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Isolation of full-length cDNAs encoding abundant adult human skeletal muscle mRNAs
Gene, 38 (1985) 177-188
177
Elsevier GENE
1398
Isolation of full-length cDNAs encoding abundant adult human skeletal muscle mRNAs (Recombinant DNA; gene library; fibroblasts; cardiac muscle; multiple transcripts; myogenesis; development; mitochondrial transcripts)
James C. Garrisons,*, Edna Hardemanb*, Robert Wade”, Larry Kede+** and Peter Gunning” 0 Department of Medicine, the MEDIGEN Project, and bDepartment of Biology, Stanford University and Veterans Administration Medical Center, Palo Alto, CA 94305 (U.S.A.) Tel. (415)493-2321 (Received
April 8th, 1985)
(Revision
received
and accepted
June 25th, 1985)
SUMMARY
We have used a method [Gunning et al., Mol. Cell. Biol. 3 (1983) 787-7951 of cDNA clone isolation from a cDNA library that selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length inserts. We screened an adult human skeletal muscle (skm) cDNA library and have isolated 46 cDNA clones which correspond to different mRNAs expressed at significant levels in adult skm. Of these cDNA clones 17 appear to be muscle-specific. Eleven are expressed in both cardiac muscle and skm but six are expressed primarily in skm. The remainder are expressed in muscle as well as in human fibroblasts. Comparison of cDNA insert size with mRNA size for the 17 clones expressed only in skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.
INTRODUCTION
* Permanent
address
University
of
(J.C.G.):
Virginia,
Department
Charlottesville,
of Pharmacology, VA
(U.S.A.)
Tel.
(804) 924-9976. * Current ford
address
Medical
(E.H.):
School,
Department Palo
of Pharmacology,
Alto,
CA
94305
Stan-
(U.S.A.)
Tel.
(415)497-5571. **To
whom
correspondence
and reprint
requests
should
be
addressed. Abbreviations:
Ap, ampicillin;
complementary
to mRNA;
bp, base pair(s);
EtBr, ethidium
pairs or 1000 bp; mt, mitochondrial; tified open reading skeletal
muscle;
frame;
cDNA,
bromide;
R, resistance;
SDS, sodium
URF, uniden-
dodecyl
sulfate;
SSC, 0.15 M NaCl, 0.015 M Na . citrate,
8; ::, novel joint.
0378/l 119/85/$03.30
0
1985 Elsevier
Science
DNA
kb, kilobase
Publishers
skm, pH 7-
The onset of myogenesis is accompanied by the differential regulation of a large number of genes. Analysis of mRNA complexity in pre-fusion myoblasts, myotubes and adult muscle indicates that a subset of these genes (20-50) - silent in myoblasts but actively transcribed in myotubes at very high levels - may account for 20-30x of muscle mRNA (Affara et al., 1980; Paterson and Bishop, 1977; Devlin and Emerson, 1978). The process of myogenesis is thought to involve the coordinate induction of transcription of these and presumably many other, less abundant, muscle-specific genes (Devlin and Emerson, 1978; 1979; Hastings and Emerson,
178
serts. This method is based on unique features of the
1982). However, studies using gene-specific DNA probes and protein-specific antibodies demonstrate
Okayama
the complex
not
Differential
temporal
curs at a number including fusion
properties
expression
of this process.
of muscle-specific
of stages of muscle
during (i) the formation
of myoblasts,
fibers,
(iii) neonatal
muscle
maturation
genes ocdevelopment
of myotubes
(ii) the innervation development, (Gartinkel
and Shimada, 1983). This complex pattern
and
agents
of muscle (iv) late
et al., 1982; Toyota
require
the
use
of
of muscle gene expression
as suggested
by recent
regulated studies
in-
volving mouse muscle-human non-muscle heterokaryons (Blau et al., 1983). A corollary of such a model is that genes activated at different times would respond to different inducing agents, whereas coordinately activated genes would respond to the same agent. With the recent development of an assay for gene sequences which compete for the same regulatory agents (Scholer and Gruss, 1984), it has become possible to test such models and to determine if muscle gene regulation is controlled by a small number of activating agents. To do this, it is first necessary to isolate genes which are regulated both coordinately and differentially during myogenesis. A reliable means of isolating genes which are regulated during myogenesis is to isolate first cDNA clones corresponding to regulated mRNAs. Such cDNAs can then be used to isolate the corresponding gene. To facilitate subsequent characterization of the genes - and in particular c&acting regulatory regions and transcription initiation sites - it is equally desirable to identify full-length cDNA clones. This may be particularly relevant for muscle-specific genes since a number of those examined to date, for example the genes encoding skeletal and cardiac Zactins and at least one encoding myosin heavy chain, contain introns in their 5’-untranslated regions (Buckingham and Minty, 1983; Mahdavi et al., 1984). The location of transcription initiation sites at the 5’ end of the first exon is greatly facilitated in such instances by the availability of full-length cDNAs. We have used a method of cDNA clone isolation from a cDNA library which selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length in-
radiolabeled
probes,
+ / - screening (St. John and Davis, 1979), or competition hybridization (Sargent and Dawid, 1983). We applied this method in screening skm cDNA
library
which correspond significant
an adult human
and have isolated to different mRNAs
46 cDNAs expressed
levels in adult skm. Of these cDNAs,
appear to be muscle-specific.
may well reflect the existence of temporally inducing
from
and Berg, (1982) cloning vehicle and does
at 17
Eleven are expressed in
both cardiac muscle and skm, but six are expressed primarily
in skm. The remainder
are expressed
muscle as well as in human fibroblasts.
in
Comparison
of cDNA inserts size with mRNA size for the 17 clones expressed only in skeletal or skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.
MATERIALS
AND METHODS
(a) Isolation of RNA and DNA Total RNA was isolated from adult human skm, the heart of the human heart-transplant patient and from HeLa cells exactly as described previously (Gunning et al., 1983a). Total HeLa cell DNA was isolated as described (Ponte et al., 1983). Plasmid DNA was isolated from 1.5 or 10 ml overnight bacterial cultures by the method of Holmes and Quigley (1981). (b) Restriction enzyme digestion and DNA electrophoresis DNA was digested with restriction enzymes under conditions specified by the supplier (New England BioLabs) using a 2-3-fold excess of enzyme. DNA fragments were separated by electrophoresis on agarose gels in 89 mM Tris, 89 mM borate, 2.5 mM EDTA (pH 8.4). Size-fractionated DNA was transferred to nitrocellulose paper (Millipore) by the method of Southern (1975).
179
(c) Recovery of DNA from agarose gels and transformation
RESULTS
(a) Rationale Linearized cDNA was visualized under UV light (305 nM) in gels by staining with EtBr and isolated from gel slices (Vogelstein and Gillespie, 1979). The extracted DNA was recircularized using the ligation procedure described previously (Gunning et al., 1983b) with the DNA concentration below 1 pg/ml to favor intra- over intermolecular ligation. Escherichia coli was transformed with the resulting plasmids and ApR colonies were selected (Gunning et al., 1983b). (d) cDNA insert isolation Plasmid DNA was isolated from selected clones and digested with the restriction endonucleases PvuII + PstI releasing the cDNA insert from the vector (Okayama and Berg, 1982). The digested DNA was electrophoresed on a 1.2% agarose gel and the largest cDNA insert fragment was isolated as described in section c above. The 850-bp PvuII fragment of the human skeletal actin cDNA clone pHMcrA-1 (Gunning et al., 1983b) was also prepared and used as a hybridization standard. (e) RNA hybridization analysis Serial dilutions of RNA were spotted directly onto nitrocellulose exactly as described by Thomas (1980). Total RNA was electrophoresed on 1% agarose, 2.2-M formaldehyde gels and transferred to nitrocellulose as described previously (Gunning et al., 1984). Each DNA preparation (1 pg) was nick-translated (Rigby et al., 1977) in parallel with the human skeletal actin cDNA probe (see section d above). Nitrocellulose blots were pre-hybridized and hybridized to radiolabeled DNA probes exactly as described previously (Gunning et al., 1984). The blots were washed with 0.5 x SSC, 0.1% at 65°C with five changes of solution and then subjected to radioautography with preflashed Kodak XAR-5 film.
We have previously described the construction of an adult human skm cDNA library using the Okayama and Berg (1982) method (Gunning et al., 1983b). In this procedure, the cDNA is initially synthesized using an oligo(dT)-tailed plasmid vector and the length of the cDNA insert present in the final cDNA clone is equal to the length of the initial reverse transcript. We have previously reported that, if plasmid DNA isolated from such a cDNA library is linearized with an enzyme which cuts once in the plasmid vector, the pattern of size-fractionated molecules consists of a number of discrete bands superimposed upon a background smear (Gunning et al., 1983b). Analysis of two such cDNA libraries further revealed that actin cDNA inserts fell into discrete size classes reflecting several preferred stopping points for reverse transcriptase including fulllength clones (Gunning et al., 1983b). We have devised a method for the isolation of cDNAs from such bands and the identification of the abundant class of cDNAs responsible for the band. (b) Direct visualization of the linearized cDNA library The adult human skm cDNA library in the form of plasmid DNA was linearized with one of four restriction enzymes which cut once in the vector, HindIII, ClaI, PvuI and PvuII. The resulting digests were analyzed on a 0.7% agarose gel and the DNA stained with EtBr. Fig. 1 shows that all four enzymes generate a large number of bands seen above a background smear. The DNA fragments vary in size from 2.5 kb (corresponding to the vector alone) up to 9 kb and greater. Bands are also observed migrating faster than the vector alone. These are presumably derived from cDNAs which are cleaved by the enzymes within the cDNA insert as well as in the vector. Analysis of the plasmid DNA from the library after digestion with different restriction enzymes supports the idea that many of the observed bands are composed of one major sequence type. Many of the bands are seen with 2 or 3 of the enzymes used but not with one of the other enzymes (Fig. 1). The
180
A B C D
a major sequence
class and a corresponding
unique
mRNA.
