controlled in a
cell-type-specific manner.

Many characteristics of RNA differ between eucaryotes
and procaryotes. Almost all the major types of RNA
synthesized by cellular DNA-dependent RNA polymerase undergo
changes before they can carry out their functions. Two
types of changes are usually distinguished. The first is
modification, which involves additions to or alterations of
existing bases or sugars. The other is processing, which
involves phosphodiester bond cleavage and loss of certain
nucleotides (Burgess et al., 1988). All three classes of
RNA (mRNA, tRNA and rRNA) have precursors in eucaryotes:
however, in procaryotes the mRNA is a mature transcript, and
ready for translation. Only the rRNA and tRNA have
precursors. These precursors must undergo modifications and
processing before they become functional types of RNA. A
summary of RNA modification and processing is presented in
Table 2.

The precursor of eucaryotic mRNA, heterogeneous nuclear
RNA (hnRNA), is synthesized by RNA polymerase II. The early

kinetic labeling experiments to determine the relationship

 

 

 

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7
between hnRNA and mRNA were ambiguous and indicated that not
more than 10-20% of hnRNA of higher organisms migrates to
the cytoplasm after processing (Lewin et al., 19753, 1975b).
These data suggested that degradation of hnRNA occurs in the
nucleus and the majority of the degradation products return
to an acid-soluble pool (Houssais and Attardi, 1966: Warner
et al., 1966). Due to difficulties encountered in
conducting effective pulse-chase experiments and the
inability to fractionate hnRNA from mRNA, the studies on
relationship between hnRNA and mRNA have resulted in
conflicting data (Naora, 1977: Adams et al., 1986). The
estimated half-life of hnRNA varies greatly, depending on
the species, cell types and the method of measurement. For
instance, Gefter and Mory (1977) state that hnRNA is
characterized by a high turnover rate, 5% of the genome
corresponds to hnRNA and about 35% of hnRNA undergo post-
transcription processing to mature mRNA.

It is now established that eucaryotic mRNA is the
product of the maturation of longer precursors which arises,
at least in part, from the high molecular weight nuclear RNA
known as the hnRNA. There is a several fold difference in
the size of hnRNA and mRNA. HnRNA can have a length of up
to 50,000 base pairs (bp) whereas mRNAs, in general, are not
larger than 7,000 bp. Almost all mammalian mRNAs fall
within the size range of 400 to 4,000 nucleotides. In order
to become mature mRNA, eucaryotic hnRNA must be altered by a

process composed of three main steps: 5'-capping, 3'-

8

polyadenylation and the removal of introns. CAP addition
occurs in the nucleus immediately after the 5'-end of the
polyribonucleotide chain has been synthesized. It is an
addition of a guanylate (GMP) through a 5'-5’ triphosphate
linkage to the first nucleotide, with methylation on the N7
position of the additional guanosine to become CAP 0
(”zero"). Further methylation on the 2'-0 position of the
ribose of the first two nucleotides become CAP 1 and CAP 2,
respectively (Adams et al., 1986). Polyadenylation occurs
when polymerase II proceeds to the cleavage signal (AAUAAA)
and a specific ribonuclease that can recognize the signal
then cleaves 10 to 30 nucleotides downstream of this signal.
Poly A polymerase catalyzes the addition of a 200 to 250
adenylate (AMP) residues to the 3' end. This poly A
polymerase does not require a DNA template to synthesize the
poly A tail (Burgess et al., 1988, Edmonds, 1990).

Intron excision and splicing involves a RNA-protein
complex called a spliceosome. This protein-RNA complex
composed of five small nuclear RNAs (snRNAs), four small
nuclear riboprotein complexes (snRNPs) called U1, U2, U5 and
U4/6, is assembled with hnRNA. After assembly of the
spliceosome, the reaction occurs in two steps: the first
step is lariat formation and cleavage at the 5' splice site
of intron and the second step is cleavage at the 3’ splice
site, and subsequent exon ligation (Burgess et al., 1988:
Mattaji et al., 1990: Siebel and Rio, 1990: Brow and

Guthrie, 1990). The whole process occurs in the nucleus,

9

starting with capping addition, followed by initiation of
intron excision and splicing, and then addition of the poly
A tail and completion of intron excision and splicing.
Mature mRNA is transported from the nucleoplasm to the
cytoplasm where the mRNA is translated into protein on
ribosomes. The abundance of mRNA, in general, is about 2-5%
of total RNA which is the lowest abundance of the three
types of RNAs. The variety of different mRNAs, however is
the greatest of all the three types of RNA. That is due to

a triple code required for one amino acid signal.

2. SENEBAL IEIQBNAIIQN Q! BEE!LAIIQE:QI.E§ELEIAL NHEQLB

.BBQIEIN BIEIEEEIE
Thick and thin myofilaments make up myofibrils in

skeletal muscle fiber. 0n the surface of a fiber there is a
lipoprotein bilayer, and nuclei are located along the muscle
fiber underneath the plasmalemma called sarcolemma.
Satellite cells are located between sarcolemma and basal
lamina (basement membrane). Basement membrane is composed
of proteoglycan and belonged to connective tissue (collage
type 4). The sarcolemma of individual muscle fibers is
surrounded by the endomysium, which is a delicate connective
tissue. Approximately 20-40 muscle fibers, and the
associated endomysium, are grouped into primary bundles.
Several primary bundles are grouped together to form larger
bundles known as secondary bundles. Both primary and

secondary bundles are ensheathed in the perimysium, which is

10

a sheath of collagenous connective tissue. Finally, several
secondary bundles are grouped together to form a muscle,
which is itself contained in a connective tissue sheath
called the epimysium (Judge et a1. 1989). The cellular
structure and composition of skeletal muscle presents
inherent difficulty in isolating viable nuclei (Figure 1).
Muscle cell growth is a combination of muscle protein
accretion and myogenic cell proliferation. Myogenic cell
proliferation includes embryonic cell proliferation and
satellite cell proliferation. Muscle protein accretion is
the net difference between protein synthesis and protein
degradation (Figure 2). A complete discussion on muscle
cell growth is beyond the scope of this review. In this
study we are focusing on muscle protein synthesis
specifically at the level of transcription (Figure 2).
Protein accretion in postnatal muscle growth results
from muscle hypertrophy which is synthesis of muscle
specific protein. Muscle hypertrophy includes increases in
myofiber length which involves an increase in sarcromere
length, as well as sacromere number. The synthesis of
muscle specific protein involves the synthesis of myosin
heavy chains, myosin light chains and several isoforms of
actin (Minty et al., 1981, 1982). Generally, from birth to
maturity, muscle cell proliferation is low and myofiber
number only increases slightly but nuclei number and total
DNA increase 5- to lO-fold (Allen et al., 1979): however,

DNA and RNA concentration (mg/g) decreases with increasing

ll

 

mum

 

 

Imam

 

 

 

 

 

 

 

Figure 1. Schematic representation of the structure of
skeletal muscle fibers. This figure was taken from "Muscle
as Food" 1986 (Edited by Dr. P.J. Bechtel). The location of
skeletal muscle nuclei is beneath the sarcolemma.

12

MUSCLE CELL GROWTH

 

MUSCLE PROTEIN MYOGENIC CELL

 

 

ACCRETION PROLIFERATION
PROTEIN PROTEIN EMBRYONIC CELL ’SATELLITE CELL
SYNTHESIS DEGRADATION PROLIFERATION PROLIFERATION

Figure 2. A general scheme outlining cellular processes
involved in muscle growth. Muscle protein accretion is a
function of intracellular protein synthesis and protein
degradation, and myogenic cell proliferation encompasses
processes involved in the replication of muscle precursor
cells in prenatal and postnatal muscle (satellite cells)
(From Allen, Merkel, and Young, Michigan State University,
J. Anim. Sci. 49, 116).

13

age.

Using a cell-free translation assay, Devlin and Emerson
(1979) demonstrated that the coordination of protein
synthesis in culture was regulated at the level of mRNA.
Hastings and Emerson (1982) demonstrated that gene sets in
muscle development suggested by cloned cDNA should reveal
whether transcriptional or post-transcriptional mechanisms
determine contractile-protein mRNA level. Shani et al.
,(l981) have shown using well characterized cDNA probes to
skeletal muscle alpha-actin, myosin heavy chain (MHC), and
myosin light chain (MLC) that there is a strong correlation
between the abundance of mRNA and the synthesis of these
proteins during myogenesis in rat primary skeletal muscle
cell cultures. These data suggest that a common mechanism
may regulate gene expression in muscle based on coordinated
regulation of muscle gene sets. Furthermore evidence for
transcriptional regulation of muscle genes as a primary
control was provided by Affara et a1. (19803).by comparison
of the DNAse 1 hypersensitive site between myoblasts and
myotubes in muscle cell culture. An open chromatin
structure, sensitive to the activity of DNase 1, is
correlated with the potential for a gene to be transcribed.
Further indirect evidence for transcriptional regulation was
provided by transfecting a putative sequence of muscle genes
linked to a reporter gene such as CAT (chloramphenicol
acetyl transferase) into myoblasts or myotubes in culture

(Buckingham, 1989).

14

Medford et al. (1983) conducted a transcription assay
with nuclei isolated from rat L6E9 muscle cell culture using
a nonspecific myosin heavy chain probe to show that
regulation is at the transcription level. However, the
mechanism(s) by which synthesis of different proteins
characteristic of muscle is turned on and off during
myogenesis and during further maturation of muscle fibers
remains to be determined. Factors which regulate the
specific initiation, elongation, termination and subsequent
processing of muscle-characteristic RNAs and the mechanisms
that control the activation of muscle gene expression are
also unclear. Since muscle geneactivation during
development results in the accumulation of specific mRNAs,
Pearson and Epstein (1982) had suggested that the regulation
of these genes occurs primarily at the level of
transcription. Pearson and Epstein (1982) indicated that
knowledge of the processing of nascent transcripts is also
essential for understanding of active mRNA production.

The current studies will focus on the molecular
mechanism(s) related to protein synthesis of SM alpha-actin
which has been used as a myofibrillar protein marker.
Alpha-actin represents 22% of the myofibrillar proteins in
skeletal muscle (Yates et al., 1983). Skeletal muscle
alpha-actin (Garfinkel et al., 1982; Helferich et al. 1990:
Zakutetal et al., 1982) and myosin light chain (Garfinkel et
al., 1982: Smith et a1. 1989) has been used successfully as

markers of myofibrillar proteins in skeletal muscle.

