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Hormonal control of mRNA synthesis studied by nucleic acid hybridization
Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
REVIEW
10 (1978) 119-l 3 3 Scientific Publishers, Ltd.
ARTICLE
HORMONAL CONTROL OF mRNA SYNTHESIS STUDIED BY NUCLEIC ACID HYBRIDIZATION Malcolm G. PARKER Department of Hormone Biochemistry, London WC2A 3PX, U.K.
Imperial Cancer Research Fund, Lincoln’s Inn Fields,
Received 22 December 1977
The regulation of protein synthesis by steroids is thought to be due to hormonal effects primarily on mRNA concentration. Experimental evidence to support this conclusion has come largely from the use of DNA probes complementary (cDNA) to mRNA molecules or by translation of the mRNA in vitro. In this review the experimental procedures involved and the application to hormone action of cDNA hybridization will be reviewed. (1) mRNA concentrations can be assayed in tissue RNA samples by hybridization with radiolabelled complementary DNA probes (cDNA). From the rate of hybridization of an mRNA preparation to a cDNA probe it is possible to estimate specific mRNA concentrations and thereby study their hormonal regulation within tissues of subcellular fractions. (2) Rates of synthesis of a specific RNA can be measured by hybridization of pulse-labelled RNA with excess cold cDNA as illustrated in studies of the glucocorticoid induction of MMTV RNA. (3) Hormone-induced alterations of mRNA populations as a whole can be investigated. From the kinetics of hybridization of mRNA with its complementary DNA it is possible to estimate the number of different RNA sequences in tissues and to approximate the number of copies of each sequence per cell. Consequently, by comparing mRNA samples isolated from tissues of different hormonal status it is possible to demonstrate specific hormone-inducible mRNA species and, in some cases, identify their translation products. Keywords:
mRNA; transcription;
steroid hormones; protein synthesis.
Steroid hormones regulate protein synthesis in a variety of eukaryotic tissues (Tata, 1970). In principle, such regulation could be achieved at any step involved in transcription, processing and transport of RNA to the cytoplasm or at the level of translation of mRNA into protein. Thus it is important to investigate the effects of steroids on all these processes. Two types of experimental approach have suggested that the regulation is mediated mainly by alterations in the cellular mRNA
Abbreviations used in this paper: cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid;rRNA, ribosomal ribonucleic acid; tRNA, transfer ribonucleic acid; Rot, initial RNA concentration (moles of nucleotide per litre) X time (set); MMTV, mouse mammary tumour virus. 119
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M. G. Parker
concentration (O’Malley and Means, 1974; Palmiter, 1975; Schimke et al., 1975). First, mRNA activity in cell-free protein-synthesizing systems has been assayed by examining the translation products on polyacrylamide gels. Since the amount of protein synthesized is proportional to the amount of mRNA supplied, it is possible to quantitate mRNA activity by measuring the incorporation of radioactive amino acid precursors into polypeptides. This method measures only those mRNAs which are active in the particular cell-free system used. The second procedure is to assay mRNA molecules by nucleic acid hybridization using DNA probes. This method measures all mRNA molecules whether or not they are active in protein synthesis. Both approaches have been utilized in assessing hormonal effects on cellular mRNA composition, but for the purpose of this review only the use of hybridization with DNA probes will be discussed. A DNA probe which is complementary to a specific mRNA or mRNA population can be synthesized in vitro using RNA-directed DNA polymerase (reverse transcriptase) purified from RNA tumour virus particles. The cDNA product therefore can hybridize uniquely to the mRNA by Watson-Crick base pairing. By using radiolabelled precursors the cDNA probe can be labelled to a high specific activity so that the hybridization reaction can be monitored. The rate of hybridization of a small amount of labelled cDNA to an excess of mRNA depends on the mRNA concentration (Bishop et al., 1974). This forms the basis of the assay in that the higher the mRNA concentration, the faster the hybridization. Thus the effect of steroid hormones on mRNA concentration can be determined by measuring the rate of hybridization of the cDNA probe with RNA samples isolated from tissues of differing hormonal status. Rates of RNA synthesis in cells can also be measured by hybridization of radiolabelled RNA synthesized during a short time period with excess cDNA, thereby quantitating the amount of RNA synthesis per unit time. The experimental procedures required for these studies will be summarized first and these will be followed by an outline of the sorts of information that have been obtained, referring to specific examples to illustrate the points of interest.
METHODS The experimental procedures consist of (1) isolation of RNA, (2) the synthesis of a cDNA probe and (3) an assay procedure to measure the kinetics of hybridization of the RNA with the cDNA probe. Isolation of RNA
A large number of variations in the methodology used to extract RNA have been described, but the method of choice depends mostly on tissue sources, whether nuclear, cytoplasmic or total RNA is required, and whether the RNA must be isolated intact. Alternative methods have been summarized in several reviews (Brawerman, 1973; Muramatsu, 1973; Busch et al., 1976) but fall largely into three
mRNA
121
analysis by cDNA hybridization
important approaches: (a) isolation of polysomal mRNA using sucrose gradient sedimentation (Blobel and Potter, 1967; Noll, 1969; Means et al., 1971; Palacios et al., 1972); (b) isolation of nuclear RNA by citric acid extraction (Busch, 1967; Holmes and Bonner, 1973); and (c) extraction and deproteinization of total cell or cytoplasmic RNA using phenol and c~orofo~ (Penman et al., 1968). Further fractionation of RNA is necessary before it can be used as a template for cDNA synthesis. Obviously, studies of a specific mRNA molecule necessitate its purification in order to synthesize a homogeneous cDNA preparation. This has been achieved by exploiting differences in mRNA size, presence or absence of poly(A) tracts at the 3’ end of the molecule, or the ability of ~tibodies to immunoprecipitate a partially completed peptide on specific polysomes from which mRNA can be extracted (Palacios et al., 1973; Rosen et al., 1975; Wahli et al., 1976; Shapiro and Baker, 1977). Success has usually depended on whether or not the mRNA in question accounted for a large proportion of the total RNA. The purification of mRNA species present in low concentrations can be extremely difficult and time-consuming. The synthesis of cDNA probes to investigate mRNA complexity requires only that the mRNA be separated from ribosomal RNA (rRNA) and transfer RNA (tRNA). This can be achieved by dT-cellulose chromatography (Aviv and Leder, 1972) or by poly~)~epharose chromatography (Sheldon et al., 1972) which bind RNA molecules containing poly(A) tracts, a common feature of many eukaryotic mRNA molecules. This means that mRNA lacking poly(A) will elute with the rRNA and tRNA and consequently has not been studied in much detail. Synthesis
of complementary
DNA
The synthesis of DNA complementary to RNA is achieved using reverse transcriptase, first described by Temin and Mizutani (1970) and Baltimore (1970) (fig. 1). When an oligo-dT primer is hyb~dized to the poly(A) tract at the 3’ end of the mRNA molecules it is possible to extend the primer and synthesize DNA copies of the RNA molecules. The inclusion of 3H-labelled dCTP results in the synthesis of a radiolabelled cDNA probe of specific activity up to 10’ cpm/pg DNA. Typical reaction conditions are as follows: 0.1 ,uM 13H]dCTP (15-20 Cif mmol), 36 &ml actinomycin D (to inhibit DNA synthesis in which cDNA acts as a template), 50 mM Tris-HCI (pH 8.3), 10 mM dithiothreitol, 6 mM MgC12, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 5 pg/ml oligo-(dTra_rs) and 25 pg/ ml poly(A)-containing RNA, 100 units/ml avian myoblastosis virus reverse tran-
5’
AA&A 3’
reverse oligo
transcriptase dT.
dNTPs
Fig. 1. Reaction scheme for the synthesis of cDNA.
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M. G. Parker
scriptase incubated at 37°C for 30 min. The RNA is then hydrolysed with alkali and the cDNA separated from unincorporated ~3H~dCTP by gel filtration through Sephadex G-SO. After precipitation with ethanol the cDNA is stored in 0.01 M Tris-HCl (pH 7.5) at -70°C. Specific cDNA probes have now been synthesized for several mRNA molecules regulated by steroid hormones, including those for ovalbumin, vitellogenin and casein (Su~iv~ et al., 1973; Mon~an et al., 1976a; Rosen and Barker, 1976; Ryfell, 1976; Shapiro and Baker, 1977). cDNA probes to investigate mRNA complexity in chicken oviduct and rat ventral prostate have also been synthesized (Monahan et al., 1976b; Cox, 1977; Hynes et al., 1977; Parker and Mainwaring, 1977). More recently, cDNA hybridization has been carried out to investigate rates of synthesis of specific mRNA molecules in which pulse-labelled RNA is hybridized with excess cold cDNA. In this case f3HjdCTP is replaced with dCTP and the reactants increased to prepare ~.lgamounts of cDNA (Young et al., 1977).
