DEVELOPMENTAL

Diversity

BIOLOGY

69,

111-123 (1977)

of Polyadenylated Messenger RNA Sequences 12-hr Regenerating Liver’

DONALD Division

A. COLBERT,~ M. VALERIA

TEDESCHI, NELSON FAUSTO~

of Biology

and Medicine,

Brown

Received December 2,1976;

University,

VLADIMIR

Providence,

in Normal and

ATRYZEK,

Rhode Island

and

02912

accepted in revised form May 5,1977

Molecular hybridization techniques were used to examine the structural organization of the rat genome and gene expression during liver regeneration. The nonrepetitive fraction of rat DNA comprises approximately 69% of the total and has a complexity of 1.9 x lo9 nucleotide pairs. Complementary DNAs (cDNA) were synthesized using polyadenylated polysomal mRNA templates from normal and 12-hr regenerating livers. Hybridization of cDNA with a large excess of total rat DNA indicates that the proportion of polyadenylated polysomal mRNA homologous to repetitive and nonrepetitive DNA is very similar in normal and regenerating livers. Hybridization of cDNAs with excess homologous or heterologous mRNA revealed that: (a) The sequence complexity of polyadenylated polysomal mRNA is essentially the same in normal and 12-hr regenerating liver. Using these data we estimate that the maximum proportion of single-copy DNA represented in polyadenylated polysomal mRNA in livers of sham-operated rats or in 12-hr regenerating liver is 0.&X-0.90%; 6) S-90% of polysomal polyadenylated mRNA is common to normal and regenerating livers, while ll-14% is unique either to normal or 12-hr regenerating liver polysomes. We cannot discern as yet whether the polysomal polyadenylated mRNA sequences unique to normal or regenerating livers are the result of differential gene transcription and/or selective transport from the nucleus to polysomes. INTRODUCTION

Liver regeneration following partial hepatectomy is perhaps the best example of compensatory growth in adult mammalian organisms. Hepatocytes have a long life span in the adult rat, but can be made to proliferate in response to loss of liver mass (see Bucher and Malt, 1971, for review). During the regenerative process, hepatocytes, in addition to being reprogrammed to undergo mitosis, have to maintain or even increase some of their essential metabolic functions (Faust0 et al., 1975). During the first 36 hr of compensatory growth, the dramatic changes in * Supported by Grant No. AM-14706 from the NIH. * Present address: Molecular Biology Institute, University of California, Los Angeles, Calif. 3 To whom requests for reprints should be addressed.

cellular metabolism that take place in the liver occur in a relatively synchronous manner, thus allowing the study of the regenerating organ during pre- and postreplicative stages. Two phases can be recognized at the early stages of liver regeneration following partial hepatectomy in rats: hypertrophy, lasting for 12-16 hr, and hyperplasia, characterized by the initiation of DNA synthesis 14-16 hr after the operation (Grisham, 1962). The results of early filter hybridization studies by Church and McCarthy (1967) suggested that liver regeneration was mediated by short-lived RNA species not present in normal livers. However, neither the work of Drews and Brawerman (1967) nor recent studies from this laboratory support these conclusions (Greene and Fausto, 1977; Faust0 et al., 1976). The results of subsequent work (Church and 111

Copyright 0 1977by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

ISSN

0012-1606

112

DEVELOPMENTAL BIOLOGY

McCarthy, 1969) suggested that an important change in regenerating hepatocytes involves the transport to the cytoplasm of RNA transcripts which are normally confined to the nucleus (Shearer and Smuckler, 1972; Schumm and Webb, 1972; Greene and Fausto, 1977). In all of these studies, only the RNA homologous to repetitive DNA sequences was examined. It is generally accepted that most mRNA sequences are transcribed from single-copy genes (see Lewin, 1975 for review). Therefore, a more direct approach to the problem of gene expression during liver regeneration involves the characterization of polysomal mRNA populations in normal and regenerating livers. For this purpose, poly(A)+ polysomal mRNA obtained from livers of sham-operated and partially hepatectomized rats was hybridized with [3HlcDNA.4 Labeled cDNA probes were prepared in vitro with AMV reverse transcriptase using as templates polyadenylated polysomal mRNA from livers of sham-operated or partially hepatectomized rats killed 12 hr after the operation. At this time, regenerating hepatocytes are undergoing hypertrophy, but have not yet initiated DNA synthesis. In normal and 12-hr regenerating livers, we determined: (a) the sequence complexity of polyadenylated polysomal mRNA; (b) the relative abundance of mRNA sequences; and (c) the homology between polysomal mRNA populations of normal and regenerating livers. METHODS

AND

MATERIALS

Animals. Male rats, (120-160 g, Holtzmann strain, Charles River Breeding Labs.) were used in all experiments. The animals were kept in temperature-controlled rooms under 12-hr alternating light-dark cycles. Partial hepatectomies resulting in the removal of 70% of the liver were performed according to the method of 4 Abbreviations used: poly(A)+ mRNA, polyadenylated messenger RNA; HAP, hydroxyapatite; cDNA, complementary DNA.