15

Although myosin accounts for 43% of myofibrillar proteins
(Yates et al., 1983) there are up to 12 isoforms
(BuCkingham, et al., 1984, 1985: Humphries et al., 1981)
which originate from different genes. Furthermore, the rate
of expression of these genes may be altered the rate of
expression during the differentiation of myoblasts to
myotubes in the embryo. Thus, alpha-actin was selected over
myosin as a marker of SM myofibrillar proteins accretion in
growing, food-producing animals for several following
reasons. First, actin is the second-most abundant protein
in muscle representing 22% of myofibrillar proteins.
Secondly, there is only one isoform of alpha actin (Barton
et al., 1987: Caravatli et al., 1982: Ponte et al., 1983) in
postnatal skeletal muscle originating from one gene in the
species studied (,unlike the 12 isoforms of the multigene
myosin family). Finally, actin is an excellent marker of
myogenesis during growth and development and has been
demonstrated to be an early indicator of myogenesis
(Vandekerckhove et al., 1978, 1979, 1987: Sassoon et al.,
1988) in both skeletal and cardiac muscle. Since
accumulation of muscle proteins is a highly synchronous
process (Affara, et al., 198°: Obinata et al., 1981),
following the synthesis and gene expression of a single
marker (SM alpha-actin) will permit the determination of
metabolic events in the assembly of myofibrils in muscle.

Bergen et a1. (1987) have conducted experiments to

determine general changes in fractional synthesis rate (FSR)

16
of total muscle protein, total RNA, and total DNA during
development in growing pigs. FSR (defined as the percent of
the total muscle protein pool synthesized per unit time)
decreases from 20% per day in the neonate to 3% pedeay in
the mature rats. These results are consistent with data
obtained in young and old male rats (Waterlow et al., 1978).
Fractional synthesis rate (FSR) of total protein is
approximately 12% in the neonate (3d of age) and 3% in the
mature (180d of age) pig mainly due to an increase in the
muscle protein pool size. However, early results from our
laboratory indicated that relative mRNA abundance for SM
alpha-actin, increases 3- to 4-fold during this same period
(skjaerlund et al 1988). In these investigations
concentration of RNA and DNA decreased with age, whereas
RNA:DNA ratios remained similar regardless of age. Muscle
cell proteins (mostly myofibrillar proteins) per unit of DNA
increases proportionally as muscle mass increases. Since
FSR is calculated based on pool size, it is important to
distinguish between FSR (% of pool size/day) and absolute
protein synthesis rate (g/d). The myofibrillar proteins
(including SM alpha-actin) pool size is much greater in
older animals due to increased muscle mass. It is likely
that absolute synthesis rates of individual myofibrillar
proteins may not follow the general FSR described above. In
fact, absolute synthesis rates of myofibrillar proteins
including alpha-actin may increase as the animal grows from

1 day to 28 days of age to support the accretion and

17
turnover of protein in this large protein pool. In view of
the large quantities of myofibrillar protein mRNA which must
be transcribed in the muscle cell nucleus, Young and Allen
(1979) pointed out that these muscle nuclei possess a
mechanism whereby RNA polymerases have a preference to
interact with the myofibrillar protein genes to increase
their transcription. In this study, we focus on regulation
at the transcriptional level of muscle protein synthesis to
investigate the relative transcription rate of certain genes

by using skeletal muscle nuclei to conduct a transcription

assay in 2.1m-

MATERIALS AND METHODS
The first portion of the materials and methods section

of this thesis will discuss the background for the methods
used in this thesis.
I. EASEQBQEED 293 NEIEQD§.!IILIZED

Isolation of RNA is essential for both Northern blot
analysis and transcription assay. During isolation RNA must
be protected throughout the procedure from nuclease
degradation, strong shearing force, high temperature,
excessive alkalinity and acidity, and excessive ionic
strength. Enzymic degradation can be minimized by
performing all operations between 0° and.4°, and by adding
ribonuclease inhibitors (Chirgwin et al., 1979).
Ribonuclease activity can be minimized during the isolation
process by including a suitable inhibitor such as macaloid,
heparin or some anionic detergent. The secondary structure
of RNA is dependent on total ionic strength and pH of the
extraction solution. When the ionic strength is low many
RNA species undergo a certain degree of denaturation,
whereas high ionic strength results in aggregation and
precipitation of RNA.

Guanidinium, sodium sarcosinate, phenol, chloroform
and sodium dodecylsulfate (SDS) all function as dissociating

and deproteinizing agents during the dissociation and

18

19

deproteinization to release RNA. A dissociating agent
usually is an anionic detergent which is used to lyse
organelles and release RNA from protein and lipoprotein
structure, as well as to inhibit ribonuclease activity.
Deproteinizing agents can precipitate proteins that have
been released from the protein-nucleic acid complex. RNA is
further purified by ethanol precipitation at pH between 5 to
6 (Woo et al., 1975: Poulson, 1977: Wallace, 1987).

Northern blot analysis is a process by which the
isolated RNA is separated by size on a denaturing agarose
gel and transferred to a solid phase such as a
nitrocellulose membrane. The RNA of interest is then
hybridized to a radioactive-labeled single strand of cDNA.
The basic concept involved in Northern analysis is that the
denaturation of these nucleic acids (RNA and cDNA) is
followed by annealation between complementary polynucleotide
sequence. One of the simplest ways to denature nucleic
acids is by heating. The extend of denaturation at any
temperature can be readily measured by the change of in
absorbance at 260 nm of a solution of nucleic acids. The
absorbance of a DNA solution remains constant until an
elevated temperature is reached. A substantial rise in
absorbance (hyperchromic shift) accompanies the transition
from native to denatured state. At the denaturing
temperature the absorbance is increased approximately 40%
over a narrow range. This rise in temperature coincides

with the disruption of the regular base-paired structure,

20

and the two polynucleotide strands separate from one
another. The sharpness of the disruption of the regularly
hydrogen-bonded base-paired native structure may be caused
by the melting point of a pure organic compound (Marmur and
Zubay, 1988). The melting temperature (Tm) of DNA is
defined as the temperature at the midpoint of the absorption
increase. Also at this temperature, the double helix
retains 50% of the original helical structure. The midpoint
of denaturation (Tm) of naturally occurring DNAs is
precisely correlated with the average composition of the
DNA: the higher the mole percent G-C base pairs, the higher
the Tm. This is because the G-C base pair contains three
hydrogen bonds, whereas the A-T base-pair contains only two
bonds. The Tm is directly proportional to G-C mole percent
by the following equation (Doty et al., 1962):

Tm= 69.3+ 0.41 (G+C)

Nucleic acid hybridization takes place between any two
complementary single-stranded polynucleotides.when they are
annealed at Tm-25°C for a prolonged period of time to form
heteroduplexes. This is an annealed duplex structure which
does not show perfect complementarity. Since the rate of
duplex formation depends on the concentration of the
interacting complementary strands, hybridization can be used
to measure the abundance of a specific nucleic acid in a
mixture (Southern, 1979: Marmur and Zubay, 1988). In
Northern blot hybridization, RNA is separated by agarose gel

electrophoresis under denaturing conditions, immobilized

21

onto nitrocellulose membrane and is then hybridized to a
particular radioactively labeled single-stranded probe. The
membrane is washed under suitable conditions which depend on
the degree of homology between the two strands of the
heteroduplex. The homology need not be 100% as
hybridization may be between a variety of related but not
identical sequences. After hybridization, the washing is
usually performed 2 to 12°C below the Tm, and the effect of
ionic strength (u) on the melting temperature is given by
the following equation (Davidson et al., 1962):

Tmz' m1 ‘13°5 1°910(“2/“1)
Thus, to allow for the fact that the Tm of duplex nucleic
acid decreases by 1°C per 1-1.5% mismatch, the stringency of
the hybridization can be decreased by lowing the temperature
of the wash and/or by increasing the ionic strength of the
washing buffer (Adams et al., 1986). These blotting
techniques can be used to determine the size and abundance
of immobilized RNA.

The transcription assay is generally used to measure
the relative transcription rate of specific genes. This
assay is performed using isolated nuclei since the nucleus
is the major site for the regulation of transcription.
Nuclear RNA is synthesized by incubating nuclei in the
presence of radiolabeled UTP, since UTP is only incorporated
into RNA but not DNA. The nuclear RNA is purified and

hybridized to specific gene sequences which have been

immobilized on a membrane such as nitrocellulose. In this

22

in 21519 system, RNA synthesis consists of enlongation and
completion of previously initiated RNA molecules with a
negligible amount of new initiation (Reeder and Roeder,
1972). Therefore, the transcriptional reaction in this in
213:9 system is the completion of polyribonucleotide
synthesis. Within the isolated nucleus the chromatin (the
nucleoprotein fibers of eucaryotic chromosome) is maintained
in its native state. Nuclei are incubated with all four
ribonucleoside triphosphates, one of which is radiolabeled
in the transcription mixture which contains all the
essential components for RNA synthesis. The amount of a
particular RNA can be quantitated by RNA-DNA hybridization
using appropriate DNA probes. The most convenient method is
to immobilize the DNA probes as dots on a nitrocellulose
membrane. The membrane containing immobilized DNA is then
incubated in a hybridization solution with the radiolabeled
RNA transcripts. After washing to remove nonspecific
hybridization, the amount of hybridized RNA is determined
either by autoradiography followed with densitometer scan or
by excising the radiolabeled spots and quantifying by liquid
scintillation counting.

There are certain points which need to be clarified,
first is that the supercoiled plasmid DNA will not
efficiently bind to nitrocellulose, thus plasmid DNA
containing the specific cDNA sequence must be linearized
using an appropriate restriction enzyme. Second, the DNA

probe should be at least 1.0 Kb in size to ensure

23

quantitative binding to the nitrocellulose membrane. Third,
the amount of cDNA on the nitrocellulose membrane must be in
excess. It is essential that the amount of RNA hybridized
should not be limited and the quantity of DNA must be in
molar excess. In general, 5 ug plasmid containing of DNA
insert of interest/dot or 1 ug of excised DNA insert/dot is
sufficient to assure that the quantity of DNA is not
limiting. Finally, the plasmid DNA lacking the cDNA
sequence of interest must be used a negative control to
monitor for nonspecific binding (Marzluff and Huang, 1984).
The isolation of nuclei consist of disruption of cell
structure under relatively isosmotic conditions in the
presence of divalent cations to stabilize the nuclei. The
major modification we made is the physical strength used to
break down muscle structure. After several low speed
centrifugation and washing steps, nuclei are recovered by
ultracentrifugation through a cushion of sucrosefof high
density. Nuclei are the most dense of the organelles in the
cell and will pellet through the sucrose cushion (Gornall et
al., 1972; Surks et al., 1974: Liew et al., 1980, 1983:
Oppenheimer et al., 1981, 1987: Marzluff and Huang, 1984:
Hanson et al., 1985: Jump et al., 1988). This allows for
separation of nuclei from other cellular organelles and

debri.