The principle of the assay is to measure the rate of hybridization of the cDNA probe with the mRNA. This is achieved by monitoring the proportion of cDNA which has reassociated with the mRNA during the hyb~d~ation reaction. A variety of hybridization reaction conditions are available, but a solution containing 0.24 M sodium phosphate buffer (pH 7.4), 1 mM EDTA and 0.05% SDS at 70°C is used extensively (Bishop et al., 1974; Ryfell and McCarthy, 1975; W~li~s and Penman, 1975). Since the hybridization rate varies with the monovalent cation concentration and temperature, comparisons of data obtained under different conditions require correction to standard conditions, and conversion tables are to be found in Britten et al. (1974). Excess RNA is mixed with radiolabelled cDNA in hybridization buffer, overlaid with paraffin oil, and incubated at the appropriate temperature. One method of assessing the extent of hybridization depends on the fact that Sl nuclease (from AspergiElus olyzae; Sigma Chemical Co.) degrades single- but not double-stranded DNA; therefore cDNA which has hyb~~zed to RNA is resistant to Sl nuclease digestion (Milcarek et al., 1974). Portions of the hybridization mix are removed at intervals ranging from 5 min to 24 h and diluted into ‘SI nuclease buffer’ (0.05 M sodium acetate (pH 4.5), 0.1 M NaCI, 0.1 mM Z&O4 and 10 pg/ml denatured DNA) which is subsequently divided into two aliquots. To one aliquot is added approximately 100 units Sl nuclease to hydrolyse u~ybridized cDNA and then both aliquots are incubated at 46°C for 2 h. The samples are cooled in ice and adjusted to contain IO @g/ml bovine serum albumin and 5% trichloroacetic acid. They are then collected on millipore filters and counted in scinti~ation fluid. The percentage cDNA resistant to digestion is taken as the percentage hybridization, Since the amount of hybridization depends on the mRNA concentration and the time ofhybridization, hybridization is usually plotted against the Ret value, which is the product of the initial RNA concentration (in moles of nucleotide per litre) and time (in seconds). The Ret value at which 50%
mRNA analysis by cDNA hybridization
123
of a given population of nucleic acid molecules have hybridized (Rot 112) is the parameter most frequently used to compare RNA samples. Thus the proportion of specific mRNA molecules in an RNA mixture can be determined by comparing the ROtl,,) values of the purified mRNA with those of the RNA samples under investigation. The ROtI,* values will increase (decreased rate of hybridization) as the mRNA concentration decreases. A reduction in the rate of hybridization, so that the Rot,,, value increased by, say, IOO-fold, would indicate that the specific RNA represented only 1% of the RNA sample.
APPLICATIONS
OF cDNA HYBRIDIZATION
of specijic n&VA concentrations The use of cDNA probes to assay specific mRNA molecules made it possible to test whether alterations in protein synthesis, induced by a hormone, are the results of alterations in the cellular mRNA concentration. Progress has been rapid, especially in studies of the hormonal regulation of ovalbumin synthesis in the chick oviduct. This was mainly because ovalbumin mRNA, the preponderant mRNA species, was relatively easy to purify and thereby synthesis of a complementary DNA probe was straightforward. Subsequently, casein and vitellogenin mRNA have also been purified and studies of their regulation by steroid hormones (Rosen and Barker, 1976; Ryfell, 1976; Shapiro and Baker, 1977) have supported the conclusions drawn for ovalbumin mRNA. The concentration of ovalbumin mRNA in the chick oviduct has been measured during growth and differentiation of the gland following primary stimulation of immature chicks with oestrogen; following withdrawal of hormone; and following a second injection of oestrogen to withdrawn chicks (secondary stimulation) (Cox et al., 1974; Harris et al., 1975; McKnight et al., 1975) (fig. 2). It was concluded that there were very few copies of ovalbumin mRNA in immature chicks but that it accumulated slowly after oestrogen administration following differentiation of tubular gland cells in which it is synthesized. Withdrawal of the hormone from the chicks resulted in a dramatic decrease in ovalbumin mRNA content, but a secondary administration of oestrogen resulted in its rapid restoration. There were discrepancies in the various measurements of the response time for this secondary induction, but alternative procedures (Chan et al., 1973; Palmiter, 1973) and further studies using ovalbumin cDNA (Palmiter et al., 1976; Groner et al., 1977) suggest that oestrogen induces the accumulation of ovalbumin mRNA in the tubular gland cells after a lag of about 3 h. It should be stressed that these determinations are measurements only of the concentrations of mRNA and say nothing about synthetic or degradative rates of RNA molecules. Therefore the conclusion that oestrogen regulates ovalbumin mRNA by regulating transcription has been drawn somewhat prematurely from these data. The failure to detect ovalbumin mRNA in the absence of hormone is usually cited as evidence of transcriptional control, and the relatively Measurement
124
M. G. Parker
-2
-1
0
1 Log
2 Rot
3
4
Fig. 2. Hybridization of ovalbumin cDNA with oviduct mRNA (curves adapted from the work of Cox et al., 1974; Harris et al., 1975; McKnight et al., 1975). At intervals the extent of hybridization was determined (resistance to Sl nuclease). Rot values are the product of RNA concentration in moles of nucleotide per litre and time of reaction in seconds.
long half-life determined for ovalbumin mRNA appeared to support this notion (Kafatos, 1972; Chan et al., 1973; Palmiter, 1973; Harris et al., 1975). However, the half-life was determined for polysomal and total cellular ovalbumin mRNA and not nuclear RNA. In fact, the observation that the ti/a for ovalbumin mRNA is longer in the presence than in the absence of oestrogen already suggests that regulation of ovalbumin mRNA levels is not restricted to transcriptional controls (Palmiter and Carey, 1974; Cox, 1977). Finally, the inability to detect the accumulation of an RNA species does not indicate that it was never synthesized. The recent observation that histone mRNA is synthesized throughout the cell cycle of HeLa cells but is only found in the cytoplasm during the period of DNA synthesis (Melli et al., 1977) suggests that RNA processing rather than transcription is the major regulatory step in this particular case. Consequently, in studies of hormone-inducible mRNA molecules it is necessary to establish rates of RNA synthesis in the presence and absence of hormone to support models based on transcriptional controls. Determination of synthetic rates for a particular RNA requires measurements of the rate of incorporation of labelled precursors into the RNA. It may be difficult to obtain labelled RNA of sufficient specific activity in whole tissue, but it is feasible in cell cultures. Recently the rate of synthesis and degradation of mouse mammary tumour viral (MMTV) RNA has been measured in mammary tumour cell lines following glucocorticoid induction (Ringold et al., 1977; Young et al., 1977). The initial rate of synthesis of MMTV RNA was measured by hybridization of pulse-labelled RNA with non-radiolabelled cDNA. The hybridized material was quantitated by two novel procedures (fig. 3) that were utilized to reduce the background interference due to non-specific hybridization of other labelled RNA
125
mRNA analysis by cDNA hybridization A
I
I
I
I
I
*
I
I
I
I
Sephadex
bead
dC dC dC dC 5'
Fig. 3. Schematic diagrams to illustrate the procedures used to quantitate MMTV RNA. Both procedures were devised because MMTV RNA comprises a small proportion of the mammary cell RNA even in the presence of glucocorticoid. Following hybridization of the labelled RNA with MMTV cDNA probes, the reaction mix was incubated with pancreatic RNase to degrade unannealed RNA, thereby reducing background interference of other labelled RNA species due to non-specific binding. A. MMTV RNA initially hybridizes to MMTV cDNA extended with poly(C) and the hybrid is then separated from cellular labelled ribonucleotides by chromatography on poly(I)-Sephadex columns (Young et al., 1977). B. MMTV RNA initially hybridizes to MMTV cDNA made double-stranded (tailed duplex) by omission of actinomycin D during the synthesis of cDNA. The hybridized material can then be separated from cellular labelled ribonucelotides by chromatography on hydroxyapatite.
species. In one (Young et al., 1977) the cDNA had been elongated with poly(dC) so that the poly(dC)-cDNA-RNA hybrid could be bound to a poly(I)-Sephadex column by virtue of the poly(dC) regions of the hybrid, thereby separating hybridized from non-hybridized RNA. In the other (Ringold et al., 1977) the cDNA was extended to form ‘tailed duplex’ DNA using DNA polymerase and deoxynucleotide triphosphates. Such double helical DNA hybridized with pulselabelled RNA could be separated from non-hybridized RNA by hydroxyapatite chromatography. Thus, by incubating mouse cell lines with [3H]uridine and then exposing the culture to dexamethasone for different periods of time, it was possible
M. G. Parker
126
Table 1 Kinetics of glucocorticoid tumour cells
induction
Time after dexamethasone addition to cell culture (mm)
of MMTV RNA
synthesis
410of total RNA synthesized cDNA probe Young
et al. (1977)
that hybridized
Ringold
0 10
0.15 0.7
0.05 _
15 20
_ 0.85
0.45 -
30
1.05
0.4
The MMTV RNA was assayed
by the procedures
described
in cultured
mouse
mammary
to MMTV RNA
et al. (1977)
in fig. 3.
