Cell, Vol.

23, 731-739, March 1981, Copyright 0 1981 by MIT

Transcriptional Control in the

Production of Liver-Specific mRNAs

Eva Derman, Kenneth Krauter, Linda Walling, script submitted) and one probable instance of control
Carey Weinberger, Martha Ray and at the level of differential processing have been estab-
James E. Darnell, Jr. lished for adenovirus mRNA production (Chow et al.,
The Rockefeller University 1979; Shaw and Ziff; 1980, Nevins and Wilson, 1981).
New York, New York 10021 To study the level at which specific cellular genes
are controlled, recombinant DNA segments comple-
mentary to specific mRNAs can be used in a manner
Summary exactly analogous to the use of recombinant bacterio-
phage DNAs and adenovirus DNA fragments in earlier
cDNA clones complementary to liver mRNA were work. In this report we describe the preparation of
prepared and used to determine transcription rates DNA segments complementary to specific mRNAs of
of specific genes in isolated nuclei from liver, brain, mouse liver, and the use of these DNA segments to
and hepatoma cells. The cDNA sequences comple- measure the rate of transcription for liver-specific
mentary to.mRNA found only (or mainly) in the liver genes in liver nuclei, brain nuclei and nuclei from
hybridize to labeled nuclear RNA only from liver cultured hepatoma cells. The results suggest that
nuclei. It appears that transcriptional events are most moderately abundant tissue-specific genes may
primarily responsible for the synthesis of these, and be regulated at the level of transcription.
perhaps most, tissue-specific moderately abundant
mRNAs. Results

Introduction Selection of Recombinant cDNA Plasmids

Mammalian liver is composed of relatively few cell
Control of gene expression through the control of DNA types. The majority of liver cells are the so-called
transcription was first demonstrated at the molecular parenchymal cells or hepatocytes, which synthesize
level for the lactose operon in E. coli (Hayashi et al., many proteins that are not made in other cell types or
1963). Those initial experiments involved molecular that are synthesized in other cell types in much smaller
hybridization of pulse-labeled RNA to purified bacte- amounts. Antibiotic-resistant E. coli plasmid DNA, re-
riophage DNA, into which the lactose operon had combined with individual segments of mouse DNA that
been recombined. The formation of P-galactosidase was copied from unfractionated mouse liver mRNA
mRNA was shown to be at least 30 times greater in “cDNA,” would likely contain sequences at fairly high
induced compared to uninduced cells. Many other frequency that represent liver-specific mRNAs. We
examples of transcriptional control, mediated by both therefore constructed recombinant pBR322 cDNA
positively acting and negatively acting regulatory pro- plasmids containing sequences derived by copying
teins, have been demonstrated in bacteria by employ- DNA from the total poly(A)+ RNA from the livers of
ing hybridization of labeled RNA to specific DNA (see, adult male mice, using reverse transcriptase, E. coli
for example, Bertrand et al., 1975; Lozeron et al., DNA polymerase I and the dG:dC tailing procedure
1976; Lee, 1978). (Villa-Komaroff et al., 1978). The mouse sequences
Our strategy in studying eucaryotic gene regulation were inserted at the single site for the restriction
has followed the lead of the successful studies of enzyme Pst I (see Experimental Procedures). Colonies
bacterial gene regulation. No molecular technique containing liver-abundant sequences were detected
other than hybridization of pulse-labeled RNA to spe- by colony hybridization screen (Young and Hogness,
cific DNA can answer the question of whether, inside 1977) of about 1000 bacterial colonies. The individual
a cell, a gene is controlled by regulating its rate of bacterial colonies were grown on replicate Millipore
transcription. Other measurements may be necessary filters, and were hybridized with labeled poly(A)’ RNA
to determine if additional levels of control are impor- from liver, hepatoma cells and mouse L cells. The
tant for any particular gene. RNA was labeled by terminal addition of 32P with
Hybridization of pulse-labeled nuclear RNA to re- polynucleotide kinase after alkali breakage to create
striction fragments of adenovirus (Weber et al., 1977; 5’ OH ends (Spradling et al., 1980).
Evans et al., 1977; Evans and Ziff, 1978) located the The result of one screening test is presented in
initiation sites for a number of adenovirus transcrip- Figure 1. Of the 1000 colonies screened in this fash-
tional units that are responsible for the production of ion, 130 colonies gave unambiguous positive signals
many different adenovirus mRNAs. Knowing the lo- only with labeled liver poly(A)+ RNA. Of these, about
cation of various promoters, the transcriptional control 80 colonies harbored plasmids complementary to the
pattern for adenovirus mRNA production could be same, very abundant liver mRNA and the remaining
firmly established (Nevins et al., 1979; Wilson et al., 50 colonies represented sequences from different
1979). In addition to various types of transcriptional “liver-specific” mRNAs (see below). Also, 50 colonies
control, two instances of control at the level of mRNA were selected that gave positive signals with RNA
turnover (M. C. Wilson and J. E. Darnell, Jr., manu- from all three cell types (“common” clones).
Cell
732

ABCDEFGHI JK
-Origin

- 1600

Figure 2. Northern Gel Analysis of Mouse Liver mRNA

Poly(A)-containing mRNA isolated from mouse liver was denatured
and subjected to electrophoresis in a 1.25% agarose-formaldehyde
gel for 5 hr at 200 V. Following electrophoresis the RNA was trans-
ferred to nitrocellulose, baked and cut into individual lanes (A-K)
representing about 8 pg of each RNA. The lanes were then hybridized
to individual cDNA clones separately by using the two-step hybridi-
zation procedure described in Experimental Procedures. At right are
the positions of single-stranded DNA markers of 1600, 1000 and 430
nucleotides.

