Embryonal Carcinoma and Committed Myogenic and. Erythropoietic Cell Lines. Nabeel A. Affara, Michel Jacquet,. Hedwige Jakob, Francois Jacob and.
Cell,
Vol. 12, 509420,
October
1977,
Copyright
0 1977 by MIT
Comparison of Polysomal Polyadenylated RNA from Embryonal Carcinoma and Committed Myogenic and Erythropoietic Cell Lines Nabeel A. Affara, Michel Jacquet, Hedwige Jakob, Francois Jacob and Francois Gros lnstitut Pasteur Department Biologie Moleculaire 25, rue du Dr. Roux 75015 Paris. France
Summary Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A)+ mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700,13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells. Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A)+ RNA sequences are common to embryonal carcinoma cell polysomes-apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A)+ RNA fails to hybridize 7-10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.
Introduction Results of recent experiments suggest that there is a gradual reduction in tne amount of genetic information expressed as development proceeds toward the formation of adult tissues. This occurs together with the maintenance of a substantial group of mRNA sequences found in common between different developmental stages and tissues, thus lending credence to the concept of “housekeeping” enzymes (Galau et al., 1976). In addition, it appears that differences in cellular phenotype may be brought about by a relatively small number of specific sequences in conjunction with quantitative modulation of sequences found in common between different cells and tissues (Ryffel and McCarthy, 1975; Galau et al., 1976; Axel, Feigelson and Schutz, 1976; Hastie and Bishop, 1976; Young, Birnie and Paul, 1976). Our interest in this paper lies in comparing the extent of gene expression occurring in a pluripotent mouse embryonal carcinoma cell (line PCCS) with that of cells committed to terminal differentiation. We attempt to define the relative complexity, the degree of homology and the extent of specificity in polysomal poly(A)-containing mRNA populations during these changes. For these studies, we have chosen cell types capable of differentiating along the myogenic and erythroid pathways, both directions of cellular differentiation known to arise during the course of embryonal carcinoma (called EC) cell development (Martin, 1975; Martin and Evans, 1975; lllmensee and Mintz, 1976). The myoblast cell lines [line PCD 2 clone T233 (Boon et al., 1974) and line Cl751 clone T984 (H. Jakob, personal communication)] are both derived from EC cells and undergo terminal differentiation upon reaching confluence during cell culture. Several markers characteristic of myogenesis are expressed in these cells. These include the muscle form of creatine phosphokinase, actin and myosin light chains, and acetylcholine receptor. Furthermore, these cells fuse to form contracting multinucleate myotube structures. A mouse Friend erythroleukemic cell was chosen as the second cell type representing, in this case, the erythropoietic pathway. The erythroid nature of this cell is supported by much evidence demonstrating that the Friend cell arises by transformation (with Friend virus complex) of a committed erythroid precursor, whose further development is then arrested at the proerythroblast stage (Frederickson et al., 1975; Tambourin and Wendling, 1975). This developmental arrest can be overcome by treating the cells with a variety of chemical agents, such as dimethylsulphoxide. Such treatment leads to terminal differentiation along the erythroid pathway
Cell 510
(Friend et al., 1971) as evidenced by cessation of DNA synthesis, increased synthesis of S-aminolevulinic acid synthetase and heme (Friend et al., 1971; Ebert and lkawa, 1974), o(- and p-globin mRNAs and protein chains (Ross,Ikawa and Leder, 1972; Boyer et al., 1972; Ostertag et al., 1972; Conkie et al., 1974), spectrin (Arndt-Jovin et al., 1977) and erythrocyte surface antigens (Furusawa, lkawa and Sugano, 1971), together with morphological changes characteristic of erythroid maturation. In the studies reported here, we have compared the committed but not yet terminally differentiated cells to the nondifferentiated EC cell type. We have examined only the polyadenylated mRNA population [called poly(A)+ RNA] from purified polysomes for several reasons. First, we are taking polysomal location as a criterion of functional activity for mRNA, our interest being in those sequences which are actually translated into protein and hence involved in determining cellular phenotype. Second, in view of parallel studies with nuclear and messenger ribonucleoprotein compartments, we wished to avoid contamination by ribonucleoprotein particles from either of these fractions (for discussion of this point, see Galau, Britten and Davidson, 1974). Results Polysomal Poly(A)+ RNA Sequences in Myoblast and Pluripotent Embryonal Carcinoma Cells Sequence Complexity A large excess of polysomal poly(A)+ RNA (prepared as described in Experimental Procedures) from mononucleate exponentially