to isolate 3H-labelled RNA which could be assayed for the presence of MMTV RNA. Both techniques suggested that dexamethasone resulted in a 3-lo-fold stimulation in viral RNA synthesis within lo-l.5 min (table 1). Prior to this study, both groups had measured MMTV RNA accumulation as described for ovalbumin, using excess cold RNA and [3H]cDNA prepared to MMTV RNA (Ringold et al., 1975; Scolnick et al., 1976). In these studies they had detected only a 2-fold increase in concentration 30 min after dexamethasone treatment. Thus, measurement of the rate of synthesis proved to be more definitive and more sensitive. Young et al. (1977) have also attempted to measure the half-life of MMTV RNA by incubating pulse-labelled cell cultures with actinomycin D for different periods and following the decay rate of the viral RNA. In the presence of dexamethasone the tr/a was greater than 8 h but unfortunately, in the absence of steroid, the rate of synthesis of MMTV-specific RNA was too low to provide an accurate determination of half-life after actinomycin D addition. Ringold et al. (1977) have pointed out that if the tr/a for MMTV RNA in the absence of steroid was considerably shorter than the labelling period it is conceivable that an increase in the apparent rate of synthesis might in fact be the result of the hormone inhibiting the degradation of the rapidly turning-over RNA species. Such a mechanism is difficult to discount and extremely difficult to test. Similar studies have not been reported for other hormone-inducible mRNA molecules, but it is necessary to establish rates of RNA synthesis in the presence and absence of hormones to support models based on transcriptional control. The mammary tumour virus system is a good example of the advantage a cell line offers over other hormone-responsive tissues in that it is relatively easy to obtain pulselabelled RNA. Analysis of mRNA populations in general It is also possible to investigate the role steroid hormones
play in regulating
mRNA analysis by cDNA hybridization
121
mRNA populations as a whole even when hormonally induced proteins have not been identified. These studies require cDNA probes complementary to the total mRNA population rather than to specific mRNA species. In fact, technically it is more straightforward than investigations of specific mRNAs because mRNA has only to be separated from rRNA and tRNA and does not require further puritication. The procedure is to synthesize cDNA using total mRNA as template and then measure the rate of hybridization using excess mRNA and a trace amount of radioactive cDNA. The rate of hybridization is determined by the sequence complexity (see below) of the template mRNA and by the relative concentrations or abundance of the different sequences. The concentration of different mRNA species in cells extends over a wide range and those present in high concentrations will hybridize faster than those in low concentrations. For instance, consider a population of mRNA in which one species accounts for 33%, 100 species each account for 0.33% (i.e. 33% in total) and 10,000 species each account for 0.0033% of the mRNA. The most abundant mRNA sequence will hybridize 100 times faster than the moderately abundant sequences, which themselves will hybridize 100 times faster than the scarce sequences. The hybridization curve will then consist of three transitions (fig. 4), the first transition representing the most abundant RNA and subsequent transitions representing classes of RNA of lower abundance. The plateau level of each transition gives the proportion of mRNA molecules in each class, and the Rot112 values of each transition by comparison with that obtained for a defined mRNA species give the sequence complexity of each class. Globin mRNA coding for both the 01and fl chains is frequently used as a standard and, containing 1300 nucleotides, has a complexity of 1300 nucleotides. By dividing the Retr/a values
Fig. 4. Hybridization curves of globin mRNA and a hypothetical tissue mRNA sample. Globin mRNA (X -X) was hybridized with its cDNA and tissue mRNA ( -) hybridized with its cDNA, the arrows indicating Rot,,, values of the transitions (see table 2).
M. G. Parker
128
obtained for each transition by 6 X 104, the RotlIZ value obtained for the hybridization of globin mRNA and globin cDNA, it is possible to calculate the sequence complexity in each class of mRNA. Initially one correction to the observed Retr/a values has to be made because each class of RNA is not a pure component but is diluted by the other classes, thereby reducing its rate of hybridization. In the example shown in fig. 4, inspection of the hybridization curve indicates that the mRNA consists of three classes of mRNA at distinct concentrations. The first transition with an observed Ret,,, value of 1.8 X 10e3 mol elitre-‘-set is corrected because it only comprises l/3 of the total mRNA and would hybridize 3fold faster were it a pure component (table 2). Division of the corrected ROtr,2 value by 6 X 10b4, the value for globin mRNA (complexity 1300 nucleotides), indicates that it too has complexity of 1300 nucleotides [(1.8 X 10m3 + 3)/(6 X 10m4) X 13001. This would be equivalent to one sequence were its number average molecular weight to be 1300 nucleotides. A similar series of calculations indicate that the second transition represents a class of RNA comprising 100 sequences and the third transition a class of RNA comprising 10,000 sequences (table 2). In fact, when tissue RNA samples are analysed, transitions are rarely as distinct as those shown in fig. 4 (for example see fig. 5) and it is preferable to estimate the number of classes and their Retr/a values by computer analysis of the data. Several programmes have been described (Hastie and Bishop, 1976; Pearson et al., 1977). Estimates of the number of copies of each sequence can be obtained from the amount of mRNA per cell. From the DNA content per cell and the RNA/DNA ratio of the tissue under investigation the approximate amount of mRNA per cell can be calculated. The calculations can only be approximates since cell heterogeneity within tissues is not taken into account and the proportion of mRNA in total RNA has usually to be estimated.
Table 2 Sequence
complexity
of hypothetical
tissue RNA
Transition
% cDNA hybridized
Ret ~2 observed (mol . litre-’ -set)
Roti/ corrected
Complexity nucleotides _
No. of sequence
I II III
33 33 33
1.8 X 1O-3 1.8 X 10-l 1.8 X lo1
6x10” 6 X 1O-2 6 X 10’
1,300 130,000 13,000,000
1 100 10,000
The data were obtained from the hybridization curves shown in fig. 4. The observed Rotr,a values were corrected for the fraction of RNA they represent. The complexity in nucleotides was derived by comparison of the corrected R 0t 112 values with that obtained for globin mRNA, to a complexity of 1300 nucleotides. The namely 6 X lo4 mol . litre-’ -set, corresponding number of sequences was calculated by dividing the complexity in nucleotides by 1300, the number average chain length of the RNA.
mRNA analysis by cDNA hybridization
129
The first studies of mRNA populations in steroid target tissues were carried out with chick oviduct mRNA. Axe1 et al. (1976) demonstrated that, in common with many other tissues and cell types (Lewin, 1975) oviduct RNA consisted of three classes of mRNA sequences present in different cellular concentrations or abundances. There was a total of 12,000-15,000 different sequences most of which were present in fewer than 10 copies per cell, but one class of oviduct mRNA consisted of only one species present in about 100,000 copies per cell and probably represents ovalbumin mRNA sequences. It is also possible to analyse the tissue specificity of mRNA sequences by hybridization of the cDNA from one tissue with mRNA from another tissue, so that cDNA which remains unhybridized is complementary to tissue-specific RNA sequences. These hybridization reactions are referred to as heterologous to distinguish them from homologous reactions in which the cDNA is synthesized from the same mRNA to which it is being hybridized. To examine whether oviduct mRNA sequences are also present in chick liver, Axe1 and co-workers hybridized oviduct cDNA with liver mRNA. Such heterologous hybridization reactions indicated that at least 85% of the mRNA species are shared. Presumably common sequences encode ‘housekeeping’ functions shared by all cell types, but they also suggested that the mRNA molecules for the egg-white proteins are also present in the liver albeit at a much reduced concentration. The effect of oestrogen on mRNA complexity was first investigated by O’Malley reactions indicated that (Monahan et al., 1976a). Homologous hybridization poly(A)-containing RNA from the oviducts of hens and diethylstilboestrol-stimulated chicks contained 20,000-25,000 different RNA sequences present in three classes of different abundance, ranging from fewer than three to many thousand copies of each sequence per cell. In contrast, there were only about 10,000 different sequences in the oviducts of chicks withdrawn from oestrogen and there was no class of RNA corresponding to the high abundance class demonstrated in hen and hormone-induced chick oviduct. These results suggested that oestrogen was regulating both the relative abundance of certain mRNA molecules and the total number of different RNA sequences. Subsequent studies using heterologous hybridization reactions in which cDNA synthesized with mRNA from hen oviduct was hybridized with mRNA from oestrogen-withdrawn chicks (Cox, 1977; Hynes et al., 1977) have confirmed that the concentration of the most abundant class of mRNA, probably containing ovalbumin mRNA, but not the total number of different sequences, is regulated by oestrogen. Caution should be exercised in allocating precise values for the number of sequences in different abundance classes (Bishop et al., 1974; Hastie and Bishop, 1976; Monahan et al., 1976a). Allowance has to be made for rRNA contamination of mRNA samples which will affect the absolute numbers of sequences in each class of RNA. This may be critical when comparing the complexity of different RNA samples but to some extent can be circumvented by using heterologous hybridization reactions.