(lanes G and H). All the other lanes show mRNA

species of different sizes.
Figure 1. Colony Hybridization with Various Labeled mRNAs In addition to this “RNA blotting” procedure, differ-
Liver, hepatoma cell and L cell poly(A) + RNA was labeled in vitro (as ent plasmids were also checked for distinct sequence
described in Experimental Procedures) and was used to screen 1000
bacterial colonies harboring recombinant plasmids. An example of
content by the Southern blotting procedure. Purified
such a screen is shown. Arrows: colonies giving autoradiographic plasmid DNA was digested with the restriction enzyme
signals with liver mRNA only (a), colonies giving the strongest signals Pst I, releasing the inserted mouse DNA sequence
with hepatoma cell mRNA (b) and colonies harboring plasmids com- (Figure 3A), and the DNA fragments were separated
plementary to mRNAs from all three sources (c). by agarose gel electrophoresis. After transfer of the
DNA to nitrocellulose filters, the plasmid DNAs were
Characterization of Clones hybridized to a single 32P-labeled plasmid DNA. Some
To ensure that the mouse sequences in the series of of the plasmids crossreacted, presumably indicating
individual plasmids represented different mRNA mol- clones containing portions of the same mouse liver
ecules, two tests were carried out. Unlabeled dena- mRNA sequence. In Figure 36, both the plasmids in
tured mouse liver mRNA (Rave et al., 1979) was lanes I and J appear to have hybridized to the same
separated by agarose gel electrophoresis and trans- labeled probe, all the other plasmids did not contain
ferred (bound) to Millipore filters (Southern, 1975). these sequences. Combining the results of the RNA
These filters were then hybridized to 32P-labeled plas- blotting and DNA blotting procedures, some 16 differ-
mid DNA, as outlined in Experimental Procedures. ent plasmids with inserts larger than 400 nucleotides
Each plasmid complementary to an mRNA of a differ- were identified.
ent size was considered a distinct sequence. In Figure Table 1 summarizes the properties of the 16 differ-
2 one example is shown, where two different recom- ent recombinant plasmids chosen for further studies.
binant plasmids hybridized to mRNAs of the same size 3”P-labeled poly(A)+ RNA from liver, brain or cultured
Transcriptional Control of Liver-Specific mRNAs
733

Figure 3. Crosshybridization Study of Mouse

ABCDEFGH I JKLMNOP Liver cDNA Clones
(A) Purified plasmids containing double-
stranded cDNA segments linked to pBR322
by dG:dC tails were digested with restriction
endonuclease Pst I to excise the insert (see
Experimental Procedures). Detection of the
inserts was accomplished by ultraviolet fluo-
rescence. Approximately 0.5pg of each digest
was loaded onto a 1.4% agarose gel and
subjected to electrophoresis for 3 hr at 200 V.
(B) Following electrophoresis. DNA in the gels
was transferred to nitrocellulose, baked and
hybridized to nick-translated cDNA clones.
The clones in lanes H and I are seen to cross-
hybridize to the H probe, but no other clones
crosshybridized with this probe.

hepatoma ceils was again prepared by alkali breakage Table 1. Characterization of liv-pBR Plasmids
and 5’ end-labeling with polynucleotide kinase. Each
Percent Steady-State Poly(A)+
plasmid DNA bound to a nitrocellulose filter was hy- RNA”
bridized to the samples of labeled mRNA, to determine Insert mRNA
the concentration of specific sequences in the three Size Size Hepatoma
different mRNA samples. The DNA was in excess of (bp)” (bpf’ Male Liver Cells Brain
the labeled RNA, as indicated by a failure to detect plivS-I 850 980 8.00 *
significant amounts of unbound RNA during a second plivS-2 1350 1700 0.40 *
exposure to filter-bound DNA. Eleven of the plasmids *
plivS-3 400 1500 0.20
were complementary to liver mRNA but hybridized to
plivS-4 300 2050 0.17 0.03
less than 10% as much labeled mRNA from brain
plivS-5 600 800 0.10 *
cells. (These were designated plivS-1 through -1 1, for
plasmids with liver-specific sequences.) Two of the plivS-6 550 2200 0.10 0.02
pliv-S plasmids hybridized to about 15-20% as much plivS-7 1720 1750 0.07 *
mRNA from hepatoma as from liver cells; the remain- plivS-8 600 1050 0.07 0.002
ing plasmids hybridized very little hepatoma cell *
plivS-9 500 1700 0.05
mRNA. Five of the cloned DNAs hybridized to about
equal amounts of mRNA from the liver, brain and plivS-I 0 ;oo 1650 0.04 0.004

hepatoma cells. (These plasmids were designated plivS-11 500 1600 0.015 *

plivC, for common sequences cloned from liver.) The plivC-I 2 700 940 0.25 0.22
concentration of mRNA (Table 1) was recorded as the
plivC-13 850 0.17 0.11
fraction of poly(AY RNA that was complementary to
a particular clone after pancreatic RNAase digestion. plivC-14 750 750 0.15 0.10

The individual clones hybridized to between 0.015% plivC-15 1100 1250 0.27 0.17
and 0.4% of the labeled liver mRNA. Because the plivC-I 6 640 735 0.35 0.30
length of the recombinant mouse DNA segments av- a The size of the insert was determined by Pst I digestion of the
erages about one half of the average mRNA length, recombinant plasmid and electrophoresis on agarose gels, as de-
the real abundance of the mRNAs ranged between scribed in Experimental Procedures and illustrated in Figure 3.
0.03 and 0.8% of total mRNA. The one exception is ’ The size of mRNA was determined by hybridizing plasmid DNA to
Northern blots of liver poly(A)+ RNA, as described in Experimental
plasmid pliv-Sl , which hybridized to as much as 8% Procedures and illustrated in Figure 2.
of mRNA from male liver. Our current evidence indi- ’ Poly(A)+ RNA was prepared and kinased in vitro, as described in
cates that this plasmid is derived from mRNA for major Experimental Procedures, and hybridized to an excess of plasmid
urinary proteins (E. Derman, unpublished results). As- DNA bound to Millipore filters. The fraction of total poly(A)+ RNA that
suming that liver cells contain 5 x lo5 mRNA mole- remains bound to plasmid DNA after pancreatic RNAase digestion is
expressed as percentage of steady-state poly(A)+ RNA; each figure
cules per cell (Hastie & Bishop, 19761, the number of is the average of at least three measurements. In all cases the
liver mRNA molecules complementary to the eleven variation was less than 0.5-l .5 times the value shown.
clones range between 150 and about 4000. The av- * ~0.001 %--that is, 10 copies per cell.
erage abundance classes estimated by Hastie and
Bishop were 15 copies per cell for 12,000 different we have selected for study cover the moderately
sequences, about 300 copies per cell for about 350 abundant range. Because the colony screening pro-
different sequences and 12,000 copies per cell for cedure with the 32P-kinased mRNA is based on DNA
about 10 different sequences. Thus, the sequences excess hybridization, the signal-to-noise ratio for
Cell
734