24

II- IAIBEIAL§.AND EEIEQDS

(1) Relative abundance of alpha-actin and beta-tubulin

IRNA in LB muscle

ANIMAL: Four 1-day-old pigs and four 28-day-old pigs
were selected from the Michigan State University swine
research center. Animals were immobilized and then
slaughtered by exsanguination and approximately 3 grams of
longissimus dorsi (LD) muscle were collected from each pig,
quickly frozen by submerging in liquid nitrogen, and stored
at -80°C until RNA extraction.

333 extrggtign Total RNA was extracted from LD muscle
by a method modified from the acid guanidinium thiocyanate-
phenol-chloroform extraction procedure of Chomczynski et al.
(1987). Briefly, 1 9 muscle was homogenized using a
Polytron homogenizer in 10 ml homogenization buffer
consisting of 4 M guandine isothiocyanate (Boehringer
Mannheim Biochem., Indianapolis, IN), 1 M sodium citrate
(Sigma Chem. Co., St. Louis, MO), 0.1 M mercaptoethanol
(Mallinckrodt, Inc., Paris, KY), 10% sodium sacrosyl
(Boehringer Mannheim Biochem., Indianapolis, IN).
Sequentially, 1 m1 2 M sodium acetate (Sigma Chem. Co., St.
Louis, MO), pH 5, 10 ml water saturated phenol and 2 m1 of a
chloroform (Fisher Scientific Co., Fair Lawn, NJ) : isoamyl
alcohol (Sigma Chem. Co., St. Louis, MO) mixture (24:1) were
added to the homogenate and mixed after the addition of each

reagent. The final suspension was vigorously shaken and

25

placed on ice for 15 minutes. The aqueous and the organic
phase was separated by centrifugation at 10,000 x g for 20
minutes. Under these conditions where the pH in the aqueous
phase is between 5 to 6, RNA would be soluble in the aqueous
phase, whereas DNA and protein would be present in the
interphase or organic phase (Wallace, 1987). The aqueous
phase was transferred to a 30 mthorex tube, mixed with an
equal volume isopropanol (Mallinckrodt, Inc., Paris, KY),
and stored at -20°C for a minimum of 2 hours to precipitate
RNA. Sedimentation of RNA was conducted by centrifugation
at 10,000 x g for 20 minutes and the pellet was dissolved in
750 ul of a pH 7 solution which was composed of 7 M
guanidine-hydrochloride (Research Organics Co., Cleveland,
OH), 0.2 M sodium acetate, 1 mM dithiothreitol (Boehringer
Mannheim Biochem., Indianapolis, IN), 1 mM EDTA (disodium
EDTA Boehringer Mannheim Biochem., Indianapolis, IN)
adjusted to pH 7 with glacial acid, and then transferred to
a 1.5 m1 microfuge tube. RNA was precipitated with 75 ul 2
M sodium acetate and 450 ul ethanol then placed at -20°C for
1 hour. The mixture was centrifuged at 12,000 g for 3
minutes and pellet was sequentially washed with 300 ul 3.0 M
sodium acetate, 10 mM iodoacetamide (Boehringer Mannheim
Biochem., Indianapolis, IN), 300 ul 30 mM sodium acetate,
0.1 mM iodoacetamide, 66% ethanol and finally with 300 ul
absolute ethanol (-20°C). Each wash consisted of
resuspension and sedimentation procedures, discarding the

supernatant after each step. The RNA pellet was dried and

26

resuspended in 100 ul.TE (10 mM Tris/1 mM EDTA, pH 8)
buffer. The concentration of RNA was determined by
absorbance at 260 nm and used for Northern blot analysis
(Thomas et al., 1980). Complete details of this procedure
are given in appendix A.

NEILAQIR plot 53311111 Messenger RNA (mRNA) abundance

of skeletal muscle alpha-actin and beta-tubulin was
determined in the LD muscle by Northern blot analysis.
Northern blot analyses were conducted as described by

Jump et a1. (1988) with the following modification.

Aliquots of total RNA were denatured and electrophoretically
separated in a 1.2% agarose gel containing 2.2 M
formaldehyde. RNA was transferred to nitrocellulose paper
(Schleicher and Schuell, Keene, NH) with 10X SSC (25x SSC is
equivalent to 3.75 M sodium chloride, 0.375 M sodium
citrate). Blots were prehybridized for 2 hours at 42°C in
50% formamide (Boehringer Mannheim Biochem., Indianapolis,
IN), 5x SSC, 5x Denhardt’s solution: 100X Denhardt’s
solution is equivalent to 2% bovine serum albumin (from
fraction V, essentially fatty acid free, Sigma Chem. Co.,
St. Louis, M0), 0.1% SDS (sodium dodecyl sulfate, Boehringer
Mannheim Biochem., Indianapolis, IN), 1 mM EDTA (disodium
EDTA, Fisher Scientific Co., Fair Lawn, NJ), 50 mM sodium
phosphate buffer (pH 6.5), and 0.5 mg/ml tRNA (Boehringer
Mannheim Biochem., Indianapolis, IN). The hybridization
solution was identical to the prehybridization solution,
except that the hybridization solution contained 1X

27

Denhardt's and 2 million cpm of appropriate cDNA probe/ml.
The human full length alpha-actin cDNA subcloned into
the Pst 1 and Pvu II sites of a modified pBR322 plasmid
(Okayama and Berg, 1982) was obtained from Dr. Kedes at
University of CA, Los Angeles (Gunning et al., 1983). A
mouse beta-tubulin cDNA (m-beta-S tubulin) subcloned into
the EcoRI site of pUC9 was obtained from Dr. Donald
Cleveland at John Hopkins University (Sullivan and
Cleveland, 1986). The cDNAs were randomly primed
(Boehringer Mannheim Biochemicals) using [32P] dCTP (3000
Ci/mmol) at a specific activity of 3.4 X 108 cpm/ ug DNA for
the alpha-actin probe and 2 X 108 cpm/ug DNA for the beta-
tubulin probe. After hybridization at 42°C for 16 hours,
blots were washed with 2X SSC and 0.1% SDS at room
temperature for 10 minutes, then washed three times for 45
minutes in 0.1x SSC and 0.1% SDS at 65°C for the alpha-actin
blots and in 0.2x SSC and 0.1% SDS at 55°C for the beta-
tubulin blots. After the last wash, the nitrocellulose
membrane was dried and exposed to x-ray film (XAR-S: Eastman
Kodak, Rochester, NY) at -80°C with two intensifying screens
(DuPont, Wilmington, DE). Following autoradiography,
hybridization of cDNA to RNA was quantified by densitometry.
The blot that contained adult pig RNA was stripped with
0.01X SSC and 0.5% SDS for 2 hours at 75°C to remove the
radiolabeled beta-tubulin cDNA, then re-hybridized with the
alpha-actin cDNA probe. Blots that contained four l-day-old

and four 28-day-old pig LD muscle RNA samples were stained

28

with methylene blue as described by Maniatis et a1. (1982)
to visualize the 18S and 28S ribosomal bands in order to
confirm that equal amounts of RNA are loaded onto the gel
and that the transfer efficiency was similar among lanes.
Details of the Northern blot analysis are presented in

appendix B.

55331131231 analysis All data were statistically

analyzed using the General Linear Models Procedures of SAS
(1987). Contrasts between means were analyzed by Bonferonni

t-test as outlined by Gill (1978).

(2) Transcription Assay

Animal; Four 1-day-old and four 28-day-old pigs were
randomly selected from the Michigan State University swine
research center.

Englg1,1§glgtign Thirty grams LD muscle from 1-day and
28-day old pigs was collected immediately following
exsanguination and ground through a fine plate (4.5 mm) meat
grinder at 4°C. The samples were mixed with buffer A (0.32
M sucrose, density gradient grade, ribonuclease free,
(Schwarz/Mann, Organgeburg, NY), 3 mM MgC12, 1 mM KHZPO4) ,
then gently homogenized with a Polytron homogenizer. The
homogenate was diluted with 1 volume buffer A and filtered
through 1 layer of cheesecloth. Nuclei and cellular debris
were pelleted by centrifugation at 700 g for 10 minutes.

The pellet was gently homogenized with 0.25% triton X-100 to

remove an outer nuclear membrane without further disruption

29

of the nuclei (Blobel and Potter, 1966) and centrifuged at
700 9. After two washes with buffer A, the pellet was
resuspended into buffer B (2.4 M sucrose, 3.mM MgClz) and
ultracentrifuged at 105,500 g for 80 minutes to separate
nuclei from other cellular organelles (Figure 3). Nuclei
were resuspended in storage buffer [pH 7.5, 40% glycerol
(Mallinckrodt, Inc., Paris, KY), 75 mM HEPES (4-[2-
Hydroxyethyl]-1-piperazine-ethanesulfonic acid, Boehringer
Mannheim Biochem., Indianapolis, IN), 60 mM KCl, 15 mM NaCl,
0.15 mM spermidine (Sigma Chem.Co., St.Louis, M0), 0.5 mM
spermine (Sigma Chem.Co., St.Louis, M0), 0.5 mM
dithiothreitol (Boehringer Mannheim Biochem., Indianapolis,
IN), 0.1 mM EDTA and 0.1 mM EGTA], examined under a phase—
contrast microscope, and quantified at 260 nm.
Concentration of nuclei were determined by converting 1
absorbance unit at 260 nm equal to 5 X 10 3 nuclei/ul.
Overall transcriptional activity was determined using [3H]-
UTP incorporation into RNA. Tritiated UTP incorporation
assays were conducted immediately after nuclei isolation.
Various nuclei concentrations were used to determine
viability of nuclei and appropriate conditions for the
transcription assay. Details of nuclei isolation are
presented in appendix C.

mum M mm mm The [BM-UTP
incorporation assay (Marzluff and Huang, 1984: Liew and
McCully, 1988) was conducted at different time points and at

various nuclei concentrations to determine appropriate assay

30

SAMPLE RPTER ULIRRCENTRIFUGHIION

   

-:ss (Mensa: c1511
debris

2-4 H sucrose-—u—

 

 

 

Figure 3. Schematic picture of nuclei separated from other

cellular organelles after ultracentrifugation at 105,500 g
for 80 minutes with 2.4 M sucrose buffer.