130
Ivl.G. Parker
Accurate estimates of the Ret,,, values of low abundance sequences are often difficult if they comprise only a small fraction of the total RNA even though they account for most of the different mRNA sequences. In the case of heterologous hybridization reactions, differences in mRNA populations are estimated from differences in the percentage saturation of homologous versus heterologous reactions. Unfortunately a difference of 5% in the percentage of saturation does not necessarily indicate that 95% ofthe sequences are similar. The mRNA complexity is determined mainly by one class of RNA, namely the slowest hybridizing class which comprises a lot of sequences represented by only a few copies per cell. If this class were to contain 10,000 sequences and account for only 20% of the RNA, then a 5% difference in saturation could be equivalent to a difference of 2500 sequences and not 500 sequences. In fact, since it is not known to which class of RNA unhybridized cDNA corresponds, such a difference in percentage saturation may be less provided it corresponded to a class containing few sequences in high abundance. In the case of oviduct RNA (Cox, 1977; Hynes et al., 1977) the percentage saturation was similar for both the homologous and heterologous hybridization reactions and therefore indicates that most, if not all, of the mRNA species present in the oviducts of laying hens are also present in the oviducts of hormone-withdrawn chicks. Comparisons of mRNA populations in steroid target tissues of different hormonal status can be extremely useful even when hormonally regulated proteins have not been identified. For example, although rat ventral prostate is a target tissue for androgenic steroids and has been used extensively to study the mechanism of action of androgens (King and Mainwaring, 1974; Mainwaring, 1977) satisfactory protein markers of hormone action had not been reported. By analysing prostatic mRNA from normal and castrated rats, a class of highly abundant RNA species that is regulated by androgens in vivo has been demonstrated (Parker and Mainwaring, 1977). Prostatic RNA isolated from normal animals comprised three abundance classes, the most abundant of which was absent after castration. The effect of androgens on this class of RNA was studied in more detail by isolating cDNA complementary only to these sequences. This was achieved by hybridizing total cellular poly(A)-containing RNA from normal animals with its complementary DNA to an Ret value of 2 X IO-’ mol . litre-’ -set when about half of the cDNA, corresponding to the abundant RNA sequences, would have annealed. This abundant cDNA was then separated from the non-abundant cDNA by hydroxyapatite chromatography and shown to be complementary to about three different poly(A)containing RNA sequences (fig. 5). Thus it was then possible to investigate the effects of androgens on mRNA levels in the ventral prostate using this fractionated cDNA probe (Parker and Scrace, 1978). Once mRNA sequences that are regulated by a hormone have been identified it would be important to determine for what proteins they are coding. In the past this has not been easy, but recently Paterson and Bishop (1977) have developed a method for relating cDNA sequences, their corresponding mRNA species and the proteins for which they code. The method is particularly suitable for abundant
131
mRNA analysis by cDNA hybridization
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Fig. 5. H~brid~ation of fractionated EDNA. Total cDNA (a) to prostatic poly(A)~ont~ning RNA was hybridized to an Ret value of 2 x IO-2 mol. litre-s -see. The hybridized material was separated from unhybridized material by hydroxyapatite chromatography. The hybridized cDNA corresponding to abundant cDNA (0) and the unbybridized cDNA corresponding to nonabundant cDNA (0) were then treated with alkali to degrade cofractionated RNA, neutralized and hybridized with total poly(A)-conning RNA.
cDNA preferably fractionated as described above. Hybridization of the mRNA with the cDNA in excess converts all the mRNA molecules complementary to the probe into a hybrid form which cannot be translated in cell-free systems. However, heat dissociation of the hybrid reinstates translations activity so that it is possible to identify which proteins are translated from the mRNA for which cDNA probes have been isolated (Paterson and Bishop, 1977). This approach could in theory be applied to investigate the role of any hormone on RNA and protein metabolism and may be especially useful in studies of target tissues in which hormone markers have not yet been identified.
CONCLUDING
REMARKS
It is clear from experiments using cDNA probes that steroid hormones regulate intracellular mRNA concentrations and thereby modulate protein synthesis. At least in the case of the hormonal control of MMTV RNA, glucocorticoids appear to increase the rate of synthesis of the RNA. Similar studies with other steroidresponsive tissues and cells are required to establish the generality of this observation. Large amounts of cDNA are required for these studies, but the current possibilities of cloning cDNA molecules in bacteria would certainly make such studies more feasible. Such cloned cDNA may ultimately be used to isolate specific genes from total cellular DNA and permit more precise studies into the mechanism of action of steroid hormones.
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A4 G. Parker
ACKNOWLEDGEMENTS I should like to thank my colleagues Drs. R.J.B. King, R. Shields, J.A. Smith, R.F. Brooks, C. Dickson, G.E. Peters, R.A. Weiss and Mr. G.T. Scrace for their suggestions during the preparation of the manuscript, and Mrs. M.C. Barker for excellent secretarial assistance.
REFERENCES Aviv, H. and Leder, P. (1972) Proc. NatI. Acad. Sci. U.S.A. 69,1408-1412. Axel, R., Feigelson, P. and Schutz, G. (1976) Cell 7,247-254. Baltimore, D. (1970) Nature (London) 226, 1209. Bishop, J.Q., Morton, J.G., Rosbash, M. and Richardson, M. (1974) Nature (London) 250, 199-204. Blobef, G. and Potter, V.R. (1967) J. Mol. Biol. 26,279. Brawerman, G. (1973) Methods Cell Biol. 7, l-22. Britten, R.J., Graham, D.E. and Ncufeld, B.R. (1974) In: Methods in Enzymology, Vol. 29, Part E, Eds.: L. Grossman and K. Moldane (Academic Press, New York and London) p. 363. Busch, H. (1967) In: Methods in Enzymology, Vol. 12, Part A, Eds.: L. Grossman and K. Mot dane (Academic Press, New York and London) pp. 434-439. Busch, H., Choi, Y.C., Daskal, Y., Liarakos, CD., Rao, M.R.S., Ro-choi, T.S. and Wu, B.C. (1976) Cancer Res. 13,101-197. Chan, L., Means, A.R. and O’Malley, B.W. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1870-1874. Cox, RF. (1977) Biochemistry 16,3433-3443. Cox, R.F., Haines, M.F. and Emtage, J.S. (1974) Eur. J. Biochem. 49,225-236. Groner,B., Hynes, N.E., Sippel, A-E., Jeep, S., Nguyen-~Iuu, M.C. and Schutz, G. (1977) .I. Biol. Chem. 252,6666-6674. Harris, S.E., Rosen, J.M.,Means, A.R. and O’Malley, B.W. (1975) Biochemistry 14,2072-2081. Hastie, N.D. and Bishop, J.O. (1976) Cell 9,761-774. Holmes, D.S. and Bonner, J. (1973) Biochemistry 12,2330-2338. Hynes, N.E., Groner, B., Sippel, AX., Nguyen-Huu, M.C. and Schutz, G. (1977) Cell 11,923932. Kafatos, F.C. (1972) In: Gene Transcription in Reproductive Tissue, Vol. 5, Ed.: E. Diczfalusy (Karolinska Institutet, Stockholm) p. 319. King, R.J.B. and Mainwaring, W.I.P. (1974) Steroid-Cell Interactions (Butterworth & Co., London) pp. 56-83. Lewin, B. (1975) Cell 4,77-93. Mainwaring, W.I.P. (1977) In: The Mechanism of Action of Androgens (Springer-Verlag, New York) pp. 43-77. McKnight, G.S., Pennoquin, P. and Schimke, R.T. (1975) J. Biol. Chem. 250,8105-8110. Means, AR., Abram, LB. and O’Malley, B.W. (1971) Biochemistry 10,1561-1570. Me& M., Spinelli, G. and Arnold, E. (1977) Cell 13,167-174. Milcarek, C., Price, R. and Penman, S. (1974) Cell 3,1-10. Monahan, J.J., Harris, S.E. and O’Malley, B.W. (1976a) J. Biol. Chem. 251,3738-3748. Monahan, J.J., Harris, SE., Woo, S.L.C., Robberson, D.L. and O’Malley, B.W. (1976b) Biochemistry 15,223-233. Muramatsu, M. (1973) Methods CelI Biol. 7,23-51. Nell, H. (1969) In: Techniques in Protein Biosyn~es~, Vol. 2, Eds.: P.N. CampbelI and J.R. Sargent (Academic Press, New York) pp. 101-179.