23.7,
(c) Isolation and identification
of abundant cDNAs
DNA was isolated from each of the bands observed in the Hind111 and CIaI digests greater than 3.4 kb in size and from the PvuI and PvuII digest
9-7,
bands
indicated
religation DNA,
6.79
Fig. 1. Visualization the restriction (D) HindIII. 0.7%
agarose
of linearized
human
muscle library,
in plasmid
endonucleases
(A) &I,
The resulting
digests
gel and stained
muscle cDNA library.
form, was digested
with
(B) PvuI, (C) PvuII and
were electrophoresed
with EtBr. The negative
on a image
resulting
from UV illumination
the ClaI
and Hind111 lanes
asterisks
in the PvuI and PvuII lanes were excised and DNA was
recovered.
of the gel is shown. Bands within and the two bands
This DNA was then recircularized
form E. coli. The positions standards
of, and transformation
12 of the resultant
(Fig. 1). Following with, the recovered
colonies
from each trans-
formation were grown and DNA was isolated by the mini-lysate procedure. We determined whether a
2*3The human
by an asterisk
are indicated
of co-electrophoresed
marked
with
and used to transI Hind111 size
and the sizes are shown in kb.
absence of a band most likely results from the presence of a second enzyme cleavage site within the cDNA insert thus reducing the band to a smaller size. This is not the pattern expected if a band were composed of a number of unrelated but similarly sized clones, since that would require the improbable coincidence that they all have the same restriction enzyme sites within their cDNA inserts. Therefore, it is highly probable that most of these bands contain
major sequence class existed in these 12 colonies by monitoring the fraction of identical clones, i.e. those displaying the same restriction pattern. We chose to rely upon restriction enzyme criteria since this analysis could be accomplished quickly and reliably. Accordingly, each group of 12 mini-lysate DNA preparations were digested with M&I, an enzyme that cuts frequently within DNA. As expected, all cDNA inserts examined were cut multiple times by this enzyme. A comparison of 12 clones from each transformation usually revealed that several have non-vector restriction fragments identical in size. It should be noted that clones derived from an identical mRNA could contain slightly nonidentical insert sizes due to differences in poly(A) tract length and 5’-end termination sites. This possible variability was taken into consideration when evaluating the restriction digest data. In most cases examined, a duplicated sequence type was identified within each group of 12 clones. Two examples of such an analysis are shown in Fig. 2. Bands common to all twelve clones represent plasmid DNA fragments. In the case of the clones derived from band H20 (Fig. 2A), it appears that clones d and 1 and clones f and j are members of two sequence classes. The remainder are unique. When band Cl0 was analyzed (Fig. 2B), it was more obvious that transformants d, e, f, h, i, j and k share internal restriction fragments and are members of a common sequence class. Again, the remaining clones are unique. Clones a, b, c and g represent clones with short inserts that have contaminated the Cl0 band. The unexpected presence of such short inserts was often observed with transformants derived from the high molecular weight size fractions. These may result from slowly migrating plasmids in
181
abcdefghi
abcdefghi
jkl
jkl
-V
-V -V
-V Fig. 2. Identification transformants A, respectively,
of cDNAs
generated
derived
by transformation
from identical
in Fig. 1. The DNA was digested
clones from each band are denoted
mRNAs.
DNA was isolated
of E. coli with DNA recovered with HinfI, electrophoresed
a-l. The position
of DNA fragments
a relaxed conformation that escaped restriction endonuclease cleavage. The overall analysis generated 72 examples of cDNA clones present two or more times among 12 independent isolates. In addition we derived over 300 apparently different cDNAs expressed in muscle but not present in multiple copies in these small samples. (d) Expression of cDNAs in adult human skeletal muscle We next tested the relative level of expression of each of the cDNA inserts we had tentatively identified as bearing abundantly expressed sequences. The amount of mRNA in adult human skm corresponding to each of the clone types was measured relative to that of the most highly expressed muscle mRNA, skeletal a-actin. cDNA insert fragments from each clone and from the human skeletal a-actin clone pHMc&-1 (Gunning et al., 1983b), were radio-labeled in parallel and hybridized to a dot panel of serial dilutions of human skm RNA. Fig. 3 shows the types of results obtained. Most of the clones hybrid-
from twelve cDNA
from band H20(A) on a 2% agarose
derived
clones chosen
and ClO(B) isolated gel and stained
from the vector
are indicated
at random
from
from lanes D and
with EtBr. The twelve by V.
ized with a similar intensity to that of actin (e.g., P2a, lane C) or within two orders of magnitude either higher (e.g., H18b, lane A) or lower (e.g., C23a, lane D). Using these comparative dot blots, we calculated the steady-state levels of transcripts related to each clone relative to that of skeletal cr-actin. The relative expression values for each clone relative to skeletal cr-actin and corrected for probe-length differences are summarized in Table I. The clones were grouped into live categories based upon these relative levels af expression. 19 clones were expressed at levels greater than that of actin. This was unexpected since actin accounts for 4-8% of skm mRNA (Schwartz and Rothblum, 1981). 40 clones were expressed at l-100% the level of actin, whereas only ten clones were expressed at < 1y0 of the level of actin mRNA. We therefore conclude that the majority of the cDNAs isolated in this way do correspond to moderately to highly abundant transcripts. After we compared the patterns generated by digestion of the clones with the restriction endonuclease HinfI, we suspected that some of the clones selected from different library bands might be trans-
182
TABLE
I
Level of expression Abundance Super
b
of cDNAs
(4 100%)
High (lo-loo%)
H6a
P,e
H7b
Psa HlOk
b
H8b HlOd ( Hllb
to actin
Moderate
b
(l-10%)
H4e ( c14g Hl2f H14d
HlOb Hl4i
Hl8f
C5a
Cl6a
C8f
Cl6b Cl7e
c9c Cllb
Hl8b
H22h
c12g
H24g
Clod
Cl5k
c21c
Clle
Cl7f
Cl3a
Cl9e C20a
Cl6c
( Cl4a Cl5b
C24b
Cl6e
No. 23
No. 19
H14f
Cl3e
Cl9b H20f
Rare (~0.1%)
c12c
Hl9f
H20d
b
(
Hl9a
Hl6i
H2lc
H8g Hllf
b
Hl6g
C24h
Low (0.1-l %)
Hl7g (
Hl5a
(
muscle relative
( Cl4d Hl6a b
H13i
b
skeletal
(% actin)
( H8c b
in adult human
C20b C2li C22d
No. 33
C22h C23a No. 64 No. 70
a The hybridization cc-actin cDNA
of each transcript b Bracketed
of cDNA
inserts to serial dilutions
(see Fig. 3). The hybridization relative
intensity
of adult human was corrected
muscle RNA was compared
with that of the skeletal
to actin and the abundance
to actin was calculated.
sets of clones are derived
from the same mRNA.
cripts from the same gene. We then tested such clones for cross hybridization to a panel of all the selected clones. Eight clones were found to be represented more than once in the population and these groups are indicated in Table I. Most of the multiply selected clones belong to the high and super abundant transcript categories. Overall, however, our cloning approach mainly generated non-redundant, presumably unique, cDNAs. (e) Characterization
skeletal
for the DNA probe length relative
of super-abundant
clones
Since actin is the most abundant mRNA within skm (Schwartz and Rothblum, 1981), the detection of clones expressed at levels greater than that of actin is most surprising. The most likely interpretation is that these clones contain repetitive sequences. Accordingly, we hybridized representatives
of each of the 12 superabundant clone types to size fractionated, EcoRI digested, total human DNA. Three types of hybridization patterns were observed in these Southern blots. Six different cDNA types (H6a, C24b, H21c, H16i, H16g, H15a). hybridized to a smear of genomic DNA fragments covering the entire length of the gel. These clones therefore correspond to highly repetitious genomic sequences some members of which are transcribed in skm. Five clones, (H18b, H13i, C16c, No. 23, No. 33), hybridized very strongly to one, or in some cases two, EcoRI band(s). The final superabundant clone, HlOd, hybridized to a large number of discrete bands. The clones which produced the two simpler hybridization patterns were further analyzed by hybridization to human DNA digested with seven restriction enzymes (Fig. 4). HlOd hybridized to a discrete number of genomic fragments with each en-
183
A
A
B
B 1234567
1234567
Fig. 4. Hybridization
of “super-abundant”
DNA
several
digested
with
restriction
cDNAs enzymes.