mRNAs of very low abundance presumab1.y would not demonstrated that the proportion of labeled hemoglo-
be large enough to allow detection of colonies har- bin-specific RNA precursor relative to total newly syn-
boring plasmids complementary to scarce mRNAs. thesized RNA is similar in intact Friend cells and
Friend cell nuclei, and that the synthesis of different
Transcription in Isolated Nuclei: Comparison of segments of the hemoglobin transcriptional unit is
Transcription Rate in Vivo and in Vitro equimolar in isolated nuclei. Nascent RNA labeled in
Having established that eleven of the piasmids con- isolated nuclei therefore should not be subject to
tained liver-specific mRNA sequences, we could de- hypothetical requlatory mechanisms involving differ-
termine the step or steps during mRNA biosynthesis ential processing of some mRNA precursors and not
responsible for this difference. Hypothetically, regu- others. Other transcriptional studies of specific polym-
lation might occur at three possible levels: transcrip- erase II products have also suggested that there exists
tion, differential processing of primary transcripts or a similarity in the action of polymerase II, in vivo and in
differential mRNA turnover. To be certain which step vitro. For example, the incorporation of 3H-uridine by
or steps might participate in gene regulation it is whole cells or 3H-UTP by isolated nuclei intd adeno-
necessary to be able to separately monitor each step virus-specific sequences was similar, both early in
leading to the formation of mature mRNA (for review infection when virus RNA represents 0.2% of the total
see Darnell, 1979). However, since the first step in arid late in infection when it is 30% of the total (Weber
the biosynthesis of mRNA is the tranScription of the et al., 1977; Evans et al., 1977; Weber, 1979).
mRNA precursor from the template DNA, the rate of In order to validate more generally the use of iso-
transcription of the liver-specific sequences was ex- lated nuclei to measure specific gene transcription
amined first. Only if transcriptional differences could rates, we have compared the percentage of pulse-
not adcount for the differential abundance of tissue- labeled nuclear RNA from isolated nuclei and from
specific mt?NA would it be necessary to investigate intact cultured CHO cells that was complementary to
further the other levels of regulation. several cloned DNA sequences containing CHO
An ideal measurement of the rate of transcription of mRNA sequences (Harpold et al., 1979; Table 2). The
a transcriptional unit should be made under conditions relative transcription rates were quite similar in whole
that ensure that the synthesis of all sequences is cells and in isolated nuclei for this set of genes. The
equimolar. This can be accomplished in vivo only in a percentage of newly labeled RNA complementary to
short pulse, by measuring the rate of transcription of each of the genes was somewhat higher in whole
yet unprocessed nascent RNA (Derman et al., 1976; cells. This may reflect a changed balance of polym-
Evans et al., 1977; Evans et al., 1979). It is not erase II activity relative to polymerases I and Ill in
possible to obtain nascent labeled RNA of specific isolated nuclei, or the fact that a 10 min pulse in vivo
activity high en?ugh to measure individual gene tran- is not an ideal pulse (see above). But we wish to
scription rates in intact mouse liver. However, isolated emphasize that the transcription ratio between differ-
nuclei from tissues can be prepared and pulse-labeled ent mRNA sequences remains the same in isolated
to yield enough radioactive RNA. .Available evidence nuclei as inside the cell.
suggests that the majority pf RNA synthesis in isolated
nuclei represents elongation of already initiated RNA Measurement of Specificity of Nuclear
chalks (Evans et al., 1977), and that RNA processing Transcription
in isolated nuclei is only partially carried out even on Isolated nuclei were prepared from mouse liver, brain
already completed labeled RNA chains (Blanchard @t and from cultured hepatoma cells; the newly’ synthe-
al., 1978). In addition, Hofer and Darnell (1981) have sized nklear RNA was labeled by incorporation of

Table 2. Transcription of Five Different mRNA Sequences in Pulse-Labeled Cells and in Isolated Nuclei

Pulse-Labeled Cells (10 min) Isolated Nuclei

Input: 40 x 1 O6 cpm RNA Input: 90 x IO6 cpm RNA

Fraction.of Relative Fraction of Relative

RNA Synthesis Transcription RNA Synthesis Transcription
Clone cpm Hybridized x 10-6 Rate cpm Hybridized x 10-6 .. Rate..
A i555 38.9 1.00 . 1336 14.8 1.00
s 844 21.1 0.54 1007 I 11.2 0.76
C 539 13.5 0.35 399 4.4 0.30
F 260 7.0 0.18, 338 2.4 0.25
D 260 6.5 0.17 218- 3.7 0.16

The CHO clones A-D, growth of CHO ceils, extraction of pulse-labeled RNA and hybridization conditionsare as described in Harpold et at. (1979).
In vitro transcription was performed in sucrose-purified nuclei incubated in the presence of 1 mCi u-32P-UTP as described in Ex.perimental
Procedures
 Transcriptional Control of Liver-Specific mRNAs
735

ol-3’P-UTP. The labeled nuclear RNA was then hy- nuclei. There was variation from experiment to exper-
bridized to excess individual plasmid DNA samples iment in the proportion of labeled RNA complementary
bound to individual nitrocellulose filters, and the to some of the clones-for example, clones 1 and 6.
RNAase-resistant hybrids were measured. These variations were not due to inadequacy of DNA
All eleven of the cloned DNAs designated as liver- on the filters, because supernatants were checked
specific hybridized to labeled nuclear RNA from liver and they did not contain substantial additional hy-
nuclei (Table 3) and hybridized to less than 10% as bridizable RNA. Variations in polymerase I, II and Ill
much, labeled RNA from brain. With one exception activity might occur, and true differential transcription
(plivS-6) the same was true for labeled nuclear RNA in different groups of animals might occur. In any case
from hepatoma nuclei. The labeled brain or hepatoma a general conclusion seems valid: for the eleven liver-
nuclear RNA that did hybridize to liver-specific clones specific mRNA sequences studied, differential expres-
above the background was only in the range of l-5% sion in different cells appears to be accounted for
of the amount that liver-specific RNA hybridized. mainly on the basis of differential transcription.
Whether this low level of hybridization (only twice Several interesting points about the transcription
background) was significant is not possible to state at rates of the liver-specific and the “common” se-
present. In contrast, all five of the clones that were quences were apparent. First, there was a general
complementary to liver, brain and hepatoma mRNAs correspondence between the rate of transcription in
(plivC12-16) hybridized to about the same proportion isolated nuclei and the cell concentration of the liver-
of labeled nuclear RNA from liver, brain and hepatoma specific sequences (plivs-1 through -11). Second,

‘Table 3. Transcription of Liver mRNA Sequences in Liver, Brain and Hepatoma Nuclei” and Hepatoma Celk?