VOLUME 59, 1977

Higgins and Anderson (1931). All control rats were sham-operated: The liver was exposed and handled but not removed. Surgical procedures were performed under continuous ether-oxygen anesthesia, a method which does not alter the hepatic levels of nucleoside triphosphates and cyclic nucleotides (Bucher and Swaffield, 1966; Faust0 and Butcher, 1976). Partially hepatectomized and sham-operated rats were killed 12 hr following the operations. Food was withdrawn after the surgical procedures. The isolation of polysomal poly(A)+ mRNA involved approximately 100 partially hepatectomized and 70 shamoperated rats. Glassware and solutions. Solutions and glassware used for extraction of nucleic acids were sterilized by autoclaving. The glassware utilized in hybridization experiments was siliconized (1% dichlorodimethylsilane in toluene) and kept at 300°C for 12 hr. Solutions used in these experiments were treated with Chelex 100 (Bio-Rad) to remove metals. Preparation of polysomes. Polysomes were prepared by the method of Munro et al. (19641 with the modifications previously described (Levin and Fausto, 1973). Livers were rapidly excised, cut into small pieces, washed, and homogenized in a solution containing 0.25 M sucrose, 0.02 M Tris buffer, pH 7.6, 0.01 M magnesium acetate, 0.04 M NaCl, 0.1 M KCl, and 0.006 M mercaptoethanol. After removal of nuclei, mitochondria, lysosomes, and cell debris by centrifugation, one-tenth volume of 10% sodium deoxycholate in 0.05 M Tris buffer, pH 8.2, was added dropwise. The extracts were layered on a discontinuous sucrose gradient (l-2 M sucrose in homogenizing solution) and were centrifuged for 4 hr at 105,OOOg in a B-60 International ultracentrifuge. Routinely, one pellet from each preparation was used to monitor the integrity of the polysomes. Polysomal profiles were obtained by centrifugation of carefully resuspended pellets on 15-30% sucrose gradients for 3 hr at 25,000 r-pm in

COLBERT ET AL.

Messenger

the SB-110 rotor of a B-60 centrifuge. The profiles were analyzed with a Hitachi spectrophotometer equipped with a flow cell. The resolution obtained was excellent as shown in Fig. 2. Extraction of poly(A)+ mRNA. Each polysomal pellet was suspended in 0.05 M Tris buffer, pH 7.4,O.Ol M EDTA, 2% SDS. The resuspended pellets were pooled and dissolved, and the concentration of NaCl was adjusted to 0.1 M. Polysomes were extracted with a mixture of phenol-chloroform saturated with TNM buffer (0.05 M Tris, 0.25 M NaC1, and 0.005 M MgC&) at room temperature using a wrist-action shaker. After centrifugation, the aqueous phase was extracted repeatedly with the same mixture until no proteinaceous interphase was observed. The final aqueous phase was extracted twice with a 24:l mixture of chloroform-isoamyl alcohol. The purity of the extracted RNA was checked by measuring its absorbance at 230, 260, and 280 nm. The RNA was loaded directly onto a poly(U)-Sepharose column, and the eluate from the first passage was reloaded. The column was washed with NETS buffer (0.01 M Tris, pH 7.6, 0.1 M NaCl, 0.01 M EDTA, and 0.2% SDS), and the poly(A)+ RNA was eluted with 90% formamide in NETS buffer. The polyadenylated RNA was precipitated with alcohol, dissolved in a small volume, and passed through a column containing Chelex 100 under Sephadex G-50. The samples were finally precipitated with alcohol and were stored frozen at -70°C until use. The degree of contamination of poly(A)+ mRNA by ribosomal RNA was determined by electrophoresis in acrylamide-agarose gel slabs both with and without 7.0 M urea. Redistilled phenol used for RNA extractions was stored in the refrigerator and was saturated with TNM buffer prior to use. Poly(U)-Sepharose columns were prepared in the laboratory by binding poly(U) (Miles Laboratories) to cyanogen bromideactivated Sepharose 4B (Pharmacia Fine Chemicals) using previously described procedures (Wagner et al., 1971; Greene and