to human 32P-labeled
cDNA inserts isolated from clones (A), H 10d and (B) H18b were hybridized tionated
to human
restriction
endonuclease
DNA. Following
filters were autoradiographed HindIII,
Fig. 3. Expression
of cDNA
clones
in human
skeletal
muscle
relative to that of actin. The cDNA inserts of the different clones were nick-translated fragment
isolated
(pHMaA-1) hybridized
in parallel from
(Gunning
the
an 850-bp skeletal
PvuII-Xbal
m-actin
cDNA
et al., 1984). The 32P-labe1ed probes were
to serial dilutions
nitrocellulose.
with
human
of human muscle RNA dotted onto
A typical autoradiogram
RNA in each dot is indicated (A), H18b; (B), skeletal
is shown. The quantity
of
in pg units. The probes used were
a-actin;
(C) P2a and (D), C23a.
zyme digest (Fig. 4A) and therefore recognizes a moderately repetitive genomic sequence. In contrast H18b hybridized very strongly to only one to three genomic fragments with each enzyme digest (Fig. 4B). However, the most complex hybridization pattern observed with H18b occurred with human DNA cut with BglII (Fig. 4B, lane 2). This pattern of DNA electrophoresis was reminiscent of that observed for a large circular DNA molecule existing in relaxed and supercoiled forms and suggested that H18b might be hybridizing to the mt genome. Furthermore, BglII does not cleave human mt DNA, whereas all the other enzymes we tested cut at least once (Anderson et al., 1981). Similar hybridization patterns were observed for cDNA clones H13i,
(4) PstI, (5) SacI,
digested,
hybridization
for 6 h. (1) EcoRI, (6) XbaI
(2) BglII,
the (3)
(7) BumHI.
The
1 Hind111 size standards
are
positions
of co-electrophoresed
indicated
and the sizes are shown
and
size-frac-
and washing,
in kb.
C16c, No. 23 and No. 33 and suggested that these might also represent mt transcripts. The presumptive mt cDNAs were positively identified by restriction mapping. The sizes of human DNA fragments hybridizing to each of these clones was determined. Comparison of these fragment sizes with the restriction map of the human mt genome allowed us to unambiguously assign each clone to a defined region. The cDNAs were then restriction mapped and compared with the corresponding region of the mt genome. The results are shown in Fig. 5. Clones H18b, H13i, C16c and No. 33 correspond to full-length cDNAs of the mitochondrial 16s rRNA, ATPase 6, URF2 and CO111 mRNAs respectively (Fig. 5). Clone No. 23 is a cDNA of the mt URFl mRNA and is about 150 bp short of full length. In summary, all of the cDNAs identified as superabundant expressers either carried highly or moderately repetitive sequences or proved to be mt transcripts. (f) Identification of full-length cDNAs
The length of the cDNA inserts in all the highly abundant and most of the moderately abundant cDNAs were compared with the corresponding
184 =
; h I
; f I
1610
1240
=
0 :: I
% I 620
d 1
310
TABLE H18b pHMMt-16s
'
II
cDNA
clone
skeletal
muscle
Clone 5’-3’
=
r
-
E
%;
I
-;
880 730
400
“;
230200
N
P,a HlOb
?
3.3
2.9
Y
HlOk
3.3
6.0, 4.3, 2.7, 1.5 ?
Hl2f
2.6
2.5, 1.5
Hl3i
1.1
0.9
Y
Hl4d
2.2
4.2
N
=
Hl6a
1.9
1.7
Y
..f #33
Hl8b
1.6
1.5
Y
Hl9a
1.3
4.2
N Y
= #23 pHMMt-URFl
pHMMt-CO3
= Clbc
chondrial
The restriction
cDNAs
genome.
are shown. The distance,
from the 3’ terminus maps and direction the human co-linear
of cDNAs
of transcription (Anderson
with a mt transcript.
in bp, of each restriction
mitosite
These restriction
were compared
with that of
et al., 1981). Each cDNA was
The cDNAs
mt gene. In addition,
and H 13i have been directly
by human
maps of the live types of mt
PvuII site is indicated.
mt genome
the corresponding
encoded
sequenced
are named to identify regions of clones Hl8b
to confirm their identity
(not shown).
mRNA
sizes to determine
Full-length
2.2, 1.8
OpHMMt-URF2
maps
(kb)
2.2
III;-
Fig. 5. Restriction
mRNA
mRNA
1.4
’
I s
(kb)
corresponding
1.7
900
z % t
cDNA
size and
P,e
0
4izo
insert
whether they represented
full-length copies. The cDNAs were linearized with the restriction endonuclease initially used to linearize the corresponding band in the cDNA library and the complete clone length was determined by co-electrophoresis with size standards. The size of the vector, 2.5 kb, was subtracted from the clone length to yield the cDNA insert size. The inserts varied in size from 0.9 kb to 4.7 kb (Table II). Size fractionated adult skm RNA was hybridized with each of the clones and the corresponding transcript sizes were measured. Table II shows that whereas a majority of the clones hybridized to a single transcript size, 12 of them hybridized to multiple (2 to 4) transcripts. For six of the clones that recognized multiple transcripts, we could not unambiguously determine whether the cDNAs were full-length. However, 16 of the remaining 26 clones tested were within 5% of being fulllength (Table II). This represents a remarkably high yield of full-length cDNAs and will simplify characterization of the corresponding genes.
H20d
1.3
1.4
1.3
6.2
N
H22h
1.0
0.9
Y
C5a
3.2
5.6, 4.2
N
C8f
4.7
4.3
Y
c9c
4.1
8.6
N
Clod
4.0
4.0, 7.5
?
ClIe
3.6
3.6
Y
Cl3a
2.8
6.5
N
c14g
3.0
2.8
Y
Cl5b
2.7
2.4
Y
Cl5k
2.7
5.0, 4.3
N
Cl6c
1.1
1.0
Y
Cl6e
2.7
2.5, 1.5
Y
C20b
1.6
4.3, 2.5, 1.2
?
C2li
1.6
2.6, 1.5
?
C23a
1.2
2.5, 1.2
?
No. 19
1.0
1.5, 1.0, 0.8
Y
No. 23
0.9
1.1
N
No. 33
0.9
0.8
Y
No. 64 No 70
1.0
4.4. 1.8
N
I 0
I0 -____
Y
3
The size of the cDNA insert and the corresponding
section
as described
(Y/N)b
Y
H20f
determined
size in
in MATERIALS
AND
mRNA was METHODS,
d.
b Y denotes cDNA inserts which are within 5% of the size of the mRNA. N denotes size of the mRNA. unambiguously
cDNA inserts which are less than 95% of the ? denotes
evaluated
cDNA
inserts
which
cannot
due to the existence ofmultiple
be
hybrid-
izing transcripts.
(g) Cell-type-specific
expression of cDNAs
Previous studies have indicated that most of the abundant transcripts in skm are specific for that tissue (Paterson and Bishop, 1977; Affara et al., 1980). We therefore tested the specificity of expres-
185
sion of the isolated cDNA clones in muscle and non-muscle cells. Radiolabeled cDNA inserts were hybridized to serial dilutions of human skm, heart and HeLa cell RNA. Fig. 6 shows examples of the three types of hybridization patterns which were observed. Some clones such as Clod hybridized exclusively with skm RNA, others like C9c hybridized only to skm and heart RNA whereas the third group hybridized to RNA of all three cell types. The expression of each of these cDNAs in heart and HeLa cells relative to that in skm was calculated. Table III shows that 11 of 13 cDNAs expressed at high abundance in skm (as defined in Table I) are expressed at very low levels, if at all, in HeLa cells. In contrast, only 6 of the 15 cDNAs expressed at moderate abundance in skm display a strong preferential expression in muscle cells. This result correlates well with the expectation that the most abundant muscle transcripts are most likely to encode muscle-specific proteins (Paterson and Bishop, 1977). We therefore conclude that we have isolated cDNAs corresponding to abundant skm mRNAs - many of which display cell-type specific expression - and have simultaneously selected for full-length cDNAs.
DISCUSSION
(a) Isolating skeletal muscle cDNA clones
We have taken advantage of the Okayama and Berg (1982) cloning system to devise a method of
A SH
B S
H
Expression
muscle
Clone
Expression Skeletal
P,a
H
H16a
H
F
*
Fig. 6. Expression inserts of (A) Clod, hybridized skeletal
in
muscle”
of cDNAs
in different
to serial dilutions
of RNA isolated
muscle (S), adult human
The quantity
cell types. The cDNA
(B) C9c and (C) C15b were “P-labeled
and
from adult human
heart (H) and HeLa cells (F).
of RNA in each dot is indicated
in pg.