Liver Nuclei Brain Nuclei Hepatoma

cpm RNA Hybridized cpm RNA Hybridized cpm RNA Hybridized

Average Average Pulse-labeled Average

Transcription Transcription Nuclei Cells Transcription
Insert Input: Input: Rate Input: Input: Rate Input: Input: Rate
Clone Size 6 X 10’ 9 x 10’ x lo-- 8~10~ 8x10’ xIO-~’ 3x 107 4x 107 x 1o-ec

plivS-I 850 6,150 18,300 165.0 10 30 0.2 20 20 0.5

plivS-2 1,350 2,805 2,995 38.6 9 49 0.3 34 7 0.6
plivS3 400 2,429 1,106 23.5 42 114 1 .o 66 77 2.0
plivS-4 300 1,273 952 14.8 71 52 0.8 22 52 1 .o
plivS-5 600 1,215 1,007 15.0 20 33 0.3 40 26 0.9
plivS-6 550 1,519 371 12.6 2 44 0.3 66 170 3.3
plivS-7 1,720 573 9.5 30 0.4 30 30 0.8
plivS-8 600 485 278 5.0 0 33 0.3 16 50 0.9
plivS-9 500 1,105 1.257 15.7 6 33 0.3 30 47 1 .o
plivS-10 700 547 248 5.3 6 33 0.3 25 37 0.9
plivS-1 1 500 301 154 3.0 0 18 0.1 20 20 0.5

plivC-12 700 221 470 4.5 223 148 2.3 152 193 5.0
plivC-I 3 81 115 1.3 213 275 3.0 198 120 4.5
plivC-14 740 112 140 1.8 74 182 1.6 40 60 1.4
plivC-I 5 1,100 183 320 3.3 269 270 3.4 470 285 10.2
plivC-16 640 167 185 2.4 80 1 .o 12 95 3.0

pBR322 (con-
trol) 27 47 38 27 30 33

a The nuclei were obtained from fresh tissues or from monolayer hepatoma cells and were incubated in the presence of 0.5-l mCi w~‘P-UTP as
described in Experimental Procedures. For one experiment, livers from 6 animals and brains from 50 animals were needed.
’ Monolayer cells were labeled for 10 min with 10 mCi 3H-uridine. Nuclear RNA was extracted, freed of DNA and hybridwed to an excess of
plasmid DNA bound to Millipore filters.
’ Average transcription rate is the fraction of total RNA synthesized in 20 min in isolated nuclei (or in 10 min in pulse-labeled hepatoma cells) that
is hybridized to excess plasmid DNA averaged over two experiments. In all experiments transcription of ribosomal RNA constituted 40-50% of
total transcription.
 Cell
736

some of the common mRNAs (for example, plivC-12, The tissue-specific mRNA pattern, or in fact differ-
-13, -14, -15 and -16) and the liver-specific mRNAs entiation itself, is often thought of as infrequently
(plivs-3-6) were present at about the same concen- reversible (Mintz, 1974). Possible exceptions to this
tration in the liver. The rates of transcription for the proposal are malignant cells. Frequently in tumor cells
liver-specific group, however, averaged about 3 to 10 the synthesis of tissue-specific proteins of the pre-
fold higher than the common group. The implication sumed cell of origin is reduced or abolished and
of this measurement is that the liver-specific mRNAs synthesis of new proteins is observed. At which level
either have a shorter half-life or are less efficiently does this “deregulation” occur? The experiments pre-
processed. Third, the concentration of the liver-spe- sented here show that the decrease in concentration
cific sequences in the total labeled RNA from isolated in hepatoma cells of the liver-specific mRNAs is par-
nuclei was about %OO of that in the liver mRNA. Even alleled by a decrease in the transcription of these
allowing for possible extra polymerase Ill action in in sequences. The origin of hepatoma cells is uncertain,
vitro nuclei and for about 50% of RNA transcription to however. If they arise from “dedifferentiation,” our
be due to polymerase I transcription of rRNA genes, experiments suggest they lose the tissue-specific
this result emphasizes the earlier conclusion (Harpold transcriptional pattern. If hepatoma cells instead arise
et al., 1979) that many RNA sequences in the nucleus by proliferation of immature stem cells, then the inter-
are not found among the stable poly(A) + cytoplasmic pretation of our measurements is that the liver-specific
mRNA. genes are not yet transcriptionally active in stem cells.
A technical limitation of our experiments should be
Discussion noted. The amount of labeled nuclear RNA from liver
cells complementary to most of the liver-specific
During animal development a pattern of differential genes was relatively small. Thus the level of confi-
gene expression is established, resulting in mRNA dence in concluding that brain and hepatoma nuclei
populations that are distinguishable from one cell type do not synthesize these RNAs is limited to a factor
to another in both qualitative and quantitative fashion between 10 and 50, based on the amount of labeled
(see, for example, Hastie and Bishop, 1976). The first nuclear RNA hybridized on the DNA filters compared
step in determining how such differential distribution with background. However, the presence of these
of mRNA is achieved is to prepare pure DNA probes sequences as mRNAs in brain and hepatoma could be
for genes expressed preferentially in one cell type. measured with confidence to be %OO to %OO of that in
With the availability of pure DNAs that are comple- liver. Thus it is possible that some low level of tran-
mentary to a broad set of tissue-specific mRNAs, it scription of all genes might proceed in all cells at all
should be possible to identify the regulatory step(s) in times. Even in bacterial cells, where reversible control
mRNA biosynthesis. of gene expression is thought of as “on” or “off,”
Our results, with a group of cloned DNA sequences there is a low level of transcription equivalent to O.l-
complementary to moderately abundant liver-specific 1% of fully induced levels for most genes (Kumer and
mRNA, show that the nuclei from brain cells either do Szybalski, 1969). In mammalian cells a low level of
not transcribe the liver-specific mRNA sequences or nuclear RNA might also be synthesized and remain
transcribe these sequences at a rate %O to %O of that unprocessed, or a low level of resulting mRNA might
in liver cell nuclei. These results demonstrate that not be stabilized. But the rate-limiting step for mRNA
transcriptional events have a primary role in establish- production, and therefore the point of control for these
ing and maintaining the differential gene expression liver-specific mRNAs, appears to be transcription.
of these two terminally differentiated cell types. That There have been a number of experiments based
transcriptional events primarily determine mRNA con- on reassociation kinetics with mammalian nuclear
centration in eucaryotic cells has been suggested RNA (Chikaraishi et al., 1978) and with sea urchin
since the first description of the Jacob-Monod model nuclear RNA, in which there is apparent extensive
(1961). For example, differential puffing patterns in overlap of sequence content of the nuclear RNA se-
insects (reviewed by Daneholt, 1975) have been in- quences. This has been reported particularly in sea
terpreted as a demonstration of differential transcrip- urchins for oocyte and blastula tissue compared with
tion activity of insect genes. More recently, with the specialized cells or gastrula cells (Kleene and Hum-
ability to measure the transcriptional activity of individ- phrey, 1976; Wold et al., 1978; Sheperd and Nemer,
ual genes, evidence has been reported for increased 1980). Several points about these results are worth
nuclear RNA synthesis of ovomucoid sequences in making in light of the apparent transcriptional control
oviduct nuclei (Tsai et al., 1978) and for a-2-globu- reported here. First, most genes may in fact be tran-
lin synthesis in rat liver nuclei (Chan et al., 1978). scribed in all cells at very low levels. Second, the
Until the present work no survey has been available scarce mRNAs that are not included in our group of
of tissue-specific RNA synthesis rates for a number of cloned sequences, and which may represent tran-
different mRNAs that have a broader range of concen- scripts of the majority of all active genes, might be
trations. regulated posttranscriptionally. Also, genes active in
Transcriptional Control of Liver-Specific mRNAs
737