RNA

in Regenerating

Liver

113

Fausto, 1974). The capacity and efficiency of the columns were checked with [3Hlpoly(A) obtained from Miles Laboratories. Preparation of complementary DNA (cDNA). Enzymatic synthesis of L3HlcDNA in vitro using AMV reverse transcriptase was performed as described by Milcarek et al. (1974) with minor modifications. The reaction was carried out for 45 min at 45°C in an incubation mixture of 20 ~1 containing: 3-6 pg of polysomal poly(A)+ mRNA, 30 pg/ml of oligo(dT),,-,, (Collaborative Research), 500 FM each of dTTP, dATP, and dGTP, 125 &i of [3H]dCTP (New England Nuclear, specific activity of 22.9 Ci/mmole), 125 pg/ml of actinomycin D, 20 fl dithiothreitol, 12 mM magnesium acetate, 0.1 M Tris buffer, pH 8.3, and 5 units of reverse transcriptase. The reaction was terminated by the addition of 0.5 ml of a solution containing 100 n&I NaCl, 1 mM EDTA, 0.5% SDS, and 10 n&I Tris buffer, pH 4. The mixture was then extracted with chloroform-isoamyl alcohol (24:l) until no proteinaceous interphase was observed. The final aqueous phase was passed through a column containing Sephadex G-50 layered over Chelex 100, and the excluded volume plus 1 ml was collected. The fractions containing cDNA were pooled, distributed in small aliquots, fast-frozen in dry ice-acetone and stored at -70°C until use. The reverse transcriptase (6200 unit/mg, specific activity of 26,956 unit/mg protein) was a gift from Dr. J. W. Beard. cDNA-mRNA hybridization. Suitable amounts of polysomal poly(A)+ mRNA from livers of partially hepatectomized or sham-operated rats were mixed with [3H]cDNA at a RNA/DNA ratio of 104-106. The nucleic acids were precipitated with ethanol, and the pellets were dissolved in a solution containing 0.24 M phosphate buffer, pH 6.9, 0.05% SDS, and 0.001 M EDTA. The reaction mixtures were dispensed into sterile siliconized 50- or lOO-~1 capillary tubes, and the ends of the tubes were sealed with a small torch. After boil-

114

DEVELOPMENTAL BIOL~CY

ing for 3-5 min, the samples were incubated at 70°C for the period of time necessary to reach the appropriate R,t value (RNA concentration in moles of nucleotide per liter x time of the reaction measured in seconds). At the end of the various incubation periods, the contents of the capillary tubes were rapidly frozen in acetonedry ice and were added to tubes containing 50 n&f sodium acetate, pH 4.5, 0.1 mM zinc sulfate, 100 mM NaCl, and 10 pg/ml of heat-denatured calf thymus DNA. From each capillary tube, two samples were processed: One was digested with 200 unit of nuclease S, from Aspergillus oryzae (Miles Laboratories) for 1 hr at 37°C; the other sample was incubated for the same period of time in buffer without nuclease. The hybrids were precipitated with TCA, collected in Whatman GF-C filters, and washed with TCA and alcohol. The filters were dried and placed in counting vials containing hyamine hydroxide and were heated at 70°C for l-2 hr. The radioactivity was determined in a Nuclear Chicago scintillation counter using Omnifluor counting solution. The percentage of hybridization was calculated as [(the counts per minute in the S-treated sample)/(counts per minute in the buffer-treated sample) x 1001. Controls were run separately to determine the efficiency of the S, digestion procedure. A background value of 6-8% was routinely observed. In all data presented, this background value has been subtracted. DNA-DNA and cDNA-DNA annealing reactions. The method described by Brit-

ten and Kohne (19681, with some minor modifications, was used for these experiments. Annealing reactions were carried out in Kontes incubation vials (DNADNA experiments) or conical tubes covered with paraffin oil (cDNA-DNA experiments). Samples boiled for 5 min were incubated at 60°C in 0.12 M phosphate buffer containing 0.4% SDS. C,t values (concentration of DNA in moles of nucleotide per liter x time of reaction in seconds), requiring very long or extremely short incubation times, were obtained by changing the

VOLUME 59, 1977

concentration of DNA or phosphate. All results were calculated in terms of equivalent C,t, using the correction factors presented by Britten and Smith (1970). The annealing reactions were terminated by immersing the vials in acetone-dry ice. When conical tubes were used, samples of the incubation mixture were taken at the appropriate times, and their salt concentrations were adjusted to 0.12 M phosphate. The samples were diluted lo-fold with 0.12 M phosphate buffer and were frozen until processed. The amount of DNA hybridized was determined by measuring the amount of 260-nm absorbing material and/or the 3H radioactivity which binds to HAP“ in 0.12 M phosphate, but is eluted by 0.5 M phosphate. The background caused by nonspecific binding to HAP was reduced by prewashing the column with 0.12 M phosphate buffer containing 0.4% SDS and by maintaining the nucleic acid concentration of the sample above 20 pg/ml of HAP (Britten et al., 1974). For this purpose, sheared Escherichia coli DNA in 0.12 M phosphate was added to the samples prior to loading onto the HAP column. With this procedure, the background observed was between 0.4 and 0.8%. DNA