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O’Malley, B.W. and Means, A.R. (1974) Science 183,610-620. Palacios, R., Palmiter, R.D. and Schimke, R.T. (1972) J. Biol. Chem. 247,2316-2321. Palacios, R., Sullivan, D., Summers, N.M., Kiely, M.L. and Schimke, R.T. (1973) J. Biol. Chem. Palmiter, R.D. (1973) .I. Biol. Chem. 248,8260-8270. Palmiter, R.D. (1975) Cell 4,189-197. Palmiter, R.D. and Carey, N. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,2357-2361. Palmiter, R.D., Moore, P.B., Mulvihill, E.R. and Emtage, S. (1976) Cell 8,557-572. Parker, M.G. and Mainwaring, W.I.P. (1977) Cell 12,401-407. Parker, M.G. and Scrace, G.T. (1978) Eur. .I. Biochem., in press. Paterson, B.M. and Bishop, J.O. (1977) Cell 12,751-765. Pearson, W.R., Davidson, E.H. and Britten, R.J. (1977) Nucleic Acids Res. 4, 1727-1738. Penman, S., Vesco, C. and Penman, M. (1968) J. Mol. Biol. 34,46-69. Ringold, G.M., Yamamoto, K.R., Tomkins, G.M., Bishop, J.M. and Varmus, H.E. (1975) Cell 6, 299-305. Ringold, G.M., Yamamoto, K.R., Bishop, J.M. and Varmus, H.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74,2879-2883. Rosen, J.M. and Barker, S.W. (1976) Biochemistry 15,5272-5280. Rosen, J.M., Woo, S.L.C., Holder, J.W., Means, A.R. and O’Malley, B.W. (1975) Biochemistry 14,69-78. Ryfell, G.U. and McCarthy, B.J. (1975) Biochemistry 14,1379-1385. Ryfell, G.U. (1976) Eur. J. Biochem. 62,417-423. Schimke, R.T., McKnight, G.S., Shapiro, D.J., Sullivan, W. and Palacios, R. (1975) Recent Prog. Horm. Res. 31,175-208. Scolnick, E.M., Young, H.A. and Parks, W.P. (1976) Virology 69, 148-156. Shapiro, A.J. and Baker, H.J. (1977) J. Biol. Chem. 252,5344-5350. Sheldon, R., Jurale, C. and Kates, J. (1972) Proc. Natl. Acad. Sci. U.S.A. 69,417. Sullivan, D., Palacios, R., Stavneger, J., Taylor, J.M., Faras, A.J., Kiely, M.L., Summers, N.M., Bishop, J.M. and Schimke, R.T. (1973) J. Biol. Chem. 248, 7430-7439. Tata, J.R. (1970) In: Biochemical Actions of Hormones, Vol. 1, Ed.: G. Litwack (Academic Press, New York and London) pp. 89-133. Temin, K.M. and Mizutani, S. (1970) Nature (London) 225, 1211. Wahli, W., Wyler, J., Weber, R. and Ryfell, G.U. (1976) Eur. J. Biochem. 66,457-465. Williams, J.G. and Penman, S. (1975) Cell 6,197-206. Young, H.A., Shih, T.Y., Scolnick, E.M. and Parks, W.P. (1977) J. Virol. 21, 139-146.
0 Elsevier/North-Holland
REVIEW
10 (1978) 119-l 3 3 Scientific Publishers, Ltd.
ARTICLE
HORMONAL CONTROL OF mRNA SYNTHESIS STUDIED BY NUCLEIC ACID HYBRIDIZATION Malcolm G. PARKER Department of Hormone Biochemistry, London WC2A 3PX, U.K.
Imperial Cancer Research Fund, Lincoln’s Inn Fields,
Received 22 December 1977
The regulation of protein synthesis by steroids is thought to be due to hormonal effects primarily on mRNA concentration. Experimental evidence to support this conclusion has come largely from the use of DNA probes complementary (cDNA) to mRNA molecules or by translation of the mRNA in vitro. In this review the experimental procedures involved and the application to hormone action of cDNA hybridization will be reviewed. (1) mRNA concentrations can be assayed in tissue RNA samples by hybridization with radiolabelled complementary DNA probes (cDNA). From the rate of hybridization of an mRNA preparation to a cDNA probe it is possible to estimate specific mRNA concentrations and thereby study their hormonal regulation within tissues of subcellular fractions. (2) Rates of synthesis of a specific RNA can be measured by hybridization of pulse-labelled RNA with excess cold cDNA as illustrated in studies of the glucocorticoid induction of MMTV RNA. (3) Hormone-induced alterations of mRNA populations as a whole can be investigated. From the kinetics of hybridization of mRNA with its complementary DNA it is possible to estimate the number of different RNA sequences in tissues and to approximate the number of copies of each sequence per cell. Consequently, by comparing mRNA samples isolated from tissues of different hormonal status it is possible to demonstrate specific hormone-inducible mRNA species and, in some cases, identify their translation products. Keywords:
mRNA; transcription;
steroid hormones; protein synthesis.
Steroid hormones regulate protein synthesis in a variety of eukaryotic tissues (Tata, 1970). In principle, such regulation could be achieved at any step involved in transcription, processing and transport of RNA to the cytoplasm or at the level of translation of mRNA into protein. Thus it is important to investigate the effects of steroids on all these processes. Two types of experimental approach have suggested that the regulation is mediated mainly by alterations in the cellular mRNA
Abbreviations used in this paper: cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid;rRNA, ribosomal ribonucleic acid; tRNA, transfer ribonucleic acid; Rot, initial RNA concentration (moles of nucleotide per litre) X time (set); MMTV, mouse mammary tumour virus. 119
120
M. G. Parker
concentration (O’Malley and Means, 1974; Palmiter, 1975; Schimke et al., 1975). First, mRNA activity in cell-free protein-synthesizing systems has been assayed by examining the translation products on polyacrylamide gels. Since the amount of protein synthesized is proportional to the amount of mRNA supplied, it is possible to quantitate mRNA activity by measuring the incorporation of radioactive amino acid precursors into polypeptides. This method measures only those mRNAs which are active in the particular cell-free system used. The second procedure is to assay mRNA molecules by nucleic acid hybridization using DNA probes. This method measures all mRNA molecules whether or not they are active in protein synthesis. Both approaches have been utilized in assessing hormonal effects on cellular mRNA composition, but for the purpose of this review only the use of hybridization with DNA probes will be discussed. A DNA probe which is complementary to a specific mRNA or mRNA population can be synthesized in vitro using RNA-directed DNA polymerase (reverse transcriptase) purified from RNA tumour virus particles. The cDNA product therefore can hybridize uniquely to the mRNA by Watson-Crick base pairing. By using radiolabelled precursors the cDNA probe can be labelled to a high specific activity so that the hybridization reaction can be monitored. The rate of hybridization of a small amount of labelled cDNA to an excess of mRNA depends on the mRNA concentration (Bishop et al., 1974). This forms the basis of the assay in that the higher the mRNA concentration, the faster the hybridization. Thus the effect of steroid hormones on mRNA concentration can be determined by measuring the rate of hybridization of the cDNA probe with RNA samples isolated from tissues of differing hormonal status. Rates of RNA synthesis in cells can also be measured by hybridization of radiolabelled RNA synthesized during a short time period with excess cDNA, thereby quantitating the amount of RNA synthesis per unit time. The experimental procedures required for these studies will be summarized first and these will be followed by an outline of the sorts of information that have been obtained, referring to specific examples to illustrate the points of interest.