Hearth skm
Fibroblastsb skm
0.025
< 0.005
< 0.005
< 0.005
H22h
H
0.025
< 0.005
H
< 0.005
< 0.005
Clle
H
0.025
C13a
H
0.05
<0.005
P,e
H
0.5
< 0.025
H20d
H
8
0.025
H20f
H
2
< 0.005
0.025
C16e
H
< 0.005
H12f
M
< 0.005
H14d
M
C9c
M
Cllb
M
C20b
< 0.005 0.25
< 0.005
M
4
< 0.025 4 0.005
0.1
C23a
M
2
No. 19
H
0.25
0.1
HlOk
H
0.5
0.5
C15b
H
0.5
H19a
M
C5a
M
C8f
M
C14g
M
C15k
M
C20a
M
C21i
M
0.5
0.2
No. 64
M
4
0.2
No. 70
M
1
HlOb
L
2 2 0.5
16
0.5
4
0.25 2
2 10
in skeletal
muscle is shown as abundance
relative
an L is low, as
defined in Table I. expression
of these
relative to their expression
‘*
0.25
relative to that in
Clod
hybridization r)
in heart and fibroblasts
to that of actin where H is high, M is moderate
*
0.5
of cDNAs
skeletal
‘The 2
III
a Expression
C F
F
TABLE
to parallel
sequences
in heart
and tibroblasts
in skeletal muscle was determined RNA serial dilutions
by
as shown in Fig. 6.
directly isolating full-length cDNA copies of abundant mRNAs. Using this method, we have isolated 13 cDNA clones which correspond to abundant skm transcripts. At least seven of these 13 cDNAs are within 5 % of being full-length clones. Normally, the recovery of full-length clones from conventional cDNA libraries is very low. We have previously re-
186
ported that representation of full-length actin clones in Okayama-Berg (1982) cDNA libraries can vary from less than 1% (for /!i-actin in a fibroblast
library)
experiments can, with certainty, quickly clones corresponding to the same mRNA. Despite the success of our screening,
it is clear that
to 25% (for skeletal actin in a muscle library) of all
the efficiency could be improved.
actin
ber of selected clones did not correspond
clones
cDNA transcript
(Gunning
inserts
et al.,
of at least
1983b).
Since
16 of 26 clones
size was determined
whose
are within 5% of full
length (Table II), it is likely that this screening thod enriches for full-length the fact that rescreening length
clones
the
me-
clones. We can attest to a cDNA
corresponding
to
library
for full-
lo-20
different
mRNAs is a major undertaking using conventional cloning and screening procedures. Previous workers who have screened muscle cDNA libraries usually used radiolabeled cDNA as a probe to identify clones corresponding to abundant mRNAs. Hastings and Emerson (1982) obtained clones corresponding to 17 abundant quail muscle mRNAs. Garfinkel et al. (1982) similarly obtained copies of twelve different rat muscle mRNAs. In contrast, Putney et al. (1983) randomly sequenced 178 rabbit muscle cDNA clones and identified clones corresponding to 13 different abundant mRNAs. Our isolation of 13 different clones compares very well with the yield in these other studies and suggests that between these four collections, all the abundant vertebrate skm mRNAs have probably now been isolated as cDNA clones. Interestingly, except for one cDNA copy of myosin heavy chain in our isolates, we have found (R.W., unpublished) that none of our abundant clones cross-hybridize with any of those from a quail muscle library (Hastings and Emerson, 1982) kindly provided by Dr. Charles Emerson. (b) Methodology One of the major problems associated with conventional cDNA screening is the failure to identify clones encoding the same mRNA since such cDNA clones may cover nonoverlapping mRNA regions. In such situations, positive identification usually requires (1) sequencing of the cDNAs and comparison with a known protein sequence or (2) comparison of genomic Southern blot and RNA Northern blot data. This problem is completely eliminated with the Okayama and Berg (1982) vector since all clones start at the same position, the 3’ end of the mRNA. Thus restriction analysis and/or cross-hybridization
dant mRNAs.
Conversely,
of clones corresponding overlooked.
identify
A significant
num-
to abun-
it is likely that a number to abundant
mRNAs
were
This is clearly true for actin, which is the
most abundant
muscle mRNA
and yet was not iso-
lated. In this case however, the failure may be due to an intrinsic Although
difficulty
in generation
actin accounts
for 4-8%
of actin clones. of total skm po-
ly(A) + mRNA (Schwartz and Rothblum, 198 l), only 0.6% of the cDNAs in our muscle library encode actin (Gunning et al., 1983b). To improve the efficiency of isolation of abundant cDNA clones, it would be useful to introduce a preliminary screening test for their abundance. 20-30 colonies obtained from transfections with each recircularized DNA fraction could be hybridized first to radiolabeled muscle poly(A) + mRNA. Clones from each fraction which hybridize strongly could then be compared using the HinfI plasmid DNA digestion test to identify identical clone types. Since all Okayama-Berg (1982) cDNA clones contain a poly(A): : poly(T) 3 ’ segment, such a screening test would need to be carried out with radiolabeled poly(A) + mRNA divested of poly(A)+ -containing fragments prior to hybridization (Garfinkel et al., 1982). (c) Isolation cDNAs
and identification
of mitdchondrial
Considering the abundance of mt transcripts in adult skm, it is not surprising that a number of corresponding cDNAs were isolated by this method. In fact, the initial size selection of the cDNAs probably prevented us from isolating even more of them since mt mRNAs are less than 1.0 kb in size (Anderson et al., 1981) and our size cuts eliminated clones with cDNA inserts less than 0.9 kb. Mt cDNAs are certainly well represented in cDNA libraries constructed from total tissue poly(A)+ mRNA. Such clones can be quickly identified by hybridization of cDNA inserts to size-fractionated human DNA which has been digested with an enzyme which is anoncutter for the mt genome (Fig. 4). In addition, it is simple to quickly determine the identity of such clones using only a few restriction enzyme digests.
187
(d) Tissue-specific expression of abundant skeletal muscle mRNAs
Anderson,
S., Banker,
Coulson,
B.A., Sanger,
Hybridization of the cDNA probes to human RNA preparations revealed that most of the abundant muscle mRNAs are at least sarcomeric-musclespecific. Using human myoblast RNA, we have more recently confirmed that the expression of these muscle-specific mRNAs is induced during human myogenesis (P.G., E.H. and R.W., unpublished). This extends results previously reported using total mRNA hybridization experiments (Paterson and Bishop, 1977; Devlin and Emerson, 1978; Affara et al., 1980). Since heart and skrn contain similar structural proteins and enzymes, it is surprising to find that one third of the sacromeric-muscle-specific cDNAs are specific to skm alone. This most likely reflects the differential utilization of diverged members of multigene families between heart and skm (Buckingham and Minty, 1983). We are currently sequencing these muscle-specific cDNAs to help identify the corresponding protein products.
and Young,
We thank Doris Hosmer for help in the preparation of this manuscript and Peter Evans for providing excellent technical assistance. We also thank Adrian Minty, Hiroto Okayama and Helen Blau for many helpful discussions. This work was supported in part by grants to L.K. from the National Institutes of Health (HD17031), Veterans Administration and the Muscular Dystrophy Association. E.H. is a fellow of the National Institutes of Health and R.W. is a fellow of the Muscular Dystrophy Association. J.C.G. was supported in part by a Research Career Development Award from the National Institutes of Health (AM00491).
F., Schreier, genome.
of human
nuclear
deBruijn,
genes
D.P., Roe,
A.J.H.,
and organization
Nature
M.H.L.,
I.C., Nierlich,
P.H., Smith,
LG.: Sequence
mitochondrial
B.G.,
J., Eperon,
Staden,
R.
of the human
290 (1981) 457-465.
Blau, H.M., Chiu, C-P. and Webster,
C.: Cytoplasmic
in stable
activation
heterokaryons.
Cell 32
(1983) 1171-1180. Buckingham,
M.E. and Minty, A.J.: Contractile
Maclean,
N., Gregory,
Devhn,
London,
Activity
R.A. (Eds.), Eukaand Regulation.
But-
1983, pp. 365-395.
R.B. and Emerson,
contractile
protein genes, in
S.P. and Flavell,
ryotic Genes: Their Structure, terworth,
protein
C.P.: Coordinate
mRNAs
accumulation
during myoblast
of
differentaition.
Cell 13 (1978) 599-611. Devlin,
R.B. and Emerson,
contractile
protein
Develop. Gartinkel,
C.P.: Coordinate
mRNAs
during
accumulation
myoblast
of
differentiation.
Biol. 69 (1979) 202-216. L.I., Periasamy,
and characterization pomyosin
and
M. and Nadal-Ginard, of cDNA
sequences
cc-actin. J. Biol. Chem.
B.: Cloning
corresponding
I,2 and 3, troponin-C,
myosin light chains
to
troponin-T,
a-tro-
275 (1982)
11078-
11086. Gunning,
P., Ponte,
P., Blau, H. and Kedes,
cc-cardiac actin genes are co-expressed tal muscle Gunning,
ACKNOWLEDGEMENTS
A.T., Barrell,
A.R., Drouin,
and heart.
P., Ponte,
Kedes,
L.:
cDNA
clones
L.: a-skeletal
in adult human
Mol. Cell. Biol. 3 (1983a)
P., Okayama,
Isolation
and
for human
and skele-
1783-1791.
H., Engel, J., Blau, H. and
characterization alpha-,
beta-
mRNAs:
skeletal but not cytoplasmic
terminal
cysteine
of full-length and
gamma-actin
actins have an amino
that is subsequently
removed.
Mol. Cell.
Biol. 3 (1983b) 787-795. Gunning,
P., Mohun, T., Ng, S., Ponte, P. and Kedes, L.: Evolu-
tion of the human
sacromeric-actin
genes: evidence
of selection within the 3’ untranslated
for units
regions ofthe mRNAs.
J. Mol. Evol. 20 (1984) 202-214. Hastings,
K.E.M. and Emerson,
co-regulated proteins. Holmes,
mRNAs
Proc. Natl. Acad.
D.S. and Quigley,
preparation
C.P.: cDNA clone analysis of six
encoding
of bacterial
skeletal
muscle
contractile
Sci. USA 79 (1982) 1553-1557.