extraction. Rat embryos or liver nuclei were suspended in a buffer containing 1% SDS and were extracted with a chloroform-isoamyl alcohol mixture (24:l). The aqueous phase was repeatedly extracted with phenol-chloroform and was precipitated overnight. After resuspension, the sample was incubated with predigested pronase and was reextracted with phenol-chloroform and chloroform-isoamy1 alcohol. The DNA was then incubated with boiled pancreatic RNase for 2 hr at 37°C. Predigested pronase (500 pg/ml) was added, and the incubation was continued for 2 hr, after which the mixture was extracted twice with phenol-chloroform and finally was precipitated with ethanol. DNA shearing and determination of fragment size. Shearing of DNA to an ap-

proximately

250-nucleotide

fragment

COLBERT ET AL.

Messenger

length was done in a Virtis 60 homogenizer as described by Britten et al. (1974). The sizes of the fragments were determined on the basis of their electrophoretic mobilities in polyacrylamide-agarose gels and were compared with appropriate standards as described by Colbert et al. (1976). RESULTS

Sequence Complexity of Rat DNA Figure 1 shows a reassociation analysis of rat DNA. Sheared rat liver DNA was incubated at 60°C for various periods of time, and the percentage of DNA reassociated was determined by HAP chromatography. The best fit to the experimental data for an ideal curve with second-order kinetics and the apparent CotI,z values were determined with a computer program. Approximately 69% of the genome consists of nonrepetitive sequences. A class of repetitive sequences corresponding to approximately 8% of the genome reassociates very rapidly. These values are simi-

RNA

in Regenerating

115

Liver

lar to those reported by Holmes and Bonner (1974). Preparation of Polysomes, PoZy(A)+ mRNA, and Synthesis of cDNA

The polysome profiles from normal and regenerating livers were virtually identical and indicate that the methods used caused little polysome degradation (Fig. 2). The total polysome preparations obtained contained a higher proportion of free polysomes than was present in the cell. This occurs because, in these procedures, free polysomes are recovered almost entirely, while 2550% of the membranebound polysomes may be lost during cell fractionation (Adelman et al., 1973). Such losses affected equally the polysomes obtained from livers of sham-operated or partially hepatectomized rats. RNA extracted from polysomes with phenol-chloroform was fractionated by

4 FIG. 1. Reassociation of rat DNA. DNA was isolated from either adult rat liver or from l7-day rat embryos and was prepared as described in Methods and Materials. The reactions were carried out at 60°C in 0.06-1.0 M phosphate buffer with DNA concentrations of 53-3200 pg/ml. The percentage of DNA reassociated was determined by chromatography on HAP. The curve was drawn with the aid of a computer program. The following parameters were obtained: (a) fast repetitive = 8.5%, COtlit 2.8 x 10e3; (b) middle repetitive = 22.0%, C,,t,,, = 2.8; (c) nonrepetitive = 69.0%, COtlit = 2.5 x 103. The first component of the curve may represent a “foldback” fraction. The nonrepetitive fraction (69%) may have been underestimated because of repetitive sequence interspersion.

DTTOM

TO

FIG. 2. Polysomal profile from 12-hr regenerating liver. Polysomes were prepared from postmitochondrial extracts as described in Methods and Materials. Polysomes were centrifuged on a 1530% sucrose gradient, and the absorbance at 260 nm was recorded. The direction of sedimentation is from right to left. The first large peak on the right side of the gradient represents ribosomal dimers. Peaks containing up to 10 ribosomal”units can be identified. Polysomal profiles from livers of sham-operated rats were virtually identical to those presented in the figure.

116

DEVELOPMENTAL BIOLOGY

chromatography on poly(U)-Sepharose columns. The two RNA fractions obtained were analyzed by electrophoresis on polyacrylamide-agarose gels (Fig. 3A, B). The fraction eluted from the column with NETS buffer displayed sharp peaks corresponding to 28 and 18s RNA. In addition, this fraction showed the presence of four “minor bands” of RNA species with molecular weights ranging from 8 x lo5 to 1 x 106.These bands are generally included in the “18s peak” of conventional sucrose gradients and have been analyzed in detail in previous work (Levin and Fausto, 1973). The poly(A)+ mRNA fraction eluted from poly(U)-Sepharose columns with formamide displayed a broad mobility range averaging about 20s and corresponding to a molecular weight of 7.6 x lo5 daltons or approximately 2100 nucleotides. This size was identical for normal and regenerating liver preparations. No contaminating 28, 18, or 4s RNA was detectable in the poly(A)+ RNA fraction. Complementary DNA (cDNA) was prepared using as template the liver polysomal poly(A)+ mRNA from sham-operPDLY (A,’