METHODS The experimental procedures consist of (1) isolation of RNA, (2) the synthesis of a cDNA probe and (3) an assay procedure to measure the kinetics of hybridization of the RNA with the cDNA probe. Isolation of RNA
A large number of variations in the methodology used to extract RNA have been described, but the method of choice depends mostly on tissue sources, whether nuclear, cytoplasmic or total RNA is required, and whether the RNA must be isolated intact. Alternative methods have been summarized in several reviews (Brawerman, 1973; Muramatsu, 1973; Busch et al., 1976) but fall largely into three
mRNA
121
analysis by cDNA hybridization
important approaches: (a) isolation of polysomal mRNA using sucrose gradient sedimentation (Blobel and Potter, 1967; Noll, 1969; Means et al., 1971; Palacios et al., 1972); (b) isolation of nuclear RNA by citric acid extraction (Busch, 1967; Holmes and Bonner, 1973); and (c) extraction and deproteinization of total cell or cytoplasmic RNA using phenol and c~orofo~ (Penman et al., 1968). Further fractionation of RNA is necessary before it can be used as a template for cDNA synthesis. Obviously, studies of a specific mRNA molecule necessitate its purification in order to synthesize a homogeneous cDNA preparation. This has been achieved by exploiting differences in mRNA size, presence or absence of poly(A) tracts at the 3’ end of the molecule, or the ability of ~tibodies to immunoprecipitate a partially completed peptide on specific polysomes from which mRNA can be extracted (Palacios et al., 1973; Rosen et al., 1975; Wahli et al., 1976; Shapiro and Baker, 1977). Success has usually depended on whether or not the mRNA in question accounted for a large proportion of the total RNA. The purification of mRNA species present in low concentrations can be extremely difficult and time-consuming. The synthesis of cDNA probes to investigate mRNA complexity requires only that the mRNA be separated from ribosomal RNA (rRNA) and transfer RNA (tRNA). This can be achieved by dT-cellulose chromatography (Aviv and Leder, 1972) or by poly~)~epharose chromatography (Sheldon et al., 1972) which bind RNA molecules containing poly(A) tracts, a common feature of many eukaryotic mRNA molecules. This means that mRNA lacking poly(A) will elute with the rRNA and tRNA and consequently has not been studied in much detail. Synthesis
of complementary
DNA
The synthesis of DNA complementary to RNA is achieved using reverse transcriptase, first described by Temin and Mizutani (1970) and Baltimore (1970) (fig. 1). When an oligo-dT primer is hyb~dized to the poly(A) tract at the 3’ end of the mRNA molecules it is possible to extend the primer and synthesize DNA copies of the RNA molecules. The inclusion of 3H-labelled dCTP results in the synthesis of a radiolabelled cDNA probe of specific activity up to 10’ cpm/pg DNA. Typical reaction conditions are as follows: 0.1 ,uM 13H]dCTP (15-20 Cif mmol), 36 &ml actinomycin D (to inhibit DNA synthesis in which cDNA acts as a template), 50 mM Tris-HCI (pH 8.3), 10 mM dithiothreitol, 6 mM MgC12, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 5 pg/ml oligo-(dTra_rs) and 25 pg/ ml poly(A)-containing RNA, 100 units/ml avian myoblastosis virus reverse tran-
5’
AA&A 3’
reverse oligo
transcriptase dT.
dNTPs
Fig. 1. Reaction scheme for the synthesis of cDNA.
122
M. G. Parker
scriptase incubated at 37°C for 30 min. The RNA is then hydrolysed with alkali and the cDNA separated from unincorporated ~3H~dCTP by gel filtration through Sephadex G-SO. After precipitation with ethanol the cDNA is stored in 0.01 M Tris-HCl (pH 7.5) at -70°C. Specific cDNA probes have now been synthesized for several mRNA molecules regulated by steroid hormones, including those for ovalbumin, vitellogenin and casein (Su~iv~ et al., 1973; Mon~an et al., 1976a; Rosen and Barker, 1976; Ryfell, 1976; Shapiro and Baker, 1977). cDNA probes to investigate mRNA complexity in chicken oviduct and rat ventral prostate have also been synthesized (Monahan et al., 1976b; Cox, 1977; Hynes et al., 1977; Parker and Mainwaring, 1977). More recently, cDNA hybridization has been carried out to investigate rates of synthesis of specific mRNA molecules in which pulse-labelled RNA is hybridized with excess cold cDNA. In this case f3HjdCTP is replaced with dCTP and the reactants increased to prepare ~.lgamounts of cDNA (Young et al., 1977).
The principle of the assay is to measure the rate of hybridization of the cDNA probe with the mRNA. This is achieved by monitoring the proportion of cDNA which has reassociated with the mRNA during the hyb~d~ation reaction. A variety of hybridization reaction conditions are available, but a solution containing 0.24 M sodium phosphate buffer (pH 7.4), 1 mM EDTA and 0.05% SDS at 70°C is used extensively (Bishop et al., 1974; Ryfell and McCarthy, 1975; W~li~s and Penman, 1975). Since the hybridization rate varies with the monovalent cation concentration and temperature, comparisons of data obtained under different conditions require correction to standard conditions, and conversion tables are to be found in Britten et al. (1974). Excess RNA is mixed with radiolabelled cDNA in hybridization buffer, overlaid with paraffin oil, and incubated at the appropriate temperature. One method of assessing the extent of hybridization depends on the fact that Sl nuclease (from AspergiElus olyzae; Sigma Chemical Co.) degrades single- but not double-stranded DNA; therefore cDNA which has hyb~~zed to RNA is resistant to Sl nuclease digestion (Milcarek et al., 1974). Portions of the hybridization mix are removed at intervals ranging from 5 min to 24 h and diluted into ‘SI nuclease buffer’ (0.05 M sodium acetate (pH 4.5), 0.1 M NaCI, 0.1 mM Z&O4 and 10 pg/ml denatured DNA) which is subsequently divided into two aliquots. To one aliquot is added approximately 100 units Sl nuclease to hydrolyse u~ybridized cDNA and then both aliquots are incubated at 46°C for 2 h. The samples are cooled in ice and adjusted to contain IO @g/ml bovine serum albumin and 5% trichloroacetic acid. They are then collected on millipore filters and counted in scinti~ation fluid. The percentage cDNA resistant to digestion is taken as the percentage hybridization, Since the amount of hybridization depends on the mRNA concentration and the time ofhybridization, hybridization is usually plotted against the Ret value, which is the product of the initial RNA concentration (in moles of nucleotide per litre) and time (in seconds). The Ret value at which 50%
mRNA analysis by cDNA hybridization
123
of a given population of nucleic acid molecules have hybridized (Rot 112) is the parameter most frequently used to compare RNA samples. Thus the proportion of specific mRNA molecules in an RNA mixture can be determined by comparing the ROtl,,) values of the purified mRNA with those of the RNA samples under investigation. The ROtI,* values will increase (decreased rate of hybridization) as the mRNA concentration decreases. A reduction in the rate of hybridization, so that the Rot,,, value increased by, say, IOO-fold, would indicate that the specific RNA represented only 1% of the RNA sample.
APPLICATIONS
OF cDNA HYBRIDIZATION
of specijic n&VA concentrations The use of cDNA probes to assay specific mRNA molecules made it possible to test whether alterations in protein synthesis, induced by a hormone, are the results of alterations in the cellular mRNA concentration. Progress has been rapid, especially in studies of the hormonal regulation of ovalbumin synthesis in the chick oviduct. This was mainly because ovalbumin mRNA, the preponderant mRNA species, was relatively easy to purify and thereby synthesis of a complementary DNA probe was straightforward. Subsequently, casein and vitellogenin mRNA have also been purified and studies of their regulation by steroid hormones (Rosen and Barker, 1976; Ryfell, 1976; Shapiro and Baker, 1977) have supported the conclusions drawn for ovalbumin mRNA. The concentration of ovalbumin mRNA in the chick oviduct has been measured during growth and differentiation of the gland following primary stimulation of immature chicks with oestrogen; following withdrawal of hormone; and following a second injection of oestrogen to withdrawn chicks (secondary stimulation) (Cox et al., 1974; Harris et al., 1975; McKnight et al., 1975) (fig. 2). It was concluded that there were very few copies of ovalbumin mRNA in immature chicks but that it accumulated slowly after oestrogen administration following differentiation of tubular gland cells in which it is synthesized. Withdrawal of the hormone from the chicks resulted in a dramatic decrease in ovalbumin mRNA content, but a secondary administration of oestrogen resulted in its rapid restoration. There were discrepancies in the various measurements of the response time for this secondary induction, but alternative procedures (Chan et al., 1973; Palmiter, 1973) and further studies using ovalbumin cDNA (Palmiter et al., 1976; Groner et al., 1977) suggest that oestrogen induces the accumulation of ovalbumin mRNA in the tubular gland cells after a lag of about 3 h. It should be stressed that these determinations are measurements only of the concentrations of mRNA and say nothing about synthetic or degradative rates of RNA molecules. Therefore the conclusion that oestrogen regulates ovalbumin mRNA by regulating transcription has been drawn somewhat prematurely from these data. The failure to detect ovalbumin mRNA in the absence of hormone is usually cited as evidence of transcriptional control, and the relatively Measurement
124
M. G. Parker
-2
-1
0
1 Log
2 Rot
3
4
Fig. 2. Hybridization of ovalbumin cDNA with oviduct mRNA (curves adapted from the work of Cox et al., 1974; Harris et al., 1975; McKnight et al., 1975). At intervals the extent of hybridization was determined (resistance to Sl nuclease). Rot values are the product of RNA concentration in moles of nucleotide per litre and time of reaction in seconds.