early embryonic cells such as those studied in sea and terminal transferase were purchased from BRL. Si nuclease was
urchin development may possibly be regulated by a a gift from J. Logan.
Double-Stranded cDNA Synthesis
different mechanism than those active later in devel- Poly(A) + RNA from adult male mouse liver was used as a template
opment in the adult. However, to distinguish between for synthesis of double-stranded cDNA essentially as described by
transcriptional and posttranscriptional events in the Efstradiatis et al. (1976).
regulation of mRNA production, the transcription rate cDNA was synthesized in a reaction containing 10 pg poly(A)+
RNA, 25 kg/ml oligo(dT), 30 kg/ml actinomycin 0, 50 mM Tris, pH
of individual gene sequences must be studied. Only
8.3, 10 mM MgCI? and 100 U reverse transcriptase in a final volume
when it is established that the rate of synthesis of RNA of 200 ~1 at 42’C for 1 hr. After digestion of template RNA with heat-
from a gene is the same in two tissues but that the inactivated pancreatic RNAase, cDNA served as a template for the
mRNA is differentially present can posttranscriptional second-strand synthesis. The reaction in 1 ml total volume contained
control be invoked. 1 1-19cDNA, 70 mM KCI, 10 mM MgC12, 0.5 mM dXTPs, 0.1 M Hepes
6.5, 10 mM DTT and 100 U Klenow fragment of DNA polymerase I,
We have scored the rate of synthesis of RNA com- and was carried out at 15°C for 20 hr.
plementary to cDNA segments. The position of these After Si digestion of hairpin loops the double-stranded cDNA was
segments within the transcription units relative to the sedimented through a 15-30% sucrose gradient. Molecules larger
5’ end of the mRNAs was not established. For this than 400 bp were collected. Selected double-stranded DNA was then
extended by about 50 nucleotides with dC tails in a 50 ~1 reaction
reason these studies are unable to distinguish be-
containing 200 ng double-stranded DNA,. IO U terminal transferase,
tween two known modes of modulating transcription 100 mM potassium cacodylate, pH 7.4, 2 mM CoC12, 1 mM EDTA, 50
rates. The mechanisms to be considered are a change pM dCTP at 37°C for 30 min.
in the frequency of initiation of RNA chains, and a Fifty nanograms of dC-tailed double-stranded DNA were annealed
change in the proportion of premature termination to 350 ng of Pst l-cut pBR322 plasmid that had been “tailed” with 20
dG residues. Annealed molecules were used to transfect E. coli
products relative to complete transcripts (see for ex- Xl 776 as described by Villa-Komaroff et al. (1978). except that E.
ample Szybalski et al., 1970; Bertrand et al., 1975). coli were heat-shocked at 37°C. Ninety percent of transformants
carried recombinant plasmids-that is, their phenotype was Tet+
Experimental Procedures Amp-.

Animals
Selection of Liver-Specific Recombinant pBR322 Plasmids
Inbred NCS Swiss white male mice (25-30 g) were obtained from the About 1000 clones were picked and inoculated onto nitrocelulose
mouse colony maintained at the Rockefeller University.
filters (Millipore) that were placed on selective nutrient agar plates.
After colonies formed, replicates were made as described by Villa-
Cell Lines
Komaroff et al. (1978). The colonies were screened in duplicate
Hepatoma cells Hepa I (Darlington et al., 1980), a gift of G. Darlington,
according to the method of Young and Hogness (1977) with in vitro-
were maintained in monolayers in MAB medium supplemented with
labeled poly(A)+ RNA from male mouse liver, hepatoma cells and L
10% fetal calf serum. CHO cells were grown in minimal Eagle’s
cells.
medium, 7% calf serum and 1% nonessential amino acids.