31

conditions. Three nuclei concentrations, 1X107, 2X107, and
3X107, and 7 time points were evaluated in assays conducted
at 26°C. The composition of transcription mixture is
described in Table 3. One hundred microliters of nuclei
preparation at three different nuclei concentrations listed
above were added to 300 ul of the transcription mixture and
incubated for 0, 5, 10, 15, 20,430, and 60 minutes.
Following the incubation, triplicate samples aliquots (17
ul) of the reaction mixture (transcription mixture with
nuclei) were'transferred to 13 X 100 mm test tubes
containing 100 ul 1 mg/ml tRNA. The reaction was stopped
with 100 ul 20% TCA (trichloroacetic acid, J.T.Baker Inc.,
Phillipsburg, NJ) which disrupts the nuclear membrane and
precipitates the RNA at each sampling time point. Each tube
was rinsed three times with cold 10% TCA and the suspension
was subsequently filtered through glass microfiber filters
using a vacuum manifold (FH224VM, Hoefer Scientific
Instruments). After the filtration procedure was complete,
filters were placed into scintillation vials containing 10
m1 of an aqueous scintillation solvent. Radioactivity was
determined using a Beckman, LS-lOOC liquid scintillation
counter. Inhibition of RNA polymerase II was tested by
including 0.05 ug/ml alpha-amanitin at 3X107 nuclei
concentration in the reaction mixture to conduct the
experiment. Details of tritiated UTP incorporation assay

are given in appendix D.

fitgtigtiggl Analysis All data were statistically

 

32

Table 3 Composition of transcription mixture for
tritium incorporation assay

 

glycerol 21.1%
NaCI 7.95
EDTA 0.053
DTT 0.265
spermidine 0.0795
CTP 0.5
GTP 0.5
s-adenosyl 0.25
methionine

EEEEEEE

HEPES 39.75 mM
spermine 0.265 mM
EGTA 0.053 mM
KCl 31.8 mM
ATP 0.5 mM
UTP 0.5 mM

3H-UTP 60 ucia

RNase- 1 U/ul
inhibitor

 

aFor transcription ass
utilized instead of [

3

y 100 uci [32P]-UTP were

HJ-UTP.

33

analyzed using the General Linear Models Procedure of SAS
(1987). A split-split-plot with repeated measurements
design was utilized (Gill, 1987). Significance of age
effect (factor A) was tested using variation of pigs within
age (P/A as error 1). Significance of nuclei concentration
effect (factor B) and age-nuclei concentration interaction
(A x B) were tested using variation of interaction between
nuclei concentration and pigs within animal (B X P/A as
error 2). Significance of sampling time effect (factor C)
and age-sampling time interaction (A X C) were tested using
variation of interaction between sampling time and pigs
within age (C x P/A as error 3). Significance of
interaction between nuclei concentration and sampling time
(B X C), and significance of interaction among age, nuclei
concentration and time point (A X B X C) were tested using
variation of interaction among nuclei concentration, time
point and pigs within age (B X C X P/A as error 4). Effect
of nuclei concentration was analyzed by Bonferonni t-test.
Effect of sampling time was analyzed by Stepwise-regression

(Gill, 1978).

34

Transg:ip§ign_in,zitrg The transcription assay was
developed as a modification of that described by Jump et al.
(1988) and Marzluff and Huang (1984). The transcription
mixture composition is the same as that used in the
tritiated UTP incorporation assay (Table 3) except that 100
uCi of [32P1-UTP were used instead of [3H]-UTP. Nuclei
number of 3X107 was incubated in a 1.5 m1 microfuge tube at
26°C for 20 minutes in a 400 ul volume of transcription
mixture. Two microliters DNase I (30 U/ul, final
concentration equal to 1 unit/ml) (Boehringer Mannheim
Biochem., Indianapolis, IN) were added to stop the reaction
by digesting DNA at 26°C for 3 minutes. At the same time,
12 ul 10 mg/ml tRNA were added to increase the amount of RNA
for RNA extraction. Subsequently, 4 ul 0.2 M EDTA, 14 ul
10% SDS, and 12 ul 25 mg/ml proteinase K (Boehringer
Mannheim Biochem., Indianapolis, IN) were added and the
incubation continued for 2 hours at 65°C to disrupt the
nuclear membrane and digest the nuclear proteins. RNA
isolation began by adding 600 ul phenol: chloroform:isoamyl
alcohol (25:24:1) and centrifuging at 12,000 g for 3
minutes. The aqueous phase was collected and mixed with 600
ul chloroform:isoamyl alcohol (24:1) and centrifuged at
12,000 g. The aqueous phase was transferred to a microfuge
tube containing 80 ul 5 M ammonia acetate (pH 5) and 1 ml
ethanol and allowed to stand at -20°C for at least 1 hour.
RNA precipitates were recovered by centrifugation and

resuspend into 100 ul TE buffer, pH 8 in a microfuge tube.

35
Another DNase-1 digestion was conducted at 37°C for 10
minutes and stopped with 3 ul 0.2 M EDTA. The reaction
mixture was incubated at 65°C for 5 minutes chilled on ice
and the RNA was sequentially precipitated with ammonia
acetate/isopropanol for 20 minutes at -20°C for three
cycles. Then the pellet was washed with 200 ul 70% ethanol
and vacuum dried. Synthesized RNA was quantified and equal
amounts of 32P RNA transcripts were hybridized to
immobilized plasmid cDNA on nitrocellulose paper at 42°C for
72 hours. The immobilized plasmid cDNAs on nitrocellulose
membrane were prehybridized in 50% formamide, 5X SSC, 5X
Denhardt's, 0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate
buffer (pH 6.5) and 0.5 mg/ml tRNA for 16 hours at 42°C.
The hybridization solution was identical to the
prehybridization solution, except that the hybridization
solution contained 1X Denhardt's solution and [32F] labeled
transcripts. Blots were washed at 65°C with 0.1 X SSC and
0.1% SDS for three cycles and dried for autoradiography.
Hybridization was quantified by densitometry. In addition,
the dots containing cDNA were cut and radioactivity was
determined by a scintillation counting. Details of the

transcription assay are presented in appendix E.

Results

In order to determine whether skeletal muscle alpha-
actin and beta-tubulin mRNA change in relative abundance
Northern blots were conducted. Preliminary tests using
Northern blot analysis of RNA isolated from 1-day-old (n=l),
28-day-old (n=1) and adult pigs (n-l) indicated that mRNA
abundance for SM alpha-actin mRNA increases with age in the
pig (Figure 4), and there is larger difference in actin mRNA
between 1-day-old and 28-day-old pigs compared to that
between 28-day-old and adult pigs (Table 4). On the
contrary, beta-tubulin mRNA abundance decreases with age in
the pig (Figure 4), and there is larger difference between
28-day-old and adult pigs compared to that between 28-day
and 1-day-old pigs (Table 4). In addition, the Northern O
blots containing only 1-day-old and 28-day-old LD muscle RNA
show that there is a developmental increase of SM alpha-
actin mRNA abundance and a developmental decrease of beta-
tubulin mRNA abundance between these two ages (Figure 5).
Densitometer scans of these autoradiograms indicated a 2.2-
fold increase (P<0.03) in alpha-actin mRNA and no
significant change (P>0.1) in beta-tubulin mRNA between 1
and 28-day-old pigs (Table 5). Methylene blue staining
visualized the 18S and 288 ribosomal bands which indicated

that equal amounts of total RNA were loaded and transferred

36

37

A B

Alpha-actin Beta-tubulin
28-day l-day 6-month 28-day 1-day 6-month
l— — —| | — _l
1 2 3 4 5 6 1 2 3 4 5 6
28$> 288)

. o.
139” O. .. 1ss> .. .. b.

Figure 4. Autoradiogram of alpha-actin (A) and beta-tubulin
(B) mRNA abundance in LB muscle from l-day-old, 28-day-old
and adult pigs. RNA was isolated and analyzed via a Northern
blot. The mouse M35 tubulin cDNA hybridized to mRNA species
of 1800 and 2800 nucleotides- -(B), then the blot was stripped
with 0. 01X SSC and 0.1% SDS for 2 hours at 70° C and re-
hybridized with a human alpha-actin cDNA. A single band of
1650 nucleotide was observed (A). Northern blot of beta-
tubulin (B) was washed with 0. 2x ssc and 0.1% SDS at 55°C and
exposed for 24 hours. Northern blot of oalpha-actin was
washed with 0.1x SSC and 0.1% SDS at 65° C and exposed for 30
minutes. Lane 1: 10 ug 28-day-old pig LD RNA, lane 2: 20 ug
28-day-old pig LD RNA: lane 3: 10 ug 1-day-old pig LD RNA,
lane 4: 20 ug l-day-old pig LD RNA: lane 5: 10 ug adult pig
LD RNA, lane 6: 20 ug adult pig LD RNA.

38

Table 4. Relative abundance of alpha-actin and beta-tubulin
mRNA among 1-day-old, 28-day-old and adult pigs. Data was
obtained by densitometry scan of autoradiograms shown in
figure 3.a

 

Total RNA loaded

 

 

10 ug 20 ug 10 ug 20 ug
Age alpha-actin beta-tubulin
l-day-old 1.589 2.504 ' 4.258 10.78523
28-day-old 4.145 10.291 1.437 10.134b
Adult 4.454 10.774 0.555 1.599

 

aAbundance of alpha-actin mRNA is expressed as densitometer
units. RNA was isolated from three animals, one animal per
each age.

bThese bands were out of the linear portion of the
densitometer.