long half-life determined for ovalbumin mRNA appeared to support this notion (Kafatos, 1972; Chan et al., 1973; Palmiter, 1973; Harris et al., 1975). However, the half-life was determined for polysomal and total cellular ovalbumin mRNA and not nuclear RNA. In fact, the observation that the ti/a for ovalbumin mRNA is longer in the presence than in the absence of oestrogen already suggests that regulation of ovalbumin mRNA levels is not restricted to transcriptional controls (Palmiter and Carey, 1974; Cox, 1977). Finally, the inability to detect the accumulation of an RNA species does not indicate that it was never synthesized. The recent observation that histone mRNA is synthesized throughout the cell cycle of HeLa cells but is only found in the cytoplasm during the period of DNA synthesis (Melli et al., 1977) suggests that RNA processing rather than transcription is the major regulatory step in this particular case. Consequently, in studies of hormone-inducible mRNA molecules it is necessary to establish rates of RNA synthesis in the presence and absence of hormone to support models based on transcriptional controls. Determination of synthetic rates for a particular RNA requires measurements of the rate of incorporation of labelled precursors into the RNA. It may be difficult to obtain labelled RNA of sufficient specific activity in whole tissue, but it is feasible in cell cultures. Recently the rate of synthesis and degradation of mouse mammary tumour viral (MMTV) RNA has been measured in mammary tumour cell lines following glucocorticoid induction (Ringold et al., 1977; Young et al., 1977). The initial rate of synthesis of MMTV RNA was measured by hybridization of pulse-labelled RNA with non-radiolabelled cDNA. The hybridized material was quantitated by two novel procedures (fig. 3) that were utilized to reduce the background interference due to non-specific hybridization of other labelled RNA
125
mRNA analysis by cDNA hybridization A
I
I
I
I
I
*
I
I
I
I
Sephadex
bead
dC dC dC dC 5'
Fig. 3. Schematic diagrams to illustrate the procedures used to quantitate MMTV RNA. Both procedures were devised because MMTV RNA comprises a small proportion of the mammary cell RNA even in the presence of glucocorticoid. Following hybridization of the labelled RNA with MMTV cDNA probes, the reaction mix was incubated with pancreatic RNase to degrade unannealed RNA, thereby reducing background interference of other labelled RNA species due to non-specific binding. A. MMTV RNA initially hybridizes to MMTV cDNA extended with poly(C) and the hybrid is then separated from cellular labelled ribonucleotides by chromatography on poly(I)-Sephadex columns (Young et al., 1977). B. MMTV RNA initially hybridizes to MMTV cDNA made double-stranded (tailed duplex) by omission of actinomycin D during the synthesis of cDNA. The hybridized material can then be separated from cellular labelled ribonucelotides by chromatography on hydroxyapatite.
species. In one (Young et al., 1977) the cDNA had been elongated with poly(dC) so that the poly(dC)-cDNA-RNA hybrid could be bound to a poly(I)-Sephadex column by virtue of the poly(dC) regions of the hybrid, thereby separating hybridized from non-hybridized RNA. In the other (Ringold et al., 1977) the cDNA was extended to form ‘tailed duplex’ DNA using DNA polymerase and deoxynucleotide triphosphates. Such double helical DNA hybridized with pulselabelled RNA could be separated from non-hybridized RNA by hydroxyapatite chromatography. Thus, by incubating mouse cell lines with [3H]uridine and then exposing the culture to dexamethasone for different periods of time, it was possible
M. G. Parker
126
Table 1 Kinetics of glucocorticoid tumour cells
induction
Time after dexamethasone addition to cell culture (mm)
of MMTV RNA
synthesis
410of total RNA synthesized cDNA probe Young
et al. (1977)
that hybridized
Ringold
0 10
0.15 0.7
0.05 _
15 20
_ 0.85
0.45 -
30
1.05
0.4
The MMTV RNA was assayed
by the procedures
described
in cultured
mouse
mammary
to MMTV RNA
et al. (1977)
in fig. 3.
to isolate 3H-labelled RNA which could be assayed for the presence of MMTV RNA. Both techniques suggested that dexamethasone resulted in a 3-lo-fold stimulation in viral RNA synthesis within lo-l.5 min (table 1). Prior to this study, both groups had measured MMTV RNA accumulation as described for ovalbumin, using excess cold RNA and [3H]cDNA prepared to MMTV RNA (Ringold et al., 1975; Scolnick et al., 1976). In these studies they had detected only a 2-fold increase in concentration 30 min after dexamethasone treatment. Thus, measurement of the rate of synthesis proved to be more definitive and more sensitive. Young et al. (1977) have also attempted to measure the half-life of MMTV RNA by incubating pulse-labelled cell cultures with actinomycin D for different periods and following the decay rate of the viral RNA. In the presence of dexamethasone the tr/a was greater than 8 h but unfortunately, in the absence of steroid, the rate of synthesis of MMTV-specific RNA was too low to provide an accurate determination of half-life after actinomycin D addition. Ringold et al. (1977) have pointed out that if the tr/a for MMTV RNA in the absence of steroid was considerably shorter than the labelling period it is conceivable that an increase in the apparent rate of synthesis might in fact be the result of the hormone inhibiting the degradation of the rapidly turning-over RNA species. Such a mechanism is difficult to discount and extremely difficult to test. Similar studies have not been reported for other hormone-inducible mRNA molecules, but it is necessary to establish rates of RNA synthesis in the presence and absence of hormones to support models based on transcriptional control. The mammary tumour virus system is a good example of the advantage a cell line offers over other hormone-responsive tissues in that it is relatively easy to obtain pulselabelled RNA. Analysis of mRNA populations in general It is also possible to investigate the role steroid hormones
play in regulating
mRNA analysis by cDNA hybridization
121
mRNA populations as a whole even when hormonally induced proteins have not been identified. These studies require cDNA probes complementary to the total mRNA population rather than to specific mRNA species. In fact, technically it is more straightforward than investigations of specific mRNAs because mRNA has only to be separated from rRNA and tRNA and does not require further puritication. The procedure is to synthesize cDNA using total mRNA as template and then measure the rate of hybridization using excess mRNA and a trace amount of radioactive cDNA. The rate of hybridization is determined by the sequence complexity (see below) of the template mRNA and by the relative concentrations or abundance of the different sequences. The concentration of different mRNA species in cells extends over a wide range and those present in high concentrations will hybridize faster than those in low concentrations. For instance, consider a population of mRNA in which one species accounts for 33%, 100 species each account for 0.33% (i.e. 33% in total) and 10,000 species each account for 0.0033% of the mRNA. The most abundant mRNA sequence will hybridize 100 times faster than the moderately abundant sequences, which themselves will hybridize 100 times faster than the scarce sequences. The hybridization curve will then consist of three transitions (fig. 4), the first transition representing the most abundant RNA and subsequent transitions representing classes of RNA of lower abundance. The plateau level of each transition gives the proportion of mRNA molecules in each class, and the Rot112 values of each transition by comparison with that obtained for a defined mRNA species give the sequence complexity of each class. Globin mRNA coding for both the 01and fl chains is frequently used as a standard and, containing 1300 nucleotides, has a complexity of 1300 nucleotides. By dividing the Retr/a values
Fig. 4. Hybridization curves of globin mRNA and a hypothetical tissue mRNA sample. Globin mRNA (X -X) was hybridized with its cDNA and tissue mRNA ( -) hybridized with its cDNA, the arrows indicating Rot,,, values of the transitions (see table 2).
M. G. Parker
128
obtained for each transition by 6 X 104, the RotlIZ value obtained for the hybridization of globin mRNA and globin cDNA, it is possible to calculate the sequence complexity in each class of mRNA. Initially one correction to the observed Retr/a values has to be made because each class of RNA is not a pure component but is diluted by the other classes, thereby reducing its rate of hybridization. In the example shown in fig. 4, inspection of the hybridization curve indicates that the mRNA consists of three classes of mRNA at distinct concentrations. The first transition with an observed Ret,,, value of 1.8 X 10e3 mol elitre-‘-set is corrected because it only comprises l/3 of the total mRNA and would hybridize 3fold faster were it a pure component (table 2). Division of the corrected ROtr,2 value by 6 X 10b4, the value for globin mRNA (complexity 1300 nucleotides), indicates that it too has complexity of 1300 nucleotides [(1.8 X 10m3 + 3)/(6 X 10m4) X 13001. This would be equivalent to one sequence were its number average molecular weight to be 1300 nucleotides. A similar series of calculations indicate that the second transition represents a class of RNA comprising 100 sequences and the third transition a class of RNA comprising 10,000 sequences (table 2). In fact, when tissue RNA samples are analysed, transitions are rarely as distinct as those shown in fig. 4 (for example see fig. 5) and it is preferable to estimate the number of classes and their Retr/a values by computer analysis of the data. Several programmes have been described (Hastie and Bishop, 1976; Pearson et al., 1977). Estimates of the number of copies of each sequence can be obtained from the amount of mRNA per cell. From the DNA content per cell and the RNA/DNA ratio of the tissue under investigation the approximate amount of mRNA per cell can be calculated. The calculations can only be approximates since cell heterogeneity within tissues is not taken into account and the proportion of mRNA in total RNA has usually to be estimated.