Preparation of Poly(A)+ RNA from Tissues and Cell Lines Preparation of 32P-Labeled RNA with Polynucleotide Kinase
Total cellular RNA was extracted essentially as described by Ullrich About 5 pg of poly(A)+ RNA was partially hydrolyzed in 100 pl of 50
et al., (1977), in order to avoid nuclease action. Because in cultured mM Tris. pH 9.5, 5 mM glycine. 100 pM spermidine and 10 pM EDTA
cells most if not all poly(A)+ nuclear RNA is represented in the at 90°C for 30 min. Broken RNA was reacted with 150 IhCi Y-~*P-
cytoplasmic mRNA (Herman et al., 1976), over 90-95% of the poly(A) ATP (1000-3000 Ci/mmole; New England Nuclear) as substrate and
is cytoplasmic (Puckett et al., 1975) and finally the size of the poly(A)’ 10 U T4 polynucleotide kinase (P. L. Eiochemicals). RNA of specific
molecules that hybridize to our clones is appropriate for mRNA [not activity l-2 X 1 O7 cpm/pg was routinely obtained (Spradling et al.,
poly(A)+ hnRNA], we assume we have cloned sequences present in 1980).
cytoplasmic poly(A)’ mRNAs. Tissues were homogenized in 5 M
guanidinium thiocyanate (Fluka-Tridom). 0.1% lithium dodecyl sul- Preparation of Nuclei
fate, 50 mM lithium citrate, pH 7.0, and 0.1 m P-mercaptoethanol in Nuclei were obtained from mouse liver and brain essentially as
a high speed blender. Nucleic acids were precipitated by adjusting described by Tata (1974). One gram of tissue was homogenized in 5
the homogenate to 30 mM acetic acid and subsequently adding an ml of cold 0.32 M sucrose, 3 mM MgCl*, 1 mM Hepes, pH 6.8, in a
equal volume of 95% ethanol. After chilling at -2O’C, the precipitate motor-driven loose Potter homogenizer. The crude nuclear pellet was
was collected and dissolved in 7.5 M guanidine hydrochloride, 25 resuspended in 2.1 M sucrose, 1 mM MgCI?, 1 mM Hepes, pH 6.8,
mM lithium citrate, 1 mM dithiothreitol. RNA was then precipitated by and spun for 60 min at 20,000 rpm in an SW40 rotor. The nuclear
addition of acetic acid to a concentration of 50 mM and half the pellet was washed with 0.25 M sucrose, 1 mM MgCl*, 1 mM Hepes,
volume of 95% ethanol at -20°C. Precipitated RNA was dissolved in pH 6.8, and then with the reaction buffer used for in vitro transcription.
10 mM EDTA, 10 mM Tris, pH 7.4, and freed of impurities by
extraction with 4:l (v/v) chloroform:isobutanol. Poly(A)+ RNA was
obtained by two passages of RNA through an oligo(dT)-cellulose In Vitro Transcription in Isolated Nuclei
column (Aviv and Leder, 1972). Poly(A)+ RNA from the cytoplasm of We incubated 3 X 10’ nuclei for 20 min at 25°C in 2 ml of total
hepatoma and CHO cells was prepared as described by Harpold et volume with 0.5-l mCi w3’P-UTP (400 Ci/mmole; New England
al. (1979). Nuclear) in a reaction mixture containing 5 mM MgClz, 1 mM MnCI,.
10 mM Tris, pH 8.0, 0.14 M KCI, 14 mM P-mercaptoethanol, 10%
Construction of cDNA Recombinant Clones glycerol, 1 mM each of ATP, GTP, CTP. The incorporation for the
Reagents different cell nuclei was similar. Different preparations from different
pBR322 DNA was purified from E. coli HBlOl according to the sources varied over a range of approximately 2 fold.
method of Norgard et al. (1979). Reverse transcriptase was supplied Nuclear RNA was extracted as described previously (Soeiro and
by J. W. Beard. The Klenow fragment of E. coli DNA polymerase I Darnell, 19691, except that proteinase K (200 pg/ml) was added to
was purchased from New England Biolabs; Pst I restriction enzyme the lysed nuclei prior to phenol extraction of RNA.
Cell
738