M.: A rapid boiling method plasmid.
Anal. Biochem.
for the
114 (1981)
193-197. Mahdavi,
V., Chambers,
and B-myosin
Proc. Natl. Acad. Okayama,
A.P. and Nadal-Ginard,
heavy
Paterson,
a-
in tandem.
Sci. USA 8 1 (1984) 2626-2630.
H. and Berg, P.: High-efftciency
cDNA.
B.: Cardiac
chain genes are organized
cloning of full-length
Mol. Cell. Biol. 2 (1982) 161-170. B.M. and Bishop, J.O.: Changes
tion of chick myoblasts
during
in the mRNA popula-
myogenesis
in vitro. Cell 12
(1977) 751-765. Ponte,
P., Gunning,
multicopy Affara.
N.A..
Robert,
Gros, F.: Changes entiation, pressed 441-458.
P., Blau, H. and Kedes,
genes are single copy for a-skeletal
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differex-
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for /?- and are isotype
y-cytoskeletal
L.: Human
and a-cardiac genes:
specific but are conserved
actin
actin but
3’ untranslated in evolution.
Mol. Cell. Biol. 3 (1983) 1783-1791. Putney,
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W.C. and Schimmel,
P.: A new troponin
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Nature
302 (1983) 718-721.
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DNA to high specific activity in vitro by nick-translation DNA polymerase Sargent,
I. J. Mol. Biol. 113 (1977) 237-251.
T.D. and Dawid,
I.B.: Differential
of Xenopus luevis. Science
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Scholer, H.R. and Gruss, cer-containing
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Cell 46
(1984) 403-411. Schwartz,
R.J.
myogenesis;
and
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Rothblum,
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separated
(1975) 503-517.
Gene
switching
of the actin
in
multigene
20 (1981) 4122-4129.
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fragments
K.N.:
expression of specific
sequences
by gel electrophoresis.
DNA sequences plaque
gene expression
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St. John, T.P. and Davis, R.W.: Isolation
Thomas,
DNA
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Cell 16 (1979) 443-452. of denatured
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Sci. USA 77 (1980) 5201-5205.
Toyota,
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N. and Shimada,
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Elsevier GENE
1398
Isolation of full-length cDNAs encoding abundant adult human skeletal muscle mRNAs (Recombinant DNA; gene library; fibroblasts; cardiac muscle; multiple transcripts; myogenesis; development; mitochondrial transcripts)
James C. Garrisons,*, Edna Hardemanb*, Robert Wade”, Larry Kede+** and Peter Gunning” 0 Department of Medicine, the MEDIGEN Project, and bDepartment of Biology, Stanford University and Veterans Administration Medical Center, Palo Alto, CA 94305 (U.S.A.) Tel. (415)493-2321 (Received
April 8th, 1985)
(Revision
received
and accepted
June 25th, 1985)
SUMMARY
We have used a method [Gunning et al., Mol. Cell. Biol. 3 (1983) 787-7951 of cDNA clone isolation from a cDNA library that selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length inserts. We screened an adult human skeletal muscle (skm) cDNA library and have isolated 46 cDNA clones which correspond to different mRNAs expressed at significant levels in adult skm. Of these cDNA clones 17 appear to be muscle-specific. Eleven are expressed in both cardiac muscle and skm but six are expressed primarily in skm. The remainder are expressed in muscle as well as in human fibroblasts. Comparison of cDNA insert size with mRNA size for the 17 clones expressed only in skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.
INTRODUCTION
* Permanent
address
University
of
(J.C.G.):
Virginia,
Department
Charlottesville,
of Pharmacology, VA
(U.S.A.)
Tel.
(804) 924-9976. * Current ford
address
Medical
(E.H.):
School,
Department Palo
of Pharmacology,
Alto,
CA
94305
Stan-
(U.S.A.)
Tel.
(415)497-5571. **To
whom
correspondence
and reprint
requests
should
be
addressed. Abbreviations:
Ap, ampicillin;
complementary
to mRNA;
bp, base pair(s);
EtBr, ethidium
pairs or 1000 bp; mt, mitochondrial; tified open reading skeletal
muscle;
frame;
cDNA,
bromide;
R, resistance;
SDS, sodium
URF, uniden-
dodecyl
sulfate;
SSC, 0.15 M NaCl, 0.015 M Na . citrate,
8; ::, novel joint.
0378/l 119/85/$03.30
0
1985 Elsevier
Science
DNA
kb, kilobase
Publishers
skm, pH 7-
The onset of myogenesis is accompanied by the differential regulation of a large number of genes. Analysis of mRNA complexity in pre-fusion myoblasts, myotubes and adult muscle indicates that a subset of these genes (20-50) - silent in myoblasts but actively transcribed in myotubes at very high levels - may account for 20-30x of muscle mRNA (Affara et al., 1980; Paterson and Bishop, 1977; Devlin and Emerson, 1978). The process of myogenesis is thought to involve the coordinate induction of transcription of these and presumably many other, less abundant, muscle-specific genes (Devlin and Emerson, 1978; 1979; Hastings and Emerson,
178
serts. This method is based on unique features of the
1982). However, studies using gene-specific DNA probes and protein-specific antibodies demonstrate
Okayama
the complex
not
Differential
temporal
curs at a number including fusion
properties
expression
of this process.
of muscle-specific
of stages of muscle
during (i) the formation
of myoblasts,
fibers,
(iii) neonatal
muscle
maturation
genes ocdevelopment
of myotubes
(ii) the innervation development, (Gartinkel
and Shimada, 1983). This complex pattern
and
agents
of muscle (iv) late
et al., 1982; Toyota
require
the
use
of
of muscle gene expression
as suggested
by recent
regulated studies
in-
volving mouse muscle-human non-muscle heterokaryons (Blau et al., 1983). A corollary of such a model is that genes activated at different times would respond to different inducing agents, whereas coordinately activated genes would respond to the same agent. With the recent development of an assay for gene sequences which compete for the same regulatory agents (Scholer and Gruss, 1984), it has become possible to test such models and to determine if muscle gene regulation is controlled by a small number of activating agents. To do this, it is first necessary to isolate genes which are regulated both coordinately and differentially during myogenesis. A reliable means of isolating genes which are regulated during myogenesis is to isolate first cDNA clones corresponding to regulated mRNAs. Such cDNAs can then be used to isolate the corresponding gene. To facilitate subsequent characterization of the genes - and in particular c&acting regulatory regions and transcription initiation sites - it is equally desirable to identify full-length cDNA clones. This may be particularly relevant for muscle-specific genes since a number of those examined to date, for example the genes encoding skeletal and cardiac Zactins and at least one encoding myosin heavy chain, contain introns in their 5’-untranslated regions (Buckingham and Minty, 1983; Mahdavi et al., 1984). The location of transcription initiation sites at the 5’ end of the first exon is greatly facilitated in such instances by the availability of full-length cDNAs. We have used a method of cDNA clone isolation from a cDNA library which selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length in-
radiolabeled
probes,
+ / - screening (St. John and Davis, 1979), or competition hybridization (Sargent and Dawid, 1983). We applied this method in screening skm cDNA
library
which correspond significant
an adult human
and have isolated to different mRNAs
46 cDNAs expressed
levels in adult skm. Of these cDNAs,
appear to be muscle-specific.
may well reflect the existence of temporally inducing
from
and Berg, (1982) cloning vehicle and does
at 17
Eleven are expressed in
both cardiac muscle and skm, but six are expressed primarily
in skm. The remainder
are expressed
muscle as well as in human fibroblasts.
in
Comparison
of cDNA inserts size with mRNA size for the 17 clones expressed only in skeletal or skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.
MATERIALS
AND METHODS
(a) Isolation of RNA and DNA Total RNA was isolated from adult human skm, the heart of the human heart-transplant patient and from HeLa cells exactly as described previously (Gunning et al., 1983a). Total HeLa cell DNA was isolated as described (Ponte et al., 1983). Plasmid DNA was isolated from 1.5 or 10 ml overnight bacterial cultures by the method of Holmes and Quigley (1981). (b) Restriction enzyme digestion and DNA electrophoresis DNA was digested with restriction enzymes under conditions specified by the supplier (New England BioLabs) using a 2-3-fold excess of enzyme. DNA fragments were separated by electrophoresis on agarose gels in 89 mM Tris, 89 mM borate, 2.5 mM EDTA (pH 8.4). Size-fractionated DNA was transferred to nitrocellulose paper (Millipore) by the method of Southern (1975).