Table 2 Sequence
complexity
of hypothetical
tissue RNA
Transition
% cDNA hybridized
Ret ~2 observed (mol . litre-’ -set)
Roti/ corrected
Complexity nucleotides _
No. of sequence
I II III
33 33 33
1.8 X 1O-3 1.8 X 10-l 1.8 X lo1
6x10” 6 X 1O-2 6 X 10’
1,300 130,000 13,000,000
1 100 10,000
The data were obtained from the hybridization curves shown in fig. 4. The observed Rotr,a values were corrected for the fraction of RNA they represent. The complexity in nucleotides was derived by comparison of the corrected R 0t 112 values with that obtained for globin mRNA, to a complexity of 1300 nucleotides. The namely 6 X lo4 mol . litre-’ -set, corresponding number of sequences was calculated by dividing the complexity in nucleotides by 1300, the number average chain length of the RNA.
mRNA analysis by cDNA hybridization
129
The first studies of mRNA populations in steroid target tissues were carried out with chick oviduct mRNA. Axe1 et al. (1976) demonstrated that, in common with many other tissues and cell types (Lewin, 1975) oviduct RNA consisted of three classes of mRNA sequences present in different cellular concentrations or abundances. There was a total of 12,000-15,000 different sequences most of which were present in fewer than 10 copies per cell, but one class of oviduct mRNA consisted of only one species present in about 100,000 copies per cell and probably represents ovalbumin mRNA sequences. It is also possible to analyse the tissue specificity of mRNA sequences by hybridization of the cDNA from one tissue with mRNA from another tissue, so that cDNA which remains unhybridized is complementary to tissue-specific RNA sequences. These hybridization reactions are referred to as heterologous to distinguish them from homologous reactions in which the cDNA is synthesized from the same mRNA to which it is being hybridized. To examine whether oviduct mRNA sequences are also present in chick liver, Axe1 and co-workers hybridized oviduct cDNA with liver mRNA. Such heterologous hybridization reactions indicated that at least 85% of the mRNA species are shared. Presumably common sequences encode ‘housekeeping’ functions shared by all cell types, but they also suggested that the mRNA molecules for the egg-white proteins are also present in the liver albeit at a much reduced concentration. The effect of oestrogen on mRNA complexity was first investigated by O’Malley reactions indicated that (Monahan et al., 1976a). Homologous hybridization poly(A)-containing RNA from the oviducts of hens and diethylstilboestrol-stimulated chicks contained 20,000-25,000 different RNA sequences present in three classes of different abundance, ranging from fewer than three to many thousand copies of each sequence per cell. In contrast, there were only about 10,000 different sequences in the oviducts of chicks withdrawn from oestrogen and there was no class of RNA corresponding to the high abundance class demonstrated in hen and hormone-induced chick oviduct. These results suggested that oestrogen was regulating both the relative abundance of certain mRNA molecules and the total number of different RNA sequences. Subsequent studies using heterologous hybridization reactions in which cDNA synthesized with mRNA from hen oviduct was hybridized with mRNA from oestrogen-withdrawn chicks (Cox, 1977; Hynes et al., 1977) have confirmed that the concentration of the most abundant class of mRNA, probably containing ovalbumin mRNA, but not the total number of different sequences, is regulated by oestrogen. Caution should be exercised in allocating precise values for the number of sequences in different abundance classes (Bishop et al., 1974; Hastie and Bishop, 1976; Monahan et al., 1976a). Allowance has to be made for rRNA contamination of mRNA samples which will affect the absolute numbers of sequences in each class of RNA. This may be critical when comparing the complexity of different RNA samples but to some extent can be circumvented by using heterologous hybridization reactions.
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Accurate estimates of the Ret,,, values of low abundance sequences are often difficult if they comprise only a small fraction of the total RNA even though they account for most of the different mRNA sequences. In the case of heterologous hybridization reactions, differences in mRNA populations are estimated from differences in the percentage saturation of homologous versus heterologous reactions. Unfortunately a difference of 5% in the percentage of saturation does not necessarily indicate that 95% ofthe sequences are similar. The mRNA complexity is determined mainly by one class of RNA, namely the slowest hybridizing class which comprises a lot of sequences represented by only a few copies per cell. If this class were to contain 10,000 sequences and account for only 20% of the RNA, then a 5% difference in saturation could be equivalent to a difference of 2500 sequences and not 500 sequences. In fact, since it is not known to which class of RNA unhybridized cDNA corresponds, such a difference in percentage saturation may be less provided it corresponded to a class containing few sequences in high abundance. In the case of oviduct RNA (Cox, 1977; Hynes et al., 1977) the percentage saturation was similar for both the homologous and heterologous hybridization reactions and therefore indicates that most, if not all, of the mRNA species present in the oviducts of laying hens are also present in the oviducts of hormone-withdrawn chicks. Comparisons of mRNA populations in steroid target tissues of different hormonal status can be extremely useful even when hormonally regulated proteins have not been identified. For example, although rat ventral prostate is a target tissue for androgenic steroids and has been used extensively to study the mechanism of action of androgens (King and Mainwaring, 1974; Mainwaring, 1977) satisfactory protein markers of hormone action had not been reported. By analysing prostatic mRNA from normal and castrated rats, a class of highly abundant RNA species that is regulated by androgens in vivo has been demonstrated (Parker and Mainwaring, 1977). Prostatic RNA isolated from normal animals comprised three abundance classes, the most abundant of which was absent after castration. The effect of androgens on this class of RNA was studied in more detail by isolating cDNA complementary only to these sequences. This was achieved by hybridizing total cellular poly(A)-containing RNA from normal animals with its complementary DNA to an Ret value of 2 X IO-’ mol . litre-’ -set when about half of the cDNA, corresponding to the abundant RNA sequences, would have annealed. This abundant cDNA was then separated from the non-abundant cDNA by hydroxyapatite chromatography and shown to be complementary to about three different poly(A)containing RNA sequences (fig. 5). Thus it was then possible to investigate the effects of androgens on mRNA levels in the ventral prostate using this fractionated cDNA probe (Parker and Scrace, 1978). Once mRNA sequences that are regulated by a hormone have been identified it would be important to determine for what proteins they are coding. In the past this has not been easy, but recently Paterson and Bishop (1977) have developed a method for relating cDNA sequences, their corresponding mRNA species and the proteins for which they code. The method is particularly suitable for abundant
131
mRNA analysis by cDNA hybridization
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cDNA preferably fractionated as described above. Hybridization of the mRNA with the cDNA in excess converts all the mRNA molecules complementary to the probe into a hybrid form which cannot be translated in cell-free systems. However, heat dissociation of the hybrid reinstates translations activity so that it is possible to identify which proteins are translated from the mRNA for which cDNA probes have been isolated (Paterson and Bishop, 1977). This approach could in theory be applied to investigate the role of any hormone on RNA and protein metabolism and may be especially useful in studies of target tissues in which hormone markers have not yet been identified.
CONCLUDING
REMARKS
It is clear from experiments using cDNA probes that steroid hormones regulate intracellular mRNA concentrations and thereby modulate protein synthesis. At least in the case of the hormonal control of MMTV RNA, glucocorticoids appear to increase the rate of synthesis of the RNA. Similar studies with other steroidresponsive tissues and cells are required to establish the generality of this observation. Large amounts of cDNA are required for these studies, but the current possibilities of cloning cDNA molecules in bacteria would certainly make such studies more feasible. Such cloned cDNA may ultimately be used to isolate specific genes from total cellular DNA and permit more precise studies into the mechanism of action of steroid hormones.
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A4 G. Parker
ACKNOWLEDGEMENTS I should like to thank my colleagues Drs. R.J.B. King, R. Shields, J.A. Smith, R.F. Brooks, C. Dickson, G.E. Peters, R.A. Weiss and Mr. G.T. Scrace for their suggestions during the preparation of the manuscript, and Mrs. M.C. Barker for excellent secretarial assistance.
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