Hybridization of RNA to Excess DNA

Plasmid DNA was boiled for 5 min in 0.1 N NaOH and bound to 0.45
Millipore HA nitrocellulose filters as described by Melli et al. (1975). E. D. would like to thank Dr. A. Bothwell for his generous advice
Hybridization of labeled RNA to at least a 10 fold excess of comple- concerning the constructions of recombinant cDNA clones and Dr.
mentary DNA was performed in 2X TESS buffer (0.01 M TES, pH Allan Spradling for suggesting the use of kinased RNA. We would
7.4, 0.3 M N&I. 0.01 M EDTA, 0.2% SDS) at 65°C for 40 hr or in like also to acknowledge the help of Marianne Salditt-Georgieff in
30% formamide (BRL), 0.1 M Pipes, pH 7.0, 0.3 M NaCI, 10 mM performing experiments presented in Table 1. This work was sup-
EDTA, 0.2% SDS at 45’C for 72 hr. Carrier yeast RNA (100 pg/ml) ported by grants from the National Institutes of Health and the
and poly(A) (100 pg/ml) were added to the hybridization buffers. American Cancer Society.
Following hybridization, filters were washed extensively at 65°C and Eva Derman was supported by a Helen Hay Whitney Fellowship
then briefly at 70°C in 2X TESS buffer and treated with pancreatic and an NIH training grant. Ken Krauter is an American Cancer Society
RNAase in the same buffer minus EDTA and SDS and finally with Fellow and Linda Bourque is a recipient of an National Institutes of
proteinase K (200 pg/ml). On occasion the supernatants of hybridi- Health fellowship.
zation were rehybridized to ensure completeness of hybridization. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
Gel Electrophoresis of DNA and RNA
solely to indicate this fact.
Purified cDNA-containing plasmids were digested with restriciton
endonuclease Pst I according to the instructions of the supplier.
Digests were then adjusted to 10% glycerol, 0.001% bromophenol Received October 23, 1980: revised December 9, 1980
blue, and subjected to agarose gel electrophoresis in 1.25% gels
using 0.05 M Tris-borate, pH 8.3, 1 mM EDTA buffer for 3 hr at 200
V.
Poly(A)-containing mRNA was ethanol-precipitated and resus- Aviv, H. and Leder. P. (1972). Purification of biologically active globin
pended at 0.5 mg/ml in 50% formamide, 6% formaldehyde, 0.02 M messenger RNA by chromatography on oligothymidylic acid cellulose.
borate, pli 8.3, 10% glycerol, 0.2 mM EDTA. Samples were heated Proc. Nat. Acad. Sci. USA 69, 1408-l 412.
to 65’C for 2 min to fully denature the RNA (Rave et al., 1979), then Bertrand, K., Korn, L., Lee, F., Platt, T., Squires, C. L., Squires C.
loaded onto 1.25% agarose gels buffered with 0.02 M borate, pH and Yanofsky, C. (1975). New features of the regulation of the
8.3, 0.2 mM EDTA, 3% formaldehyde as the electrode buffer. Elec- tryptophan operon. Science 789, 22-26.
trophoresis was for 5 hr at 200 V. 32P-labeled DNA restriction frag-
Blanchard, J. M., Weber, J., Jelinek, W. and Darnell, J. E., Jr. (1978).
ments were run in a separate well as size markers.
In vitro RNA-RNA splicing in adenovirus 2 mRNA formation. Proc.
Nat. Acad. Sci. USA 75, 5344-5348.
Blotting and Hybridization of DNA and RNA
Chan, K. M., Kurtz, D. T. and Feigelson, P. (1978). Transcription of
Size-fractionated DNA was transferred from agarose gels to nitrocel-
the ol2-globulin gene in male rat liver nuclei in vitro. Biochemistry 7 7,
lulose sheets as described by Southern (1975). After baking blots at
3092-3096.
80°C in vacua. blots were prehybridized for 6-20 hr in 6 x SSC, 1 X
Chikaraishi, D. M., Deeb, S. and Sueoka, N. (1978). Sequence
Denhardt’s solution (0.02% BSA, 0.02% Ficoll, 0.02% polyvinyl pyr-
complexity of nuclear RNA in adult rat tissues. Cell 13, 11 l-l 20.
rolidone), 50 pg/ml denatured salmon sperm DNA, 50 pg/ml poly(A),
50 pg/ml poly(C) at 65°C. Hybridization was performed for 24-36 hr Chow, L. T., Lewis, J. B. and Broker, T. R. (1979). RNA transcription
at 65’C in the same buffer plus 50,000 cpm/ml of denatured nick- and splicing at early and intermediate times after adenovirus-2 infec-
translated specific plasmid (spec. act. 5 x 10’ cpm/pg; Maniatis et tion. Cold Spring Harbor Symp. Quant. Biol. 44, 401-414.
al., 1975). Daneholt. B. (1975). Transcription in polytene chromosomes. Cell 4,
RNA fractionated on formaldehyde gels was transferred to nitro- l-9.
cellulose in the same manner as DNA, except the RNA gels required Darlington, G. J., Bernhard, H. P., Miller, R. A. and Ruddle, F. H.
no alkali pretreatment nor salt equilibration. 10 x SSC was used as (1980). Expression of liver phenotypes in cultured mouse hepatoma
the blotting buffer. RNA of sizes ranging from about 100-6000 cells. J. Nat. Cancer Inst. 64, 809-819.
nucleotides transferred to the nitrocellulose quantitatively, as assayed
Darnell, J. E. (1979). Steps in processing of mRNA: implications for
by the transfer of labeled RNA (data not shown; B. Seed, unpublished
gene regulation. In Gene to Protein: Information Transfer in Normal
observations).
and Abnormal Cells. T. R. Russell, K. Brew, H. Faber and J. Schultz,
Hybridization of RNA blots to specific cDNA clones was accom-
eds. (New York: Academic Press), pp. 207-208.
plished by a modification of the sandwich hybridization procedure of
Dunn and Hassell (1977). Nitrocellulose sheets onto which the de- Denhardt, D. (1966). A membrane-filter technique for the detection of
natured RNA was transferred were baked for 2 h in vacua at 80°C. complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-
Filters were then prehybridized in 50% formamide, 5 X SSC, 1 x 646.
Denhardt’s solution (Denhardt, 1966), 50 mM NaPO,. pH 7.4, 50 Kg/ Derman. E., Goldberg, S. and Darnell, J. E., Jr. (1976). hnRNA in
ml salmon sperm DNA, 50 pg/ml poly(A), 50 pg/ml poly(C) for 4-20 HeLa cells: distribution of transcript sizes estimated from nascent
hr at 45°C. After prehybridization, filters were incubated with 1 pg/ molecular profile. Cell 9, 465-472.
ml of specific cDNAs cloned in pBR322 in the same buffer at 45°C Dunn, A. R. and Hassel, J. (1977). A novel method to map transcripts:
for 12 hr. Following the first round of hybridization, the blots were evidence for homology between an adenovirus mRNA and discrete
washed 3 times in 2 x SSC, 0.2% SDS for 5 min at room temperature, multiple regions of the viral genome. Cell 12, 23-36.
then incubated with 5 X IO5 cpm/ml of nick-translated pBR322 Efstradiatis. A., Kafatos, F. C., Maxam, A. M. and Maniatis, T. (1976).
(spec. act. 1 X 10’ cpm/pg) in the prehybridization buffer at 37°C
Enzymatic in vitro synthesis of globin genes. Cell 7, 279-288.
for 16 hr.
Evans, R. M. and Ziff, E. (1978). The promoter and capped 5’
All filters were washed after hybridization 4 times in 2 X SSC,
terminus of RNA from the adenovirus-2 major late transcription unit.
0.2% SDS for 15 min each at room temperature, followed by two 15
Cell 7.5, 11463-l 475.
min washes in 0.1 X SSC, 0.2% SDS at 50°C. Damp filters were
wrapped in plastic wrap and autoradiographed using DuPont Cronex Evans, R. M., Fraser, N., Ziff, E., Weber, J., Wilson, M. and Darnell
Lightning-plus intensifying screens. RNA species representing about J. E.. Jr. (1977). The initiation sites for RNA transcription in Ad2 DNA.
0.015% of total poly(A) + cellular RNA could easily be detected with Cell 72, 733-739.
a few days of exposure under these conditions. Evans, R., Weber, J., Ziff. E. and Darnell, J. E., Jr. (1979). Premature
Transcriptional Control of Liver-Specific mRNAs
739

termination during adenovirus transcription. Nature 278, 367-370. fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-
Harpoid, M. H., Evans, R. M., Salditt-Georgieff, M. and Darnell, J. E., 51 7.
Jr. (1979). Production of mRNA in Chinese hamster cells: relationship Spradling, A. C., Bigan, M. E., Mahowald. A. P., Scott, M. and Craig,
of the rata of synthesis to the cytoplasmic concentration of nine E. A. (1980). Two clusters of gene for major chorion proteins in
specific mRNA sequences. Cell 7 7, 1025-l 035. Drosophila melanogaster. Cell 79, 905-914.
Hastie, N. D. and Bishop, J. 0. (1976). The expression of three Swaneck, G. E., Nordstrom, J. L., Kreuzaler, F.. Tsai, M.-J. and
abundance classes of messenger RNA in mouse tissues. Cell 9, 761- O’Malley, 8. (1979). Effect of estrogen on gene expression in chicken
774. oviduct: evidence for transcriptional control of ovalbumin gene. Proc.
Hayashi, M., Spiegelman, S., Franklin, N. C. and Luria. S. E. (1963). ‘Nat. Acad. Sci. USA 76, 1049-l 053.
Separation of the RNA message transcribed in response to a specific Szybalski, W., Bovre, K., Fiandt. M., Hayes, S., Hradecna, Z., Kumar.
inducer. Proc. Nat. Acad. Sci. USA 49, 729-736. S.. Lozeron. H. A., Nijkamp. H. J. J. and Stevens, W. F. (1970).
Herman, R. C., Williams. J. G. and Penman, S. (1976). Message and Transcriptional units and their controls in Escherichia co/i phage:
non-message sequences adjacent to poly(A) in steady state hnRNA operons and scriptors. Cold Spring Harbor Symp. &ant. Biol. 35,
of HeLa cells. Cell 7, 429-438. 341-353.