39

A B
Alpha-actin Beta-tubulin
1-day 28-day l-day 28-day
I I
l 2 3 4 l 2 3 4 1 2 3 4 1 2 3 4.
28$> . 28$> ‘
V- . ~11“

1as> oocm O... 188> .0“ w

Figure 5. Autoradiograms of alpha-actin and beta-tubulin
mRNA abundance in LD muscle from four 28-day-old and four 1-
day-old pigs. Each lane contains 10 ug of RNA isolated from
LD muscle from each animal. Animals are number 1 to 4 for
each age group evaluated. The human alpha-actin cDNA
hybridized to mRNA species of 1650 nucleotides (A). The
Northern blot of alpha-actin was washed with 0.1X SSC and
0.1% SDS at 65°C then exposed for 30 minutes. The mouse M85
tubulin cDNA hybridized to mRNA species of 1800 and 2800
nucleotides (B), Northern blot of beta-tubulin (B) was washed
with 0.2x ssc and 0.1% SDS at 55°C then exposed for 24 hours.

40

Table 5. Relative abundance of alpha-actin mRNA and
beta-tubulin mRNA in LB muscle from l-day-old and 28-day-old
pigs.a Data were obtained by degsitometry scan of
autoradiograms shown in figure 4

 

mRNA abundance

 

 

Protein 1-day-old SEM 28-day-old SEM
Alpha-actinc 2.247 0.655 4.493 0.321

Beta-tubulind 3.459 0.649 2.523 0.769

 

aTotal number of animals is 8 (n=4 for each age group).
bNorthern blot of alpha-actin was exposed for 30 minutes.
Northern blot of beta-tubulin was exposed for 24 hours.
cThese autoradiograms were scanned by densitometry.

cAge effect for change of alpha-actin is significant

(P<0. 03).
dA Age effect for change of beta-tubulin is not statistically
significant (p>0.1).

41

to nitrocellulose membrane from each lane (Figure 6).

Visual inspection at 400x magnification under the
microscope indicated that the nuclei containing an intact
nuclear membrane were isolated from 1- and 28-day-old pigs
(Figure 7). Total transcriptional activity was determined
by conducting [3H]-UTP incorporation assays. The results of
the microscopic evaluation (Figure 7) and tritiated UTP
incorporation data (Table 6) indicated that isolated nuclei
were of high quality and able to synthesize RNA (Figure 8).
Larger quantities of total RNA appeared to be synthesized by
1-day-old pig SM nuclei than 28-day-old pig SM nuclei (Table
6, Figure 8); however, the difference was not statistically
significant (P>0.25) between 1-day-old and 28-day-old pig SM
nuclei (Table 7). Both the effects of nuclei concentration
(P<0.0001) and sampling time point (P<0.0001) have
significant effects on synthesis of total RNA as indicated
from the [3HJ-UTP incorporation data (Table 7). Therefore,
the effect of nuclei concentrations must be tested
conditionally upon sampling time points. At 5 minutes,
there was a significant difference (P<0.02) between nuclei
concentration of 1X107 in contrast to 3X107, but there was
no difference (P>0.05) between either 1X107 in contrast to
2x107 or 2x107 in contrast to 3x107. At 10, 15 and 20
minutes, there were significant differences (P<0.01) between
nuclei concentration both 1X107 in contrast to 2X107 and
1X107 in contrast to 3X107, but there were no differences

(P>0.05) between 2x107 in contrast to 3x107. At 30 and 60

42

Alpha-actin Beta-tubulin

1-day 28—day' l—day 28-day

 

 

l
1234 1234

.4.- _,<._ . ~. . -
' ‘ J. _ .. -

288>

 

   

Figure 6. Northern blots of alpha-actin and beta-tubulin
stained with methylene blue to visualize the 188 and 288

ribosomal bands.

43

  
  

 
  
 

. t.
'r; '2
"fi ..‘ H

    

al-

Figure 7. Photograph of Nuclei (400x magnification) using a
phase-contrast microscope. Nuclei were isolated by
differential centrifugation as described in Materials and
Methods.

44

Table 6. Total RNA synthesized by isolated skeletal muscle
nuclei. Tritiated UTP incorporated into RNA (tritium
incorporation assay) was conducted as describgd in Materials
and Methods.a Data are expressed as CPM X 10 for each time
point

 

1-daysold pig SM Nuclei

 

Nuclei Concentrationb

 

 

 

 

Time(min) 1x107 SEM 2x107 SEM 3x107 SEM
0 0 0 0 0 0 0

5 3.925 1.539 6.925 2.395 8.000 1.096

10 '6.250 2.661 13.075 5.393 11.233 2.598

15 7.425 2.436 14.175 5.322 14.225 2.301

20 8.775 3.612 14.150 3.992 16.925 3.957

30 10.650 4.471 15.450 4.804 21.200 4.887

60 13.733 5.985 19.550 5.462 27.725 7.004

28-day-old pig SM Nuclei
Nuclei concentration”

Time(min) 1x107 SEM 2x107 SEM . 3x107 SEM
0 0 0 0 0 0 0

5 2.475 0.309 5.400 0.187 8.150 0.202

10 4.200 0.430 8.225 0.743 10.100 1.125

15 6.033 0.418 11.150 1.282 10.375 0.557

20 ‘6.433 0.367 12.050 1.596 13.450 0.932

30 6.800 1.278 12.600 1.568 16.750 1.050

60 7.167 0.767 16.867 1.487 18.000 0.569

 

:Nuclei were isolated from 8 animals and 4 animals/age.
Nuclai concentration are 1X10 /400 ul, 2X10 /400 ul and
3x10 /400 01.

45

Tritium Incorporation Assay
Age- -C0ncentrati0n Combination

O A as I we “0 an .A an ID am
ET 257 187 $7 .27 £7

 

25 '

 

 

 

1000
A)
O'
I

15- A

10" /f/.

 

\\

 

 

 

o m l a 1 L m I l m
C) 10 20 30 4O 50 60
Tine

Figure 8. Effects of age and nuclei concentration on
transcription in yitzg (Tritiated UTP incorporated into RNA).
Incorporation assay was conducted as described in Materials
and Methods. Skeletal muscle nuclei isolated from l-day-old
and 28-day-old pig LD muscle and 3 different nuclgi
concgntrations (total nuclei number equal to 3X10 2X107 and
1X10 nuclei per 400 ul assay volume) for each age group were
evaluated.

Table 7.

46

ANOVA of tritium incorporation assaya.

The

experimental design is a split-split-plot with repeated

measurements design

 

 

Source of Variation DF Mean Squares F Value Prob.
Age of pig (A)D 1 290.397 0.73 >0.25
Pigs/age (P, E1) 6 395.175

Nuclei Concentration (8)c 2 630.604 29.25 <0.0001
Age*Conc (AB) 2 4.404 0.20 >0.5
Conc*Pigs/age (PB, 82) 12 21.556

Sampling Time (0)e 6 646.100 28.87 <0.0001
Age*Time (AC) 6 18.197 0.81 >0.5
Time*Pigs/age (PC, E3) 36 22.379

Conc*Time (80)8 12 37.126 11.50 <0.0001
Age*Conc*Time (ABC)h 12 4.783 1.48 >0.1
Conc*Time*Pigs/age (PBC, E4) 621 2.780

 

_aExperiment was conducted using 8 animals, 4 animals/age.
Nuclei concentration and sampling time were described as in

Material and Method.

bAge effect is not statistically significant (P>0.25).
cEffect of nuclei concentration is highly significant

< O .
d(P 0 0001)

e(P>0.5).

f
(p>0.5).

9Effect of concentration*time interaction is highly
significant (P<0.0001).
Effect of age*concentration*time interaction is not

h

.statistically significant (P>0.1).
1DF decrease from 72 to 62 because there were 10 missing data

of 168 data set.

Effect of age*concentration interaction is not significant

Effect of sampling time is highly significant (P<0.0001).
Effect of age*sampling time interaction is not significant

47

minutes, there are significant difference (P<0.02) among all
contrasts (Table 8). These polynomial curves (Figure 9) of
nuclei concentration and sampling time effects were
calculated by Stepwise-regression. Tritiated UTP
incorporation is represented by Y and sampling time is
represented by X, the equation for the 1X107 nuclei
concentration is Y=0.748+0.444X-0.005X2 and R square equals
to 0.976. The equation for the 2X107 nuclei is
Y=2.270+0.691X-0.007X2 and R square equals to 0.909. The
equation of 3X107 nuclei is Y=1.873+0.855X-0.008x2 and R
‘square equals to 0.973. The maximal value of Y for equation
1 and 2 is 45 minutes and for equation 3 is 40 minutes. The
appropriate assay condition for maximum incorporation is 45
minutes incubation time using a nuclei concentration of
7.5x104 nuclei/ul in a 400 ul assay volume (3x 107 nuclei
per assay) (Table 8, Figure 9). The isolated nuclei were
sensitive to alpha-amanitin, a polymerase II inhibitor.
Using a nuclei concentration of 3X107/400 ul,-there is a
decreased trend of the synthesis of total RNA after addition
of alpha-amanitin to a final concentration equal to 0.05
ug/ml (Figure 10). No hybridization was detected using
transcription assay when the assay was conducted in the
presence of alpha-amanitin (data not shown). These results
indicate that RNA polymerase II was functional in these
nuclei preparations.

Determination of relative transcriptional rate was

evaluated by hybridization of the [32PJ-RNA synthesized by

48

Table 8. Effects of nuclei concentration and sampling time
on total RNA synthesized in vitroa . Data are expressed as
CPM X 103 for each sampling time point

 

Nuclei ConcentrationD

 

 

1x107 2x107 3x107
Time

0 0 0 0
5 3.20:0.78x 6.16:1.15xy 8.0810.52Y
10 5.23:1.31x 10.6512.68y 10.59:1.17Y
15 6.83il.34x 12.6612.60y 12.30.451.32y
20 7.77:1.99x 13.1012.03Y 15.19:1.99Y
30 9.00:2.56x 14.03:2.40Y 19.72r3.24z
60 10.45r3.07x 18.40r3.02Y 23.56r4.23z

 

aThis experimental design is Split-Split-Plot with Repeated
Measurements (V325.8, f =37.5) and using Bonferroni-t test to
compare these means. W1thin a row means followed by the
different letter (x, y, z) indicated that the contrast is
ifferent.
ucl; 1 concentration are 1X107/400 ul, 2X107/400 ul and
3X10g /400 ul.