I PDENYLATED RNA 165

6 2es

- I

VOLUME 59, 1977

ated or partially hepatectomized rats. The resulting [3H]cDNAs had specific radioactivities of approximately 1 x lo7 cpm/ pg and a modal length distribution of 860 nucleotides, as estimated by their mobility on 4% polyacrylamide gels containing 7.0 M urea (Fig. 4). Seventy five percent of the cDNA synthesized from normal or regenerating liver RNAs fell within a range of 750-1,050 nucleotides. Complementary bridization

DNA:

1

I6S

I

k-4 -

Hy-

mRNA

0

50 DISTANCE

.!,

DNA

For convenience we will refer to the cDNA made from template regenerating liver polysomal poly(A)+ mRNA as “HepcDNA.” When poly(A)+ mRNA from the livers of sham-operated rats is used as template, the resulting cDNA product is referred to as “Sham-cDNA.” “Hep-cDNA” and “Sham-cDNA” were hybridized with a lo4 excess of total sheared rat DNA. The percentage of cDNA hybridized was determined by HAP chromatography. The results of the hybridization of total rat DNA with “Hep-cDNA” or “Sham-cDNA” have been normalized to 100% hybridization and are combined into

0

I-v -

Total

.

I

FIG. 3. Gel electrophoresis of polysomal RNA. Polyadenylated RNA (A) and nonadenylated RNA (B) were prepared as described in Methods and Materials. RNA was analyzed on gel slabs containing 2.5% polyacrylamide and 0.5% agarose. After electrophoresis, the slabs were stained with Stains-All (Dahlberg et al., 1969) and were scanned at 570 nm.

loo

150

0

FROM ORIGIN (mm)

FIG. 4. Electrophoretic analysis of cDNA size. Labeled “Hep-cDNA” (0-O) and “Sham-cDNA” (O-0) were prepared with AMV reverse transcriptase as described in Methods and Materials. Electrophoresis was carried out on 4% acrylamide gels with 7 M urea. The gels were stained with Stains-All and were cut into 3-mm slices for radioactivity determination. The left ordinate represents the counts per minute in Sham-cDNA; the right ordinate corresponds to the counts per minute in Hep-cDNA. The markers used were single-stranded fragments of A-DNA obtained by digestion with Hind II and III.

COLBERT ET AL.

Messenger

one figure (Fig. 5). The actual final extent of hybridization was between 85 and 90% for both reactions. The similarity between the curves obtained for the hybridization of “ShamcDNA” and “Hep-cDNA” with total rat DNA is striking. The following conclusions can be reached from inspection of these curves: (a) The very rapid reassociating portion of rat DNA comprising 8% of the total genome does not appear to contribute to the sequences found in polysomal poly(A)+ mRNA in normal and 12-hr regenerating livers; (b) approximately 18% of polysomal poly(A)+ mRNA of normal and 12-hr regenerating livers is homologous to the intermediate repetitive portion of the genome; (c) the proportion of polysomal poly(A)+ mRNA which anneals to nonrepetitive DNA is similar in normal and 12hr regenerating livers. Polyadenylated tion

RNA-cDNA

Hybridiza-

The hybridization of cDNA with its homologous mRNA permits the calculation of: (i) the sequence complexity of the mRNA population, i.e., the total number of different sequences present; (ii) the distribution of the mRNA population in different abundance classes; (iii) the RNA sequence complexity for each component class; (iv) the number of copies per cell of the various sequences, provided that the absolute amount of mRNA per cell is known (Bishop et al., 1974; Getz et al., 1975; Ryffel and McCarthy, 1975; Williams and Penman, 1975; Axe1 et al., 1976; Getz et al, 1976; Hastie and Bishop, 1976; Monahan et al, 1976; Young et al., 1976). The hybridization of “Sham” and “Hep” cDNA with homologous mRNA is shown in Figs. 6 and 7 respectively. These figures show the best fit to the experimental data for ideal curves with pseudo-first-order kinetics. For these analyses, two programs were used, one developed by Dr. J. 0. Bishop and another designed by H. Ribeiro and M. V. Tedeschi (unpublished). Hy-

RNA : I

P

in Regenerating

117

Liver

IOO-

60

+---Q

SHAM

LIVER

.---.

HEP. LIVER

,H

‘H-CDNA

,f

‘H-cDNA

;y

F: i

60.

2 E

40.