179
(c) Recovery of DNA from agarose gels and transformation
RESULTS
(a) Rationale Linearized cDNA was visualized under UV light (305 nM) in gels by staining with EtBr and isolated from gel slices (Vogelstein and Gillespie, 1979). The extracted DNA was recircularized using the ligation procedure described previously (Gunning et al., 1983b) with the DNA concentration below 1 pg/ml to favor intra- over intermolecular ligation. Escherichia coli was transformed with the resulting plasmids and ApR colonies were selected (Gunning et al., 1983b). (d) cDNA insert isolation Plasmid DNA was isolated from selected clones and digested with the restriction endonucleases PvuII + PstI releasing the cDNA insert from the vector (Okayama and Berg, 1982). The digested DNA was electrophoresed on a 1.2% agarose gel and the largest cDNA insert fragment was isolated as described in section c above. The 850-bp PvuII fragment of the human skeletal actin cDNA clone pHMcrA-1 (Gunning et al., 1983b) was also prepared and used as a hybridization standard. (e) RNA hybridization analysis Serial dilutions of RNA were spotted directly onto nitrocellulose exactly as described by Thomas (1980). Total RNA was electrophoresed on 1% agarose, 2.2-M formaldehyde gels and transferred to nitrocellulose as described previously (Gunning et al., 1984). Each DNA preparation (1 pg) was nick-translated (Rigby et al., 1977) in parallel with the human skeletal actin cDNA probe (see section d above). Nitrocellulose blots were pre-hybridized and hybridized to radiolabeled DNA probes exactly as described previously (Gunning et al., 1984). The blots were washed with 0.5 x SSC, 0.1% at 65°C with five changes of solution and then subjected to radioautography with preflashed Kodak XAR-5 film.
We have previously described the construction of an adult human skm cDNA library using the Okayama and Berg (1982) method (Gunning et al., 1983b). In this procedure, the cDNA is initially synthesized using an oligo(dT)-tailed plasmid vector and the length of the cDNA insert present in the final cDNA clone is equal to the length of the initial reverse transcript. We have previously reported that, if plasmid DNA isolated from such a cDNA library is linearized with an enzyme which cuts once in the plasmid vector, the pattern of size-fractionated molecules consists of a number of discrete bands superimposed upon a background smear (Gunning et al., 1983b). Analysis of two such cDNA libraries further revealed that actin cDNA inserts fell into discrete size classes reflecting several preferred stopping points for reverse transcriptase including fulllength clones (Gunning et al., 1983b). We have devised a method for the isolation of cDNAs from such bands and the identification of the abundant class of cDNAs responsible for the band. (b) Direct visualization of the linearized cDNA library The adult human skm cDNA library in the form of plasmid DNA was linearized with one of four restriction enzymes which cut once in the vector, HindIII, ClaI, PvuI and PvuII. The resulting digests were analyzed on a 0.7% agarose gel and the DNA stained with EtBr. Fig. 1 shows that all four enzymes generate a large number of bands seen above a background smear. The DNA fragments vary in size from 2.5 kb (corresponding to the vector alone) up to 9 kb and greater. Bands are also observed migrating faster than the vector alone. These are presumably derived from cDNAs which are cleaved by the enzymes within the cDNA insert as well as in the vector. Analysis of the plasmid DNA from the library after digestion with different restriction enzymes supports the idea that many of the observed bands are composed of one major sequence type. Many of the bands are seen with 2 or 3 of the enzymes used but not with one of the other enzymes (Fig. 1). The
180
A B C D
a major sequence
class and a corresponding
unique
mRNA.
23.7,
(c) Isolation and identification
of abundant cDNAs
DNA was isolated from each of the bands observed in the Hind111 and CIaI digests greater than 3.4 kb in size and from the PvuI and PvuII digest
9-7,
bands
indicated
religation DNA,
6.79
Fig. 1. Visualization the restriction (D) HindIII. 0.7%
agarose
of linearized
human
muscle library,
in plasmid
endonucleases
(A) &I,
The resulting
digests
gel and stained
muscle cDNA library.
form, was digested
with
(B) PvuI, (C) PvuII and
were electrophoresed
with EtBr. The negative
on a image
resulting
from UV illumination
the ClaI
and Hind111 lanes
asterisks
in the PvuI and PvuII lanes were excised and DNA was
recovered.
of the gel is shown. Bands within and the two bands
This DNA was then recircularized
form E. coli. The positions standards
of, and transformation
12 of the resultant
(Fig. 1). Following with, the recovered
colonies
from each trans-
formation were grown and DNA was isolated by the mini-lysate procedure. We determined whether a
2*3The human
by an asterisk
are indicated
of co-electrophoresed
marked
with
and used to transI Hind111 size
and the sizes are shown in kb.
absence of a band most likely results from the presence of a second enzyme cleavage site within the cDNA insert thus reducing the band to a smaller size. This is not the pattern expected if a band were composed of a number of unrelated but similarly sized clones, since that would require the improbable coincidence that they all have the same restriction enzyme sites within their cDNA inserts. Therefore, it is highly probable that most of these bands contain
major sequence class existed in these 12 colonies by monitoring the fraction of identical clones, i.e. those displaying the same restriction pattern. We chose to rely upon restriction enzyme criteria since this analysis could be accomplished quickly and reliably. Accordingly, each group of 12 mini-lysate DNA preparations were digested with M&I, an enzyme that cuts frequently within DNA. As expected, all cDNA inserts examined were cut multiple times by this enzyme. A comparison of 12 clones from each transformation usually revealed that several have non-vector restriction fragments identical in size. It should be noted that clones derived from an identical mRNA could contain slightly nonidentical insert sizes due to differences in poly(A) tract length and 5’-end termination sites. This possible variability was taken into consideration when evaluating the restriction digest data. In most cases examined, a duplicated sequence type was identified within each group of 12 clones. Two examples of such an analysis are shown in Fig. 2. Bands common to all twelve clones represent plasmid DNA fragments. In the case of the clones derived from band H20 (Fig. 2A), it appears that clones d and 1 and clones f and j are members of two sequence classes. The remainder are unique. When band Cl0 was analyzed (Fig. 2B), it was more obvious that transformants d, e, f, h, i, j and k share internal restriction fragments and are members of a common sequence class. Again, the remaining clones are unique. Clones a, b, c and g represent clones with short inserts that have contaminated the Cl0 band. The unexpected presence of such short inserts was often observed with transformants derived from the high molecular weight size fractions. These may result from slowly migrating plasmids in
181
abcdefghi
abcdefghi
jkl
jkl
-V
-V -V
-V Fig. 2. Identification transformants A, respectively,
of cDNAs
generated
derived
by transformation
from identical
in Fig. 1. The DNA was digested
clones from each band are denoted
mRNAs.
DNA was isolated
of E. coli with DNA recovered with HinfI, electrophoresed
a-l. The position
of DNA fragments
a relaxed conformation that escaped restriction endonuclease cleavage. The overall analysis generated 72 examples of cDNA clones present two or more times among 12 independent isolates. In addition we derived over 300 apparently different cDNAs expressed in muscle but not present in multiple copies in these small samples. (d) Expression of cDNAs in adult human skeletal muscle We next tested the relative level of expression of each of the cDNA inserts we had tentatively identified as bearing abundantly expressed sequences. The amount of mRNA in adult human skm corresponding to each of the clone types was measured relative to that of the most highly expressed muscle mRNA, skeletal a-actin. cDNA insert fragments from each clone and from the human skeletal a-actin clone pHMc&-1 (Gunning et al., 1983b), were radio-labeled in parallel and hybridized to a dot panel of serial dilutions of human skm RNA. Fig. 3 shows the types of results obtained. Most of the clones hybrid-
from twelve cDNA
from band H20(A) on a 2% agarose
derived
clones chosen
and ClO(B) isolated gel and stained
from the vector
are indicated
at random
from
from lanes D and
with EtBr. The twelve by V.
ized with a similar intensity to that of actin (e.g., P2a, lane C) or within two orders of magnitude either higher (e.g., H18b, lane A) or lower (e.g., C23a, lane D). Using these comparative dot blots, we calculated the steady-state levels of transcripts related to each clone relative to that of skeletal cr-actin. The relative expression values for each clone relative to skeletal cr-actin and corrected for probe-length differences are summarized in Table I. The clones were grouped into live categories based upon these relative levels af expression. 19 clones were expressed at levels greater than that of actin. This was unexpected since actin accounts for 4-8% of skm mRNA (Schwartz and Rothblum, 1981). 40 clones were expressed at l-100% the level of actin, whereas only ten clones were expressed at < 1y0 of the level of actin mRNA. We therefore conclude that the majority of the cDNAs isolated in this way do correspond to moderately to highly abundant transcripts. After we compared the patterns generated by digestion of the clones with the restriction endonuclease HinfI, we suspected that some of the clones selected from different library bands might be trans-
182
TABLE
I
Level of expression Abundance Super
b
of cDNAs
(4 100%)
High (lo-loo%)
H6a
P,e
H7b
Psa HlOk
b
H8b HlOd ( Hllb
to actin
Moderate
b
(l-10%)
H4e ( c14g Hl2f H14d
HlOb Hl4i
Hl8f
C5a
Cl6a
C8f
Cl6b Cl7e
c9c Cllb
Hl8b
H22h
c12g
H24g
Clod
Cl5k
c21c
Clle
Cl7f
Cl3a
Cl9e C20a
Cl6c
( Cl4a Cl5b
C24b
Cl6e
No. 23
No. 19
H14f
Cl3e
Cl9b H20f
Rare (~0.1%)
c12c
Hl9f
H20d
b
(
Hl9a
Hl6i
H2lc
H8g Hllf
b
Hl6g
C24h
Low (0.1-l %)
Hl7g (
Hl5a
(
muscle relative
( Cl4d Hl6a b
H13i
b
skeletal
(% actin)
( H8c b
in adult human
C20b C2li C22d
No. 33
C22h C23a No. 64 No. 70
a The hybridization cc-actin cDNA
of each transcript b Bracketed
of cDNA
inserts to serial dilutions
(see Fig. 3). The hybridization relative
intensity
of adult human was corrected
muscle RNA was compared
with that of the skeletal
to actin and the abundance
to actin was calculated.