49

Figure 9. Effects of nuclei concentration and sampling time
on total RNA synthesized in 213:9. Polynomial curves were
calculated by Stepwise-rggression. Equation of 1X107 nuclei
is Y=0.748+0.444X-0.005X and R Squar 20.976. Equation of
2X107 nuclei is =2.270+0.691X-0.007X and R Squa5e=0.909.
Equation of 3X10 nuclei is Y=1.873+0.855X-0.008X and R
Square=0.973. Tritium incorporation is represented by Y and
sampling time is represented by X. The maximum of Y for
equation 1 and 2 is 45 minutes and the maximum of Y for
equation 3 is 40 minutes.

 

 

50

Nd.u

Nu.o

NM.¢

No.0

.u.¢

.N.u

.O.o

zoqna

 

 

 

trad 01 quxnaxv (m. nmmvozmma<v

trad Om nnmv.sdu2n_ m<£oor ammo mm .
trad Om mmmvuoduZmN «(zaor Cmmo um n
vroq Om rnmvuoquxmu ”(Soar cmmo mm o

 

O
u
u
4
u
o
.9
n
u
u n
6.
u
o
4 u
u n
9
n
d L
+ E
u s
n s
4 .
a s
n
4 s
d
on
a
S
O
0 0 I I rrr+rrrrrrrr+lorrrrlrorrrrllrrortrrrtrl+rIIJIrII+rrIIIrIlorrrrrrrl+r
o a .o .u no no no on so am
. qnzn
no Cum 1>o IHMMuzo <>rcnm 09 can Incomz

51

Alpha-amanitin Inhibition Curve

A - smanittn I + arnanittn

 

 

 

CPM X 1000

 

 

 

 

Time (min)

Figure 10. Effect of alpha-amanitin inhibition on
transcription in 213:9 was evaluated as described in Materials
and Methods. Experiments were conducted using nuclei
isolated from 28-day-old pig LD muscle at nuclei
concentration equal to 3X10 in a 400 ul assay volume.

52

the nuclei to immobilized cDNAs (alpha-actin and beta-
tubulin). Transcriptional assay and hybridization results
indicate that greater (P<0.02) SM alpha-actin hnRNA was
synthesized in the 28-day-old pig SM nuclei than in the 1-
day-old pig SM nuclei (Figure 11). There was a 1.9-fold
increase (P<0.02) in the ability of nuclei to synthesize
alpha-actin hnRNA from 1 to 28 days. Hybridization was
quantified by densitometry of the autoradiogram (Table 9).
Beta-tubulin hnRNA synthesis was not detected using the
current condition of this assay (Figure 11). Nonspecific
hybridization, measured with immobilized plasmid pBR322 DNA

onto nitrocellulose paper, was negligible (Figure 11).

53

28-day l-day
. . alpha-actin
A beta-tubulin

. pBR322

. . alpha-actin

B ' beta-tubulin

pBR322

. . alpha-actin .

C beta-tubulin

pBR322

Figure 11. Autoradiograms of alpha-actin hnRNA abundance and
beta-tubulin hnRNA abundance synthesized by 1-day-old and 28-
day-old pig SM nuclei. RNA was synthesized and isolated via a
transcription assay as described in Materials and Methods.
Plasmid pBR 322 was included as a negative control. Panel A,
B, and C each represent analysis of 1- and 28-day-old pigs.

54

Table 9. Relative abundance of alpha-actin hnRNA synthesized
by 1-day-old and 28-day-old porcine SM nuclei.a Data are

expressed as densitometry unit from the autoradiograms shown in
figure 10

 

Age

1-day-old SEM 28-day-old SEM

Alpha-actin hnRNAb 4.979 0.581 9.327 1.002

aBeta-tubulin hnRNA and nonspecific hybridization to pBR322

were not detectable either by scintillation counter or
bdensitometry.

Age effect is significant (P<0.02).

 

DISCUSSION

In the present study, we investigated the regulatory
mechanism(s) underlying the synthesis of skeletal muscle
proteins in pigs. We have demonstrated that there is a
developmental increase in skeletal muscle alpha-actin mRNA
abundance as the pig ages from one to twenty eight days
(Figure 5, Table 5). These results are consistent with
preliminary results (Figure 4, Table 4) from our laboratory
suggesting that the mechanism to increase muscle protein
mass during development is due, at least in part, to change
in mRNA abundance for skeletal muscle protein. This
increase in mRNA for SM alpha-actin is due either to an
increase in transcriptional rate and/or changes in stability
of the transcript. Results of experiments from this study
demonstrate that the abundance of skeletal muscle alpha-
actin mRNA increases (P<0.03) and beta-tubulin mRNA
abundance is unchanged (P>0.1). These data suggest that
message abundance of specific proteins varies during growth
and development. Gunning et a1. (1987) found that
individual patterns of transcript accumulation could be
grouped into broad categories and each gene investigated had
its own unique determinants of transcript accumulation and
no two mRNAs were identically expressed. The muscle cell

culture studies of Gunning et al.(1987) and Kedes et al.

55

 

56

(1988) indicated that abundance of mRNA parallels the levels
of myofibrillar protein synthesis and that genes
corresponding to each transcript are regulated on an
individual basis. Gunning et a1. (1987) suggest that the
quantity of mRNA is regulated by transcriptional rate;
however stability of mature mRNA or processing of pre-
existing pre-mRNA have not been elucidated. Recently, Cox
et al. (1990) reported transcriptional regulation of actin
genes during differentiation in a mouse muscle cell line
(CZ/7). Their study indicated that mRNA of skeletal alpha-
actin and cardiac alpha-actin was not detectable in
myoblasts and these genes were cotranscribed in a
developmental manner throughout the time-course of
differentiation. Northern blot analysis indicated that
skeletal alpha-actin mRNA increased 4.3-fold from 24 hours
to 48 hours and continuously increased to 21-fold from 48
hours to 120 hours. The present study was conducted to
evaluate the contribution of transcription of the skeletal
muscle alpha-actin gene to the relative increase in skeletal
muscle alpha-actin mRNA abundance in 1- and 28-day-old pigs.
In order to determine the relative transcriptional rate
of the alpha-actin hnRNA synthesis in porcine skeletal
muscle, experiments using isolated nuclei from longissimus
dorsi muscle and a radioactive nucleotide were performed.
We established a protocol for isolation of nuclei from
longissimus dorsi muscle of young pigs. The isolated nuclei

retained the capacity to perform a number of reactions as

 

57

they occur in_xixg, and provide a useful system for studying
several aspects of RNA metabolism.

In this in yitrg system RNA synthesis consists of
elongation and completion of previously initiated RNA
molecules with a negligible amount new initiation (Reeder
and Roeder, 1972). Incorporation of tritiated UTP into
initiated RNA indicated these isolated nuclei were capable
of transcript elongation. Experiments using alpha-amanitin
demonstrated a decreasing trend in total RNA elongation,
which indicates that all the nascent RNA (pre-tRNA, pre-rRNA
and hnRNA) were elongated during the transcription reaction,
because alpha-amanitin at low concentration is a specific
inhibitor for RNA polymerase II.

Our results also indicate that an appropriate nuclei
concentration is 3X107 nuclei for transcriptional analysis.
Similar concentration were obtained by McCully and Liew
(1988) using myocardial-cell nuclei for their transcription
reaction. We also found maximal incorporation of [3HJ-UTP
into RNA between 40 to 45 minutes, which is consistent with
the observations of Ridgway et a1. (1985). However,
incubation times from 5 up to 60 minutes have been used with
success in nuclei isolated from a variety of tissues (Hanson
et al., 1982, 1985: Jump et al., 1988; Manley et al., 1980:
Oppenheimer et al., 1981, 1987: Rall et al., 1986: Ridgway
et al., 1985, Casero et al., 1989). The linear portion of
the tritiated UTP incorporation study is from 5 to 45

minutes. During this interval, the ratio of various gene

58

transcripts at different time points should be constant.
Overall transcriptional activity was estimated by measuring
the incorporation of [3H]-UTP into RNA from isolated
skeletal muscle nuclei. We found that 1-day-old pig SM
nuclei synthesized a larger amount of RNA than did 28-day-
old pig SM nuclei (Table 6, Figure 6), but the difference
was not statistically significant (P>0.25). Mulvaney and
Gore, (1988) reported greater incorporation of [3HJ-UTP into
RNA of fetal bovine skeletal muscle nuclei in response to
cortisol treatment than control. In addition, they
suggested that [3H]-UTP incorporation into total RNA
decreases with age. It is possible that there is an overall
decrease in transcriptional activity and a relative increase
in mRNA abundance for myofibrillar proteins as indicated by
the relative increase of alpha-actin mRNA. Results of the
transcription assay using nuclei isolated from skeletal
muscle indicated that nuclei from 28-day-old pigs are
capable of synthesizing 1.9-fold (p<0.02) more alpha-actin
hnRNA than nuclei from 1-day-old pigs (Figure 11, Table 9).
Transcription of beta-tubulin was not detected under the
condition used in this study. The reason for the inability
to detect beta-tubulin hnRNA may be due to technical
difficulties in detecting high abundance (alpha-actin)
messages and low abundance (beta-tubulin) messages from one

incubation on one blot.

CONCLUSION

These results indicated that pig skeletal muscle alpha-
actin mRNA abundance is greater in 28-day-old than 1-day-old
pigs and there are different developmental changes between
alpha-actin and beta-tubulin mRNA abundanCe between these
two age groups. Also, nuclei can be isolated from skeletal
muscle of young pigs (l-day-old and 28-day-old pigs).
Further modification should enable us to isolate viable
skeletal muscle nuclei from older animals: however, our
preliminary studies were unsuccessful (data not shown).
Furthermore, total RNA synthesized by the SM nuclei from 1-
day and 28-day-old pigs were not significantly different
(p>0.25). The relative transcriptional rate of the SM
alpha-actin gene increased 1.9-fold in the isolated nuclei
from 1-day to 28-days of age. These results indicate that
the relative increase in mRNA abundance for SM alpha-actin
from l-day to 28-day-old pigs is due, at least in part, to
an increase in transcriptional activity of the SM alpha-

actin gene.

59

APPENDICES

60

APPENDIX A

A312 QEAEIDIEE.2EIEQL QELQBQIQEH BEA EZIBAQIIQ!.EEIEQD
(modofied from the protocol of Chomczynsk1 and Sacchi, 1987)

BEAQENI§=

1. 4 M Guanidine thiocyanate:
guanidium thiocyanate (Kodak) 94.60 g.
sodium citrate 0.74 g.
B-mercaptoethanol (14 M) 1.42 ml
10% Na sarcosyl 10.0 ml

Total volume of 200 ml: use sterile H20.