Hofer, E. and Darnell, J. E., Jr. (1981). The primary transcription unit Tata, J. R. (1974). Isolation of nuclei from liver and other tissues.
of the mouse ,&major globin gene. Cell 23, 585-593. Meth. Enzymol. 37, 253-262.

Jacob, F. and Monod, J. (1961). Genetic regulatory mechanisms in Tsai, S. Y., Roop, D. R., Tsai, M. J., Stein, P. J., Means, A. R. and
the synthesis of proteins. J. Mol. Biol. 3, 318-356. O’Maltey, B. W. (1978). Effect of estrogen on gene expression in the
chick oviduct. Regulation of the ovomucoid gene. Biochemistry 7 7,
Kleene, K. C. and Humphreys, T. (1977). Similarity of hnRNA se-
5773-5780.
quences in blastula and pluteus stage sea nuclei embryos. Cell 72,
143-155. Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Rutter, W. J. and
Goodman, H. (1977). Insulin genes: construction of plasmidscontain-
Kumer, S. and Szybalski, W. (1969). Orientation of transcription of
ing the coding sequences. Science 796, 1313-l 319.
the lac operon and its repressor gene in E. co/i. J. Mol. Biol. 40, 145-
151. Villa-Komaroff, L., Efstradiatis, A., Broome, S., Lomdico, P., Tirard,
R., Naber, S. P., Chick, W. L. and Gilbert, W. (1978). A bacterial
Lee, N. (1978). Molecular aspects of are regulation. In The Operon,
clone synthesizing proinsulin. Proc. Nat. Acad. Sci. USA 75, 3727-
J. H. Miller and W. S. Reznikoff. eds. (New York: Cold Spring Harbor
3721.
Laboratory Monograph Series), pp. 389-410.
Weber, J. (1979). Transcription and splicing of nuclear RNA adeno-
Lozeron, H. A., Dahlberg. J. E. and Szybalski, W. (1976). Processing
virus-2-infected HeLa cells. Ph.D. thesis. Rockefeller University, New
of the major leftward mRNA of coliphage lambda. Virology 77, 262-
York, New York.
277.
Weber. J., Jelinek, W. and Darnell, J. E., Jr. (1977). The definition of
Maniatis, T., Jeffrey, A. and Kleid, D. G. (1975). Nucleotide sequence
large viral transcription unit late in Ad a-infection of HeLa cells:
of the rightward operator of phage. Proc. Nat. Acad. Sci. USA 72,
mapping of nascent RNA molecules labeled in isolated nuclei. Cell
1184-l 188.
70, 611-616.
Melli, M., Ginelli, E., Corneo, G. and diGernia, R. (1975). Clustering
Wilson, M. C.. Fraser, N. W. and Darnell, J. E., Jr. (1979). Mapping
of the DNA sequences complementary to repetitive nuclear RNA of
of RNA initiation sites by high doses of UV irradiation: evidence for
HeLa cells. J. Mol. Biol. 93, 23-38.
three independent promoters within the left 11% of the Ad-2 genome.
Mintz, B. (1974). Gene control of mammalian differentiation. Ann. Virology 94, 175-t 84.
Rev. Genetics 8, 41 l-470.
Wold, B. J., Klein, W. H., Hough-Evans, 8. R., Britten, R. J. and
Nevins, J. R. and Wilson, M. C. (1981). Expression of the adenovirus- Davidson, E. H. (1978). Sea urchin embryo mRNA sequences ex-
2 major late transcription unit during early infection: regulation at the pressed in the nuclear RNA of adult tissues. Cell 74, 941-950.
level of transcription termination and RNA processing. Nature, in
Young, M. W. and Hogness. D. S. (1977). New approach for identify-
press.
ing and mapping structural genes in Drosophila melanogaster. In
Nevins, J. R.. Ginsberg, H. S., Blanchard, J. M., Wilson, M. C. and Eukaryotic Genetics System. ICN-UCLA Symposia on Molecular Bi-
Darnell. J. E., Jr. (1979). Regulation of the primary expression of the ology, G. Wilcox et al., eds. (New York: Academic Press), pp. 315-
early adenovirus transcription units. J. Viral. 32, 727-733. 331.
Norgard, M. V., Emigholz. K. and Monohan. J. J. (1979). Increased
amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia
co/i K-12 strains RR1 and Xl776 grown in the presence of high
concentration of nucleoside. J. Bacterial. 738, 270-272.
Puckett, L., Chambers, S. and Darnell, J. E., Jr. (1975). Short-lived
mRNA in HeLa cells and its impact on the kinetics of accumulation of
cytoplasmic poly(A). Proc. Nat. Acad. Sci. USA 72, 389-392.
Rave, N., Ckvenjakou. R. and Boedtker, H. (1979). Identification of
procollagen mRNAs transferred to DBM paper from formaldehyde
agarose gels. Nucl. Acids Res. 6, 3559-3567.
Shaw, A. R. and Ziff, E. B. (1980). Transcripts from the adenovirus-2
major late promoter yield a single early family of 3’ coterminal mRNAs
and five late families. Cell 22, 905-916.
Sheperd, G. W. and Nemer, M. (1980). Developmental shifts in
frequency distribution of polysomal mRNA and their post-transcrip-
tional regulation in sea urchin embryo. Proc. Nat. Acad. Sci. 77,
4653-4654.
Soeiro, R. and Darnell, J. E.. Jr. (1969). Competition hybridization by
“presaturation” of HeLa cell DNA. J. Mol. Biol. 44, 551-562.
Southern, E. M. (1975). Detection of specific sequences among DNA