sets of clones are derived
from the same mRNA.
cripts from the same gene. We then tested such clones for cross hybridization to a panel of all the selected clones. Eight clones were found to be represented more than once in the population and these groups are indicated in Table I. Most of the multiply selected clones belong to the high and super abundant transcript categories. Overall, however, our cloning approach mainly generated non-redundant, presumably unique, cDNAs. (e) Characterization
skeletal
for the DNA probe length relative
of super-abundant
clones
Since actin is the most abundant mRNA within skm (Schwartz and Rothblum, 1981), the detection of clones expressed at levels greater than that of actin is most surprising. The most likely interpretation is that these clones contain repetitive sequences. Accordingly, we hybridized representatives
of each of the 12 superabundant clone types to size fractionated, EcoRI digested, total human DNA. Three types of hybridization patterns were observed in these Southern blots. Six different cDNA types (H6a, C24b, H21c, H16i, H16g, H15a). hybridized to a smear of genomic DNA fragments covering the entire length of the gel. These clones therefore correspond to highly repetitious genomic sequences some members of which are transcribed in skm. Five clones, (H18b, H13i, C16c, No. 23, No. 33), hybridized very strongly to one, or in some cases two, EcoRI band(s). The final superabundant clone, HlOd, hybridized to a large number of discrete bands. The clones which produced the two simpler hybridization patterns were further analyzed by hybridization to human DNA digested with seven restriction enzymes (Fig. 4). HlOd hybridized to a discrete number of genomic fragments with each en-
183
A
A
B
B 1234567
1234567
Fig. 4. Hybridization
of “super-abundant”
DNA
several
digested
with
restriction
cDNAs enzymes.
to human 32P-labeled
cDNA inserts isolated from clones (A), H 10d and (B) H18b were hybridized tionated
to human
restriction
endonuclease
DNA. Following
filters were autoradiographed HindIII,
Fig. 3. Expression
of cDNA
clones
in human
skeletal
muscle
relative to that of actin. The cDNA inserts of the different clones were nick-translated fragment
isolated
(pHMaA-1) hybridized
in parallel from
(Gunning
the
an 850-bp skeletal
PvuII-Xbal
m-actin
cDNA
et al., 1984). The 32P-labe1ed probes were
to serial dilutions
nitrocellulose.
with
human
of human muscle RNA dotted onto
A typical autoradiogram
RNA in each dot is indicated (A), H18b; (B), skeletal
is shown. The quantity
of
in pg units. The probes used were
a-actin;
(C) P2a and (D), C23a.
zyme digest (Fig. 4A) and therefore recognizes a moderately repetitive genomic sequence. In contrast H18b hybridized very strongly to only one to three genomic fragments with each enzyme digest (Fig. 4B). However, the most complex hybridization pattern observed with H18b occurred with human DNA cut with BglII (Fig. 4B, lane 2). This pattern of DNA electrophoresis was reminiscent of that observed for a large circular DNA molecule existing in relaxed and supercoiled forms and suggested that H18b might be hybridizing to the mt genome. Furthermore, BglII does not cleave human mt DNA, whereas all the other enzymes we tested cut at least once (Anderson et al., 1981). Similar hybridization patterns were observed for cDNA clones H13i,
(4) PstI, (5) SacI,
digested,
hybridization
for 6 h. (1) EcoRI, (6) XbaI
(2) BglII,
the (3)
(7) BumHI.
The
1 Hind111 size standards
are
positions
of co-electrophoresed
indicated
and the sizes are shown
and
size-frac-
and washing,
in kb.
C16c, No. 23 and No. 33 and suggested that these might also represent mt transcripts. The presumptive mt cDNAs were positively identified by restriction mapping. The sizes of human DNA fragments hybridizing to each of these clones was determined. Comparison of these fragment sizes with the restriction map of the human mt genome allowed us to unambiguously assign each clone to a defined region. The cDNAs were then restriction mapped and compared with the corresponding region of the mt genome. The results are shown in Fig. 5. Clones H18b, H13i, C16c and No. 33 correspond to full-length cDNAs of the mitochondrial 16s rRNA, ATPase 6, URF2 and CO111 mRNAs respectively (Fig. 5). Clone No. 23 is a cDNA of the mt URFl mRNA and is about 150 bp short of full length. In summary, all of the cDNAs identified as superabundant expressers either carried highly or moderately repetitive sequences or proved to be mt transcripts. (f) Identification of full-length cDNAs
The length of the cDNA inserts in all the highly abundant and most of the moderately abundant cDNAs were compared with the corresponding
184 =
; h I
; f I
1610
1240
=
0 :: I
% I 620
d 1
310
TABLE H18b pHMMt-16s
'
II
cDNA
clone
skeletal
muscle
Clone 5’-3’
=
r
-
E
%;
I
-;
880 730
400
“;
230200
N
P,a HlOb
?
3.3
2.9
Y
HlOk
3.3
6.0, 4.3, 2.7, 1.5 ?
Hl2f
2.6
2.5, 1.5
Hl3i
1.1
0.9
Y
Hl4d
2.2
4.2
N
=
Hl6a
1.9
1.7
Y
..f #33
Hl8b
1.6
1.5
Y
Hl9a
1.3
4.2
N Y
= #23 pHMMt-URFl
pHMMt-CO3
= Clbc
chondrial
The restriction
cDNAs
genome.
are shown. The distance,
from the 3’ terminus maps and direction the human co-linear
of cDNAs
of transcription (Anderson
with a mt transcript.
in bp, of each restriction
mitosite
These restriction
were compared
with that of
et al., 1981). Each cDNA was
The cDNAs
mt gene. In addition,
and H 13i have been directly
by human
maps of the live types of mt
PvuII site is indicated.
mt genome
the corresponding
encoded
sequenced
are named to identify regions of clones Hl8b
to confirm their identity
(not shown).
mRNA
sizes to determine
Full-length
2.2, 1.8
OpHMMt-URF2
maps
(kb)
2.2
III;-
Fig. 5. Restriction
mRNA
mRNA
1.4
’
I s
(kb)
corresponding
1.7
900
z % t
cDNA
size and
P,e
0
4izo
insert
whether they represented
full-length copies. The cDNAs were linearized with the restriction endonuclease initially used to linearize the corresponding band in the cDNA library and the complete clone length was determined by co-electrophoresis with size standards. The size of the vector, 2.5 kb, was subtracted from the clone length to yield the cDNA insert size. The inserts varied in size from 0.9 kb to 4.7 kb (Table II). Size fractionated adult skm RNA was hybridized with each of the clones and the corresponding transcript sizes were measured. Table II shows that whereas a majority of the clones hybridized to a single transcript size, 12 of them hybridized to multiple (2 to 4) transcripts. For six of the clones that recognized multiple transcripts, we could not unambiguously determine whether the cDNAs were full-length. However, 16 of the remaining 26 clones tested were within 5% of being fulllength (Table II). This represents a remarkably high yield of full-length cDNAs and will simplify characterization of the corresponding genes.
H20d
1.3
1.4
1.3
6.2
N
H22h
1.0
0.9
Y
C5a
3.2
5.6, 4.2
N
C8f
4.7
4.3
Y
c9c
4.1
8.6
N
Clod
4.0
4.0, 7.5
?
ClIe
3.6
3.6
Y
Cl3a
2.8
6.5
N
c14g
3.0
2.8
Y
Cl5b
2.7
2.4
Y
Cl5k
2.7
5.0, 4.3
N
Cl6c
1.1
1.0
Y
Cl6e
2.7
2.5, 1.5
Y
C20b
1.6
4.3, 2.5, 1.2
?
C2li
1.6
2.6, 1.5
?
C23a
1.2
2.5, 1.2
?
No. 19
1.0
1.5, 1.0, 0.8
Y
No. 23
0.9
1.1
N
No. 33
0.9
0.8
Y
No. 64 No 70
1.0
4.4. 1.8
N
I 0
I0 -____
Y
3
The size of the cDNA insert and the corresponding
section
as described
(Y/N)b
Y
H20f
determined
size in
in MATERIALS
AND
mRNA was METHODS,
d.
b Y denotes cDNA inserts which are within 5% of the size of the mRNA. N denotes size of the mRNA. unambiguously
cDNA inserts which are less than 95% of the ? denotes
evaluated
cDNA
inserts
which
cannot
due to the existence ofmultiple
be
hybrid-
izing transcripts.
(g) Cell-type-specific
expression of cDNAs
Previous studies have indicated that most of the abundant transcripts in skm are specific for that tissue (Paterson and Bishop, 1977; Affara et al., 1980). We therefore tested the specificity of expres-
185
sion of the isolated cDNA clones in muscle and non-muscle cells. Radiolabeled cDNA inserts were hybridized to serial dilutions of human skm, heart and HeLa cell RNA. Fig. 6 shows examples of the three types of hybridization patterns which were observed. Some clones such as Clod hybridized exclusively with skm RNA, others like C9c hybridized only to skm and heart RNA whereas the third group hybridized to RNA of all three cell types. The expression of each of these cDNAs in heart and HeLa cells relative to that in skm was calculated. Table III shows that 11 of 13 cDNAs expressed at high abundance in skm (as defined in Table I) are expressed at very low levels, if at all, in HeLa cells. In contrast, only 6 of the 15 cDNAs expressed at moderate abundance in skm display a strong preferential expression in muscle cells. This result correlates well with the expectation that the most abundant muscle transcripts are most likely to encode muscle-specific proteins (Paterson and Bishop, 1977). We therefore conclude that we have isolated cDNAs corresponding to abundant skm mRNAs - many of which display cell-type specific expression - and have simultaneously selected for full-length cDNAs.