,I -

20.

ti (‘0 ;;J ,‘I f/

EQUIVALENT

Coi

FIG. 5. Annealing of 3H “Sham-cDNA” and “HepcDNA” with total DNA. Labeled “Hep-cDNA” (O---O) and “Sham-cDNA” (O- - -01, prepared as described in Methods and Materials were mixed with a lo4 excess of sheared rat DNA, boiled for 5 min, and allowed to anneal at 60°C in 0.12-1.0 M phosphate buffer. The progress of the reaction was determined by the percentage of radioactivity bound to HAP at each equivalent Cot point. The concentrations of total DNA were 0.31-3.1 mg/ml. The final extent of the annealing was 85-90% for both reactions; values plotted in the figure represent values normalized to 100%. Zero-time values of 1.5% for “Sham-cDNA” and 1.8% for “Hep-cDNA” have been subtracted. DNADNA reassociation was identical to that presented in Fig. 1.

IO'

FIG. 6. Hybridization of “Sham-cDNA” with polyadenylated mRNA from sham-operated rats. Polysomal poly(A)+ mRNA from livers of sham-operated rats and 3H-cDNA were prepared as described in Methods. For the hybridization reaction (see Methods) the mRNA concentrations were 38 and 1166 PgLgl ml; RNA/cDNA ratios were 105-106. At the end of the incubations the extent of hybridization was determined following S, nuclease digestion as described in Methods. The final extent of the hybridization reaction was 81%. The curve was drawn using a computer program and is analyzed in Table 1. The arrows indicate the Rot,,, values of the components.

118

DEVELOPMENTAL

BIOLOGY

of “Hep-cDNA” with polyaFIG. 7. Hybridization denylated mRNA from 12-hr regenerating liver. For the hybridization reactions (see Methods and Materials), the mRNA concentrations were 50 and 2100 pg/ml; RNA/cDNA ratios were 104-105. At the end of the incubations, the extent of hybridization was determined following S, nuclease digestion as described in Methods and Materials. The final extent of the hybridization reaction was 78%. The curve was drawn using a computer program and is analyzed in Table 1. The arrows indicated the R,t,,, values of the components. TABLE ANALYSIS

OF KINETICS

OF HYBRIDIZATION

RNA class cDNA hybridized (o/o) Normal

liverd

Regenerating 1iveP

VOLUME

59, 1977

bridization of the polysomal mRNA of most tissues with cDNA involves complex mRNA populations that can be resolved into distinct kinetic components by determining the number of transitions present in the hybridization curves (Bishop et al., 1974). Ideal curves having one to four components were generated from the experimental data. The introduction of a second component decreased the sum of the squares of deviations from 0.36 to 0.01. The addition of a third or a fourth component provided only marginal improvement to the fit (SSD = 0.009). Thus, the two-component solution appears to be the simplest one that fits the data, and thus, it was used for the characterization of the mRNA populations as shown in Table 1. The total mRNA complexity changes very little whether two, three, or four components are assumed to exist. Chicken globin mRNA was used as a 1

AND SEQUENCE

Rotliz (ob-

COMPLEXITY

Rizziee(dor(I

served)

DETERMINATION

Sequence ;omplexItY

Copies of each sequence per cell’

I II

27.6 53 4 A 81.0

0.125 35.48

0.043 23.37

29 15,580 15,609

10,241 37

I II

22.6 256 7 79.3

0.20 30.6

0.057 21.87

38 14,580 14,618

7,712 50

a Corrected Rot = Rot of component class if that class were present alone. These b Sequence complexity is the number of different sequences present in the mRNA population. sequences are of average 7.15 x lo5 daltons [taking into account a 5% poly(A) content]. TheRotliz for an RNA of this size is 1.5 x lo+ under our conditions. This value was determined by using chicken globin as a standard (unpublished). c Number of copies per cell = number of copies of each sequence in the class per cell, calculated according to the formula (Getz et al., 1976): poly(A)+ mRNA/cell x fraction base sequence complexity

of cDNA hybridized x 6 x 10Z3 = number of copies per cell, of RNA class x 7.15 x lo5

The values actually reflect the number of copies per cell nucleus. Normal and I2-hr regenerating livers have approximately 25-30% binucleate cells. The values used in the formula were 1.28 pg of polysomal poly(A)+ mRNA/cell in livers of sham-operated rats and 1.54 pg of polysomal poly(A)+ mRNA/cell in 12-hr regenerating livers (McGowan and Fausto, in preparation). The number of nuclei per gram of liver was estimated as 1.2 x lo* (VanLancker and Sempoux, 1958). * Hybridization of “Sham-cDNA” with polysomal polyadenylated mRNA from livers of sham-operated rats (data from Fig. 6). e Hybridization of “Hep-cDNA” with polysomal polyadenylated mRNA from 12-hr regenerating livers (data from Fig. 7).

COLRERT

ET AL.