Adjust pH to 7.0 with 1 M HCl or 1 N NaOH.

Filter through 0.45 um millipore.

Store in sterile brown bottle at room temperature.

2. Buffer II: 7 M Guanidine Hydrochloride
guanidine-HCl (Sigma, practical grade) 668.6 g.
Na-acetate (trihydrate) 2.7 g.
dithiothreitol 150.0 mg.
iodoacetamide 1.84 mg.
0.2 M EDTA (pH 8.0) 5.0 ml.

3.

Add GD-HOH to 900 ml.

Dissolve all reagents by heating in a 50°C water bath.
When all reagents are dissolved, adjust to pH 7.0 with
glacial acetic acid or 5 N NaOH.

Adjust final volume to 1 1.

Filter through 0.45 um millipore.

Store in sterile bottle in the dark at room temperature.

Buffer III: 2 M Na-acetate, pH 5.0

Dissolve 272 g. Na-acetate (trihydrate) in 600 ml sterile
GD-HOH.

Adjust to pH 5.0 with glacial acetic acid.

Adjust final volume to 1.0 1.

Filter through 0.45 um millipore.

Store at 4°C. in sterile bottle.

Buffer VI: 3 M Na-acetate, pH 5.0
Na-acetate (trihydrate) 409.8 g.
iodoacetamide 1.84 g.

Dissolve reagents in 300 ml glacial acetic acid and 300 m1
sterile H20. Adjust to pH 5.0 with glacial acetic acid.
Adjust final volume to 1.0 1. Filter through 0.45 um
millipore filter and store in sterile bottle at 4°C.

5.

61

Buffer VII: ETOH-Na-acetate, pH 5.0.

Absolute Ethanol 660 ml.
3 M Na-acetate, 10 nM iodoacetamide, (pH 5.0) 11 m1.

Filtration of this solution is not necessary, store in
sterile bottle at -20°C.

TE-8.0:
1.0 M Tris-HCl, pH 8.0 (20 C) 10.0 ml.
0.2 M EDTA, pH 8.0 5.0 m1.

Adjust final volume to 1.0 l. with sterile H20.
Store at room temperature.

10% Na-sarcosyl:

Dissolve 10 g. Na-sarcosyl (ICN) in 100 ml HOH, filter
through 0.45 millipore and store at room temperature.

flgtg: sarcosyl is a carcinogen, so take the appropriate

precautions.

THE.EXIBA§IIQN EBQIQQQLL

1.

2.

Use sterile glassware and plasticware throughout the
procedure.

Add 1 9 frozen pig LD muscle to a sterile 30 ml Corex
tube containing 10 ml 4 M guanidine thiocyanate
homogenization buffer.

Homogenize using a Polytron at a setting of 6 for 20
seconds or until the tissue is completely homogenized.

Add 1 ml Buffer III, stopper, and vortex.
Add 10 m1 H O-saturated phenol, vortex.
Add 2 ml ch oroform:isoamyl (24:1).

Mix vigorously, put on ice for 15 minutes.

Centrifuge at 10,000 x g for 20 minutes at 4°C.
Remove the aqueous phase with a sterile Pastier pipet and
put it into a sterile 30 ml Corex tube.

To the organic phase, add 2 ml 4 M guanidine thiocyanate
and 0.2 ml Buffer III and vortex.

Add 2 ml HZO-saturated phenol and 0.4 ml chloroform:
isoamyl.

Vortex and put on ice for 15 minutes.

Centrifuge at 10,000 x g for 20 minutes at 4°C.
Add this aqueous phase to the previously collected
aqueous phase. .

10.

11.

12.

13.

14.

15.

16.

17.

18.

62

Extract the aqueous composite with 5 ml HZO-saturated
phenol and 5 ml chloroform: isoamyl.

Centrifuge at 10,000 x g for 20 minutes at 4°C.
Remove the aqueous layer and put it into a sterile 30 ml
Corex tube.

Add 1 volume of isopropanol to the aqueous layer, cover
the tube with Parafilm and mix.

Store at -20°C for at least 2 hours to allow
precipitation of RNA.

Centrifuge at 10,000 x g for 20 minutes at 4°C.

Transfer pellet to a 1.5 m1 microfuge tube with 750 ul
Buffer II. Add 75 ul Buffer III and 450 ul absolute
ethanol and allow RNA to precipitate at -20°C for 1 hour.

Microfuge at 12,000 x g for 5 minutes. Decant and drain.

Resuspend the pellet in 300 ul of Buffer VI. Sediment
and drain as described above.

Resuspend the RNA pellet in 300 ul of Buffer VII and
sediment and drain as described above.

Resuspend the RNA pellet in 300 ul of absolute ethanol
and sediment and drain as described above.

Resuspend the RNA pellet in 100 ul of TE-8 buffer.

Dilute 5 ul of RNA solution to 1 ml with TE-8 and scan in
a spectrophotometer from 320 to 220 nm. Record the
optical density at 260 and 280 nm. The ratio of 260/280
should be greater than 1.8.

RNA concentration of solution in step 16 (ug/ul) is equal
to the optical density at 260 nm multiplied by 200
(dilution factor) and divided by 25 (RNA extinction
coefficient).

RNA samples should be labeled and stored at -80°C.

63

APPENDIX D

NDBIEIBH.ELQI.AEALX§I§
(modification of Jump et a1. 1988)
Reagents

1. GEL RECIPE:

lOX-MAE, pH 7.0 25 ml

LE-Agarose 3.0 9 equals 1.2% agarose
MG-HZO 181 ml

deionized formaldehyde 44 ml

(10X-MAE, pH 7.0: 0.4 M MOPS, pH 7.0; 100 mM Na-
acetate; 10 mM EDTA, pH 8.0)

£9131 EQBMALDEHXDE.I§.IQXIQI.IN.§QME IN§IANQE§ QABQINQQEN191
TAKE TEE AEEBQEBIAIE.EBEQAUIIQN§.EHEN EQBEIN§.EIIH
EQBMALQEHXQE; WEAR GLOVES, WORK IN THE FUME HOOD.

Dissolve agarose in lOX-MAE and water by heating on high
in the microwave for three minutes. Cool melted agarose to
60°C. Transfer melted agarose to fume hood, add formaldehyde
slowly while mixing, then pour gel. The gel should be in the
hood.

2. ELECTRODE BUFFER:

lOX-MAE, pH 7.0 100 ml
MQ-HOH 720 ml
deionized formaldehyde 180 ml

Mix just before use, keep in the fume hood.
3. SAMPLE PREPARATION:
Samples should contain total RNA in a total volume of

5.5 ul. Use TE-8 as a diluent.
Denaturing Mix (for 20 samples)

lOX-MAE, pH 7.0 20 ul
deionized formaldehyde 70 ul
deionized formamide 200 ul

Add 14.5 ul of denaturing mix to each RNA sample, ca
microfuge tube and heat denature for 5 minutes at 60 C.

Add 5 ul of 4X-dye mix and load all of the sample on the gel.
(4X-RNA dye: 50 % glycerol: 0.4 % bromophenol blue; 6.4 %
zylene cyanol; 1 mM EDTA)

64

Gel electrophoresis
Load samples onto the gel. Cover the gel with Saran wrap
to prevent loss of formaldehyde.
Electrophoretically separate RNA for 16 hours at 40 volts
or 65 volts for 4 hours. The entire unit should be in the
fume hood. ' -

TRANSFER TO NITROCELLULOSE:

Transfer the gel to MO-HZO and rinse briefly. Transfer the
gel to 500 ml 50 mM NaOH and soak for 30 minutes.

Cut the marker lanes off. Stain the marker lanes with TBE
buffer and EtBr (0.5 ug/ml) for two hours, then destain with

After soaking gel in NaOH, transfer gel to 500 ml 0.1 M Tris,
pH 7.5, and soak for 30 minutes.

Soak nitrocellulose paper out to the size of the gel in water
for ten minutes, then soak nitricellulose paper, wicks, and 3
small pieces 3 MM paper (cut to the size of the gel) in 10X
SSC for 15 minutes.

Put upper surface of gel next to the wicks and use a pipet to
remove any bubbles. Put nitrocellulose paper on top of the
gel and remove any bubbles with a pipet. Put 3 pieces 3 MM
paper on top of the nitrocellulose paper. Seal the edges of
the gel with Saran wrap and put a stack of paper towels on
top. Place a 500 g weight (a book works well) on the stack
of towels. Transfer overnight.

Remove the paper towel and 3 MM paper. Turn over gel with
nitrocellulose and use a pencil to mark each well.

Soak nitrocellulose in 2X SSC briefly, dry under a heat lamp,
and dry in an 80° C vacuum oven for two hours.
Prehybridization and Hybridization

Prehybridize for at least two hours at 42°C.
Prehybridization solution:

formamide 10.0 ml
25X SSC 4.0 ml
100X Denhardt’s 1.0 ml
10% SDS 0.2 ml
0. 2 M EDTA 0.1 ml
1 M NaPO4 1.0 ml

19.0 ml
tRNA (boil) J._,_Q mi

N
O
O

O
E
H

65

Random prime cDNA insert during the prehybridization.

Hybridize overnight at 42°C.

Hybridization solution:

formamide 10.0 ml

25X SSC - 4.0 ml

100X Denhardt's 0.2 ml

10% SDS 0.2 ml

0.2 M EDTA 1.0 ml

HQ'HZO 3.5.5 E].

19.0 ml

*tRNA (boil) J._,_Q mi
20.0 ml

*The tRNA should contain enough probe so that the
hybridization solution contains probe at 2 million cpm/ml.

Wash blot with 500 ml of 2X SSC 1% SDS at room temperature
for 10 minutes. Transfer blot to 0.1 or 0.2X SSC with 0.1%
SDS at 55°C or 65°C. Wash for 45 minutes and repeat twice.

66

APPENDIX C

(modification of cardiac muscle nuclei isolation method from
Oppenheimer et al., 1974)

BEAQENIE
1. Buffer A
0.32 M Sucrose 109.44 g.
3 mM MgCl 6.609 g.
1 mM RH 234 0.136 g.
Dissolve KH€PO4 in 20 ml M - O, and add 2 ml of this

KHZPO4 solu ion to 900 ml MQ- OH containing the other
chemicals. Adjust pH to 7.5 using HCl, then bring up to
1 l. with steriled H20. Filter through 0.45 um
millipore filter, then autoclave this solution.