DISCUSSION
(a) Isolating skeletal muscle cDNA clones
We have taken advantage of the Okayama and Berg (1982) cloning system to devise a method of
A SH
B S
H
Expression
muscle
Clone
Expression Skeletal
P,a
H
H16a
H
F
*
Fig. 6. Expression inserts of (A) Clod, hybridized skeletal
in
muscle”
of cDNAs
in different
to serial dilutions
of RNA isolated
muscle (S), adult human
The quantity
cell types. The cDNA
(B) C9c and (C) C15b were “P-labeled
and
from adult human
heart (H) and HeLa cells (F).
of RNA in each dot is indicated
in pg.
Hearth skm
Fibroblastsb skm
0.025
< 0.005
< 0.005
< 0.005
H22h
H
0.025
< 0.005
H
< 0.005
< 0.005
Clle
H
0.025
C13a
H
0.05
<0.005
P,e
H
0.5
< 0.025
H20d
H
8
0.025
H20f
H
2
< 0.005
0.025
C16e
H
< 0.005
H12f
M
< 0.005
H14d
M
C9c
M
Cllb
M
C20b
< 0.005 0.25
< 0.005
M
4
< 0.025 4 0.005
0.1
C23a
M
2
No. 19
H
0.25
0.1
HlOk
H
0.5
0.5
C15b
H
0.5
H19a
M
C5a
M
C8f
M
C14g
M
C15k
M
C20a
M
C21i
M
0.5
0.2
No. 64
M
4
0.2
No. 70
M
1
HlOb
L
2 2 0.5
16
0.5
4
0.25 2
2 10
in skeletal
muscle is shown as abundance
relative
an L is low, as
defined in Table I. expression
of these
relative to their expression
‘*
0.25
relative to that in
Clod
hybridization r)
in heart and fibroblasts
to that of actin where H is high, M is moderate
*
0.5
of cDNAs
skeletal
‘The 2
III
a Expression
C F
F
TABLE
to parallel
sequences
in heart
and tibroblasts
in skeletal muscle was determined RNA serial dilutions
by
as shown in Fig. 6.
directly isolating full-length cDNA copies of abundant mRNAs. Using this method, we have isolated 13 cDNA clones which correspond to abundant skm transcripts. At least seven of these 13 cDNAs are within 5 % of being full-length clones. Normally, the recovery of full-length clones from conventional cDNA libraries is very low. We have previously re-
186
ported that representation of full-length actin clones in Okayama-Berg (1982) cDNA libraries can vary from less than 1% (for /!i-actin in a fibroblast
library)
experiments can, with certainty, quickly clones corresponding to the same mRNA. Despite the success of our screening,
it is clear that
to 25% (for skeletal actin in a muscle library) of all
the efficiency could be improved.
actin
ber of selected clones did not correspond
clones
cDNA transcript
(Gunning
inserts
et al.,
of at least
1983b).
Since
16 of 26 clones
size was determined
whose
are within 5% of full
length (Table II), it is likely that this screening thod enriches for full-length the fact that rescreening length
clones
the
me-
clones. We can attest to a cDNA
corresponding
to
library
for full-
lo-20
different
mRNAs is a major undertaking using conventional cloning and screening procedures. Previous workers who have screened muscle cDNA libraries usually used radiolabeled cDNA as a probe to identify clones corresponding to abundant mRNAs. Hastings and Emerson (1982) obtained clones corresponding to 17 abundant quail muscle mRNAs. Garfinkel et al. (1982) similarly obtained copies of twelve different rat muscle mRNAs. In contrast, Putney et al. (1983) randomly sequenced 178 rabbit muscle cDNA clones and identified clones corresponding to 13 different abundant mRNAs. Our isolation of 13 different clones compares very well with the yield in these other studies and suggests that between these four collections, all the abundant vertebrate skm mRNAs have probably now been isolated as cDNA clones. Interestingly, except for one cDNA copy of myosin heavy chain in our isolates, we have found (R.W., unpublished) that none of our abundant clones cross-hybridize with any of those from a quail muscle library (Hastings and Emerson, 1982) kindly provided by Dr. Charles Emerson. (b) Methodology One of the major problems associated with conventional cDNA screening is the failure to identify clones encoding the same mRNA since such cDNA clones may cover nonoverlapping mRNA regions. In such situations, positive identification usually requires (1) sequencing of the cDNAs and comparison with a known protein sequence or (2) comparison of genomic Southern blot and RNA Northern blot data. This problem is completely eliminated with the Okayama and Berg (1982) vector since all clones start at the same position, the 3’ end of the mRNA. Thus restriction analysis and/or cross-hybridization
dant mRNAs.
Conversely,
of clones corresponding overlooked.
identify
A significant
num-
to abun-
it is likely that a number to abundant
mRNAs
were
This is clearly true for actin, which is the
most abundant
muscle mRNA
and yet was not iso-
lated. In this case however, the failure may be due to an intrinsic Although
difficulty
in generation
actin accounts
for 4-8%
of actin clones. of total skm po-
ly(A) + mRNA (Schwartz and Rothblum, 198 l), only 0.6% of the cDNAs in our muscle library encode actin (Gunning et al., 1983b). To improve the efficiency of isolation of abundant cDNA clones, it would be useful to introduce a preliminary screening test for their abundance. 20-30 colonies obtained from transfections with each recircularized DNA fraction could be hybridized first to radiolabeled muscle poly(A) + mRNA. Clones from each fraction which hybridize strongly could then be compared using the HinfI plasmid DNA digestion test to identify identical clone types. Since all Okayama-Berg (1982) cDNA clones contain a poly(A): : poly(T) 3 ’ segment, such a screening test would need to be carried out with radiolabeled poly(A) + mRNA divested of poly(A)+ -containing fragments prior to hybridization (Garfinkel et al., 1982). (c) Isolation cDNAs
and identification
of mitdchondrial
Considering the abundance of mt transcripts in adult skm, it is not surprising that a number of corresponding cDNAs were isolated by this method. In fact, the initial size selection of the cDNAs probably prevented us from isolating even more of them since mt mRNAs are less than 1.0 kb in size (Anderson et al., 1981) and our size cuts eliminated clones with cDNA inserts less than 0.9 kb. Mt cDNAs are certainly well represented in cDNA libraries constructed from total tissue poly(A)+ mRNA. Such clones can be quickly identified by hybridization of cDNA inserts to size-fractionated human DNA which has been digested with an enzyme which is anoncutter for the mt genome (Fig. 4). In addition, it is simple to quickly determine the identity of such clones using only a few restriction enzyme digests.
187
(d) Tissue-specific expression of abundant skeletal muscle mRNAs
Anderson,
S., Banker,
Coulson,
B.A., Sanger,
Hybridization of the cDNA probes to human RNA preparations revealed that most of the abundant muscle mRNAs are at least sarcomeric-musclespecific. Using human myoblast RNA, we have more recently confirmed that the expression of these muscle-specific mRNAs is induced during human myogenesis (P.G., E.H. and R.W., unpublished). This extends results previously reported using total mRNA hybridization experiments (Paterson and Bishop, 1977; Devlin and Emerson, 1978; Affara et al., 1980). Since heart and skrn contain similar structural proteins and enzymes, it is surprising to find that one third of the sacromeric-muscle-specific cDNAs are specific to skm alone. This most likely reflects the differential utilization of diverged members of multigene families between heart and skm (Buckingham and Minty, 1983). We are currently sequencing these muscle-specific cDNAs to help identify the corresponding protein products.
and Young,
We thank Doris Hosmer for help in the preparation of this manuscript and Peter Evans for providing excellent technical assistance. We also thank Adrian Minty, Hiroto Okayama and Helen Blau for many helpful discussions. This work was supported in part by grants to L.K. from the National Institutes of Health (HD17031), Veterans Administration and the Muscular Dystrophy Association. E.H. is a fellow of the National Institutes of Health and R.W. is a fellow of the Muscular Dystrophy Association. J.C.G. was supported in part by a Research Career Development Award from the National Institutes of Health (AM00491).
F., Schreier, genome.
of human
nuclear
deBruijn,
genes
D.P., Roe,
A.J.H.,
and organization
Nature
M.H.L.,
I.C., Nierlich,
P.H., Smith,
LG.: Sequence
mitochondrial
B.G.,
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ACKNOWLEDGEMENTS
A.T., Barrell,
A.R., Drouin,
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P., Ponte,
Kedes,
L.:
cDNA
clones
L.: a-skeletal
in adult human
Mol. Cell. Biol. 3 (1983a)
P., Okayama,
Isolation
and
for human
and skele-
1783-1791.
H., Engel, J., Blau, H. and
characterization alpha-,
beta-
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skeletal but not cytoplasmic
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cysteine
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Mol. Cell.
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