Messenger

standard for the sequence complexity calculations. This messenger hybridized with its cDNA with anR& of 4.2 x 10e4(data not showd under the same conditions used for liver cDNA-mRNA hybridization. The sequence complexities of polyadenylated polysomal mRNA from livers of sham-operated and partially hepatectomized rats are similar, indicating that polyadenylated polysomal mRNA populations from these livers have almost the same number of different sequences of 7.15 x lo5 average molecular weight (Table 1). The data also show that, in normal or regenerating livers, a relatively small number of sequences (Class I) is represented by many copies in the cell (5000-lO,OOO),while a large number of sequences (Class II) is represented by only 30-50 copies. The total number of polyadenylated polysomal mRNA molecules per cell in 12-hr regenerating livers is approximately 20% larger than that present in livers of sham-operated rats. Homology between Polysomal mRNA from Normal and 12-hr Regenerating Livers

While the kinetic experiments indicate that the total sequence complexities of polyadenylated polysomal mRNA in normal and 12-hr regenerating liver are similar, they do not exclude the possibility that qualitative differences exist between the two mRNA populations. To examine the homology existing between polysomal poly(A)+ mRNA populations in normal and 12-hr regenerating liver, we cross hybridized (a) “Sham-cDNA” with poly(A)+ polysomal mRNA from regenerating livers and (b) “Hep-cDNA” with poly(A)+ polysomal mRNA from normal livers. The saturation levels of these heterologous hybridizations were compared to those of the corresponding homologous reactions as shown in Figs. 8 and 9. The important factor in this kind of analysis is the final percentage of cDNA hybridized (Getz et al., 1976), but a few incubations at low R,t values were also done to ensure that the

RNA

in Regenerating

IO0 / E 80 g $ 6o 2 40 ; 20 01, 0

:

0.5

119

Liver

:-

1.0

0-

El”, -. ,xIn-‘, -

:

‘5

L

20

I

8. Homology of “Sham-cDNA” with polysomal polyadenylated mRNA from 12-hr regenerating liver. Parallel hybridization reactions were carried out between polysomal poly(A)+ mRNA from 12-h regenerating liver and “Sham-cDNA” (heterologous reaction O---O) or “Hep-cDNA” (homologous reaction 0-O). The materials used and the conditions for the reactions are described in Methods and Materials. RNA concentrations of 1500 pg/ml and RNA/DNA ratios of 104-lo5 were used in both homologous and heterologous reactions. The extent of hybridization was 89% for the homologous reaction and 75% for the heterologous hybridization. For comparative purposes, the data presented in the figure have been normalized to a 100% value for the homologous reaction. FIG.

I

I

FIG. 9. Homology of “Hep-cDNA” with polysomal polyadenylated mRNA from livers of shamoperated rats. Parallel hybridization reactions were carried out between polysomal poly(A)+ mRNA from livers of sham-operated rats and “Hep-cDNA” (heterologous reaction O---O) or “Sham-cDNA” (homologous reaction O-O). The materials used and the conditions for the reaction are described in Methods and Materials. RNA concentrations of 1800 pg/ml and RNA/DNA ratios of 104-lo5 were used in both homologous and heterologous reactions. The extent of hybridization was 79% for the homologous reaction and 69% for the heterologous hybridization. For comparative purposes, the data presented in the figure have been normalized to a 100% value for the homologous reaction.

120

DEVELOPMENTAL BIOLOGY

kinetics of the homologous reactions were similar to those presented in Figs. 6 and 7. The final extent of hybridization of both homologous reactions in Figs. 8 and 9 is greater than that of the heterologous reactions. The saturation levels reached by the heterologous reactions are approximately ll-14% below those of the respective homologous reactions. We conclude from these curves that approximately 14% of the polysomal poly(A)+ mRNA from normal liver is absent or under-represented in regenerating liver polysomes. Conversely, about 11% of the polysomal poly(A)+ mRNA from regenerating liver is not detected in normal liver polysomes.