2. Buffer B
2.4 M Sucrose 820.8 g.
3 mM MgC12 0.609 g.
Dissolve sucrose and MgCl in MQ-HZO to make one liter.
Filter through ordinary filter paper (No. 1).

3. Storage Buffer

40% glycerol 400 ml.
75 mM HEPES 17.9 g.
60 mM KCl '4.5 g.
15 mM NaCl 0.88 g.
0.15 mM Spermidine 1.2 ml.
0.5 mM Spermine 2.0 ml.
0.5 mM dithiothriotol 5.0 ml.
0.1 mM EDTA 0.5 ml.
0.1 mM EGTA 0.5 ml.

Melt 0.15 mM spermidine and 0.5 mM spermine in a 40°C
water bath. Add chemicals to MQ-H O to make 500 ml.
Filter through 0.45 um millipore filter, and add
glycerol. Adjust pH to 7.5 with HCl and add sterile
H20 to 1 liter.

NEQLEEE IEQLAIIQE REQIQQQL

1. Collect 20 g longissimus dorsi muscle in saline solution
after exsanguiation.

2. Grind with a meat grinder (some of the pieces cannot go
through the knives: out these with scissors in
Buffer A).

3. Separate the ground muscle into 10 orange-topped tubes
which each contain 10 ml Buffer A (2 g tissue/10 ml
Buffer A).

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

67

Homogenize with the Polytron tissue homogenizer at a very
low speed for 20-30 seconds.

Dilute the homogenized sample with 1 volume of Buffer A.

Filter through 1 layer cheesecloth into a polypropylene
bottle.

Spin at 700 x g for 10 minutes (in the G.S.A. rotor, 2.2
scale).

Decant supernatant, add 100 ml 0.25% X-100, resuspend
pellet by gentle homogenization.

Spin at 700 x g for 10 minutes.

Decant supernatant, add 100 ml Buffer A. Resuspend
pellet by gentle homogenization.

Spin at 700 x g for 10 minutes.
Repeat the Buffer A washing step one more time.

Decant the supernatant (removing as much of the
supernatant as possible).

Add approximately 170 ml Buffer B, resuspend pellet by
gentle homogenization.

Spin at 105,500 x g for 80 minutes in ultracentrifuge at
4 I .

Recover the pellet with storage buffer (using 0.5 ml
storage buffer at one time).

Collect the nuclei in storage buffer, put into an
Eppendorf tube. -

Spin at 7,000 x g for 3 minutes (in Beckman Microfuge, at
speed 10).

Resuspend nuclei into 1-2 ml storage buffer.

Check with a microscope (using 1 ul nuclei in 10 ul Rice
stain).

Take 10 ul storage buffer contained nuclei mix with 900
ul TE-8 and 100 ul 10% SDS.

Scan with spectrophotometry from 320 nm to 220 nm,
record the absorbange at 260 nm. DNA concentration of 1
ug/ul equal to 1X10 nuclei/ul.

Aliquate nuclei to 3X107, 2X107 and 1X107 nuclei per
tube, take one tube from each concentration to conduct

68

tritium incorporation assay. The other nuclei aliquates
store at -80°C until transcription assay.

69

APPENDIX D

1311193.INQQBEQBAIIQNCAEEAX.EBQIQQQL

1.

2.

3.

Solution A
10 ul nucleotide mixture
(50 mM ATP, 25 mM GTP, 25 mM CTP, 25 UN UTP)
10 ul S-adenosyl-erethionine
(1 mM SAM in 1 mM H2804)
80 ul s erile H20
60 ul [ H] UTP ===> Vacuum dry first

Soluiton B
0.6 M KCl 8.95 g.
12.5 mM Mg Acetate 0.536 g.

Dissolve into 200 m1 sterilized water, adjust pH 7.5 using
KOH or acetic acid, filter through 0.45 um millipore
filter paper then autoclave.

Transcription Mixture

Storage Buffer 112.7 ul
RNase Inhibitor (55 units/ul) 7.3 ul
Solution B 80.0 ul
Solution A 100.0 ul

Tritiun.1nsorneration Assay

1.

2.

Warm the transcription mixture at 26°C for 1 minute.

Add 100 ul nuclei aliquate (3x107, 2x107, and 1x107) to
the transcription mixture. IMMEDIATELY pipette out three
equal amounts (17 ul) of the reaction mixture (as 0
minute) and add to glass tubes which contained 100 ul

1 mg/ml tRNA.

Add 100 ul 20% TCA to each glass tube .

Take out sample at 5, 10, 15, 20, 30, and 60 minutes as
described in step 2 and 3.

Add about 2 ml 10% TCA to each glass tube and vortex.
Filter this solution through glass microfiber filter.

Transfer these filters to snitillation vials.
Add 10 ml safety solvent and count with scnillation
counter.

70

APPENDIX E

IBANEQBIIIIQE ASEAX IN 11139

BEAQEHIS
1. 0.4 M NaOH/l mM EDTA

400 ul of 5 M NaOH and 25 ul of 0.2 M EDTA, bring volume
to 5 ml with 4575 ul of GD-HZO.

2. Prehybridization Solution

formadine 2.5 ml.
25x SSC 1.0 m1.
100x Denhartt's 0.25 ml.
10 % SDS 0.05 ml.
1.0 M NaPO 0.25 ml.
0.2 M EDT 0.025 ml.
n20 0.675 ml.
Total 4.75 ml.
3. Hybridization Solution
formadine 2.5 ml.
25x SSC 1.0 ud.
100x Denhartt's 0.05 ml.
10% SDS 0.05 ml.
1.0 M NaPO 0.25 ml.
0.2 M EDT 0.025 ml.
H20 0.875 ml.
total 4.75 ml.

4. Solution A
10 ul nucleotide mixture
(50 mM ATP, 25 mM GTP, 25 mM CTP, 25 uM UTP)
10 ul S-adenosyl-L-methionine
(1 mM SAM in 1 mM H2804)
70 ul sSgrile H20
10 ul [ P] UTP

5. Soluiton B
0.6 M x01 8.95 g.
12.5 mM Mg Acetate 0.536 g.
Dissolve into 200 ml sterilized water, adjust pH 7.5 using
KOH or acetic acid, filter through 0.45 um millipore
filter paper then autoclave.

3. Transcription Mixture

Storage Buffer 112.7 ul
RNase Inhibitor (55 units/ul) 7.3 ul
Solution B 80.0 ul

Solution A ' 100.0 ul

71

INEQBQLIZE.£DNA

1.

5.

6.

10.

'linearize plasmid cDNA:

ul a-actin plasmid cDNA (2.5 ug/ul)-
ul psT-l

ul Buffer H

ul sterile H20

UHN-h

7 ul b-tubulin plasmid cDNA (3.75 ug/ul)
ul EcorI
ul Buffer H

3 ul sterile H20

ul psT-l
ul Buffer H
ul sterile H20

2.
2
1
4.
10 ul plasmid pBR322 (1 ug/ul)
5
7
3
Incubate at 37° C water bath for 3 hours.

Spin the Eppendrof tube for 3 minutes, add 5 ul 0.2 M

Add 10 ul of TE-8 and bring volume up to 25 ul (Not for
pBR322 digestion).

Add 1.25 ml 0.4 M NaOH/l mM EDTA and heat at 65°C for 10
minutes.

Cool on ice and add 312.5 ul 5 M NH4OAc.

Soak filter paper and nitrocellulose paper in H20 for 10
minutes, then transfer to 6X SSC for 10 minutes.

Assemble the minifold filter and rinse each well with 300
ul 6X SSC several times.

Filter the denatured plasmid DNA through nitrocellulose
paper.

Heat under a heat lamp, then bake at 80°C in a vacuum
oven for 2 hours.

Boil 0. 25 ml of 1 mg/ml tRNA for 10 minutes.
Prehybridize at 42° Cfor 17 hours.

Wessex

1. Warm up transcription mixture for 3 minutes at 26°C, then

mix with 100 ul nuclei (3X107).

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

72

Incubate at 26°C for 20 minutes. (Recommond for 42
minutes).

Add 12 ul 10 mg/ml tRNA and 2 ul DNase I (30 U/ul, final
conc.=1 unit/ml).

Incubate at 26°C for 3 minutes.

Add 4 ul 0.2 M EDTA, 14 ul 10% SDS, and 12 ul 25 mg/ml
proteinase K.

Incubate at 65°C for 2 hours.

Add 600 ul phenol:chloroform:isoamyl alcohol (25:24:1) to
the Eppendorf tube.

Vortex mix and spin for 3 minutes.

Remove aqueous layer to a new Eppendorf tube. Add 600 ul
chloroform:isoamyl alcohol (24:1).

Vortex and spin for 3 minutes.

Remove the aqueous layer to a new Eppendorf tube. Add 80
ul 5 M NH OAc and 1000 ul absolute ethanol. Percipitate
at -20°C or at least 1 hour.

Spin for 3 minutes. Resuspend pellet in 100 ul TE-8.

Add 100 ul 2x T/S-buffer, 2 ul RNase Inhibitor, 1 ul
DNase I, 10 ul tRNA. Digest at 37° C for 10 minutes.

Stop DNase I digestion with 3 ul 0.2 M EDTA. Add 20 ul
tRNA. '

Heat at 65°C for 5 minutes, then cool on ice.

Add 105 ul 5 M NH OAc and 700 ul isopropanol.
Percipitate at -2 °C for 20 minutes.

Spin for 3 minutes and resuspend pellet in 100 ul TE-8.

Add 40 oul 5 M NH4OAc and 500 ul isopropanol. Precipitate
at -20° C for 20 minutes.

Spin for 3 minutes and wash pellet with 200 ul 70%
ethanol.

Drain and dry the pellet. Resuspend in 50 ul TE-8.
Heat at 65°C for 5 minutes.

Take 1 ul transcripts, quantitated.

23.

24.

25.

26.

73

RNA-DNA hybridization at 42°C for 72 hours.

Wash dot blot with 0.1 ssc and 0.01 % SDS at 65°C for 45
minutes, three times.

Dry dot blot under heat lamp briefly.

Expose to X-ray film at -80°C for 24 hours or shorter
time.

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