VOLUME 59, 1977

in rat myoblasts appears to be transcribed from repetitive sequences. Therefore, the above calculations are maximal estimates of the proportion of nonrepetitive DNA represented in poly(A)+ polysomal mRNA. We are unaware of other data on the sequence complexity of polyadenylated polysomal mRNA from rat liver. Estimates for the sequence complexities of chicken and mouse liver mRNA (Axe1 et al., 1976; Hastie and Bishop, 1976; Young et al., 1976) are similar to those presented here. The similarity in the sequence complexity of polysomal mRNA populations in normal and 12-hr regenerating liver does not preclude the possibility that new mRNA species may be present in the polysomes of DISCUSSION regenerating livers. For this to be true it A computer analysis of the curves ob- would be necessary that either the number of these sequences be relatively small or tained from the hybridization of polyadenylated polysomal mRNAs from normal or that the appearance of new species in regenerating liver polysomes be matched by 12-hr regenerating liver with their homolthe absence of a similar number of RNA ogous cDNAs indicated that these mRNA in which mRNA populations have approximately the same sequences. Experiments with number of sequences. The sequence com- from normal liver was hybridized “Hep-cDNA” and “Sham-cDNA” was hyplexity of polyadenylated polysomal bridized with regenerating liver mRNA mRNA from normal liver is approximately showed that 15,600 sequences of 7.15 x lo5 average mo- (heterologous hybridizations) lecular weight, which corresponds to a to- the homology existing between polyadenylated polysomal mRNA from normal and retal of 11.1 x lo9 daltons. Rat DNA, which generating liver is approximately 8590%. has an analytical complexity of 1.8 x 1012 Thus, in addition to sequences shared by daltons, is comprised of approximately both mRNA populations, some RNA 69% nonrepetitive sequences (1.17 x lo’* sequences appear to be unique to normal daltons). Using these values, we estimate or regenerating liver. The proportion of that approximately 0.90% of single-copy mRNA which is not shared by the two rat DNA is represented in the polyadenylated polysomal mRNA from livers of populations is somewhat large compared to the reported differences in mRNA popusham-operated rats. A similar calculation lations between resting and growing cells for 12-hr regenerating liver indicates that in culture (Getz et al., 1976; Williams and approximately 0.85% of single-copy rat Penman, 1975). We do not yet know if the DNA is represented in polyadenylated polnonshared sequences are transcribed from ysomal mRNA. These values assume that nonrepetitive genes, or to what abundance all of the polyadenylated polysomal mRNA is transcribed from single-copy class these sequences belong. It also remains to be established whether the genes. However, this is unlikely to be the mRNA sequences unique to regenerating case since, as shown in Fig. 5, approxiliver polysomes are essential to the remately 20% of “Sham-cDNA” or “HepcDNA” hybridizes with repetitive DNA generative process. This last point can perhaps be clarified by determining the sequences. Campo and Bishop (1974) have time of appearance of these sequences shown that 20% of the mRNA population

COLBERT ET AL.

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when DNA synthesis after partial hepatectomy is delayed or inhibited by irradiation or nutritional deprivation (Faust0 et al., 1964; Hilton and Sartorelli, 1970; McGowan and Fausto, in preparation). The detection of mRNA sequences which are present only in regenerating liver polysomes does not necessarily imply that these species are transcribed by genes which are active exclusively during compensatory growth. These genes may be transcribed in normal liver, but their transcription products may be confined to the nucleus (Church and McCarthy, 1969; Shearer and Smuckler, 19721, or may reach the cytoplasm and not be incorporated into polysomes. Experiments in progress in our laboratory (Tedeschi, Colbert, and Fausto, in preparation) indicate that single-copy DNA hybridizes to a similar level with nuclear RNA from normal and 12-hr regenerating liver. In addition, “Hep-cDNA” anneals to the same extent to nuclear RNA from normal and 12-hr regenerating liver. These preliminary results suggest that the existence of polyadenylated mRNA sequences unique to polysomes of regenerating liver may be the result of post-transcriptional events. Some uncertainties about the methodology used must be kept in mind in evaluating our conclusions: (a) In preparing undegraded polysomes, some inevitable losses take place, and it is not known whether the lost polysomes represent special message classes; (b) although it is generally assumed that reverse transcriptase copies equally all of the sequences present in the mRNA population used as template, this has not yet been definitively proven (Lewin, 1975); (c) there is an inherent uncertainty in the measurement of sequence complexity of low-abundance mRNA populations by kinetic analysis. In the present experiments, sequence complexity values which diverge from each other by 20% or less cannot be considered different. This somewhat arbitrary range was established because, if one varies the RotliZ values for

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the high-complexity component of the curves by more than 20%, the resulting root mean square deviations are larger than those usually obtained in our experiments; (d) while the material used in these experiments came from large pools of animals (approximately 100 partially hepatectomized and 75 sham-operated rats), we do not know if the same degree of homology between mRNA preparations of normal and regenerating liver would be found in other experiments; (e) all of our conclusions refer to polysomal polyadenylated mRNA. It is quite likely that, as in other systems (Milcarek et al., 1974; Greenberg, 1976; Nemer et al., 1974), a sizable proportion of nonadenylated mRNA exists in rat liver. Despite these limitations, it is obvious that the pattern of gene expression in regenerating liver, 12 hr after partial hepatectomy, is very different from the pattern of gene expression during hormone-induced growth of oviducts when the complexity of the mRNA population undergoes large changes (Monahan et al., 1976). We thank Ms. Sarah Garcia-Mata for her assistance. The authors are grateful to Dr. John 0. Bishop for making available his computer program, Dr. J. W. Beard for supplying the reverse transcriptase, Dr. A. Dahlberg, Dr. A. Coleman, Dr. C. Milcarek, and MS. W. Ross for supplying materials and for help with experimental procedures, and Mr. H. Ribeiro for development of the computer programs.

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