growing T233 myoblast cells (derived from the same teratocarcinoma as’ the PCC3 cell line) and nondifferentiated EC PCC3 cells was reacted to appropriate Rot values with corresponding homologous cDNA. The hybridization kinetics, as ascertained by digestion with single-strand-specific Sl nuclease, are shown in Figure 1A and 1B. Specificity of hybridization is shown by the fact that up to 85-90% of cDNA is capable of entering into Sl nuclease-resistant hybrids with template RNA, whereas 5% was rendered Sl nuclease-resistant when reacted with E. coli ribosomal RNA (not shown). The cDNA which fails to hybridize may represent Sl nuclease-sensitive “tails” of cDNA and/or cDNA transcripts too small to form stable hybrids. As can be seen, the kinetics extend over almost four logarithmic decades, revealing the hybridization of a heterogeneous RNA population with different sequences present in classes of diverse frequency. With the use of a pure kinetic standard of
known base-sequence complexity and kinetic constant of hybridization (in this case, the reaction between adult mouse (Y- and p-globin RNA and its corresponding cDNA given in Figure lA), it is possible to resolve these curves into a number of firstorder components which, when compounded, can adequately’ describe the experimentally observed data (the theoretical basis of the analysis we have used is discussed in Experimental Procedures). Table 1 tabulates the RNA frequency classes, their kinetic constants and their base-sequence complexity in terms of nucleotides and the number of different average size mRNAs. The best-fit analysis for the PCC3 poly(A)+ RNA population indicates the presence of three abundance classes representing 0.308, 0.33 and 0.358 of the poly(A)+ RNA population and containing, respectively, base-sequence complexities of 0.111 x IO”, 0.874 x lo6 and 14.55 x 10” nucleotides. Also included in Figure IA are the results obtained with a different strain of EC cell (line PCC4; Jakob et al., 1973). The poly(A)+ RNA from this cell exhibits an identical hybridization curve to strain PCCS. In addition, PCC3 mRNA can fully hybridize PCC4 cDNA, yielding a curve indistinguishable from the PCC3 homologous curve. These data suggest that the two clones contain the same polysomal poly(A)+ RNA sequences present in the same distribution of frequencies, and that two independent multipotential cell lines, PCC3 easily differentiating in vitro and PCC4 in vivo, contain the same complement of polysomal poly(A)+ RNA sequences when maintained in the exponentially growing nondifferentiated state. For the myoblast cell line, the analysis yielding the best fit to the experimental data shows the presence of three abundance classes representing 0.30, 0.34 and 0.36 of the poly(A)+ RNA population, with complexities of 0.126 x 106, 1.224 x lo6 and 25.199 x 10” nucleotides, respectively. Very similar results were obtained with a myoblast line (T984) derived from a teratocarcinoma of different origin. The homologous Rot curve for polysomal poly(A)+ RNA from this cell is also shown in Figure IB. It can be seen that the two myoblast cell lines exhibit superimposable hybridization kinetics. Furthermore, cross-hybridization between cDNA from poly(A)+ RNA of T233 cells and poly(A)+ from T984 cells yields a Rot curve with kinetics identical to those of the two homologous curves. This shows that the two myoblast cell lines exhibit similar basesequence complexities and that at least all hybridizable cDNA sequences of T233 cells are present in T984 myoblast polysomes. It would therefore appear from the analysis of two independent myoblast cell lines that their apparent base-sequence complexity is larger than that of the PCC3 cells.
Complexity
of Poiysomal
Poly(A)+
RNA in Different
Cell Lines
511
-1
0
LOGROT Figure
1. Homologous
Hybridization
of PCC3 and
Myoblast
cDNA
to Template
RNA
Homologous hybridization kinetics of myoblast and PCC3 cDNAs with their respective polysomal poly(A)+ RNA template were followed by digestion with Sl nuclease. For Rot values up to 20, hybridization reactions contained RNA at a concentration of 200 pglml. For Rot values >20, the RNA concentration used was 1 mg/ml. At least two points of either concentration were made to overlap between the two Rot value ranges. Each point was corrected for 3-4% background of Sl nuclease-resistant cDNA obtained either by digestion of cDNA prior to incubation, by digestion of cDNA plus template RNA immediately after heat denaturation or by digestion of cDNA after incubation with E. coli ribosomal RNA. The points represent the results obtained from at least five independent experiments. Also shown is the Rot curve for the reaction between oi + p mouse globin mRNA and its corresponding cDNA. Rot is the product of RNA concentration (mole of nucleotide per liter) and time (in set). (A) EC cell lines: (O-O) PCC3 mRNA x PCC3 cDNA; (O--O) PCC4 mRNA x PCC4 cDNA; (A-A) PCC3 mRNA x PCC4 cDNA; (O-O) (Y + P-globin mRNA x 01 + @globin cDNA. (6) Myoblast cell lines: (O-O) T233 mRNA x T233 cDNA; (A-A) T984 mRNA x T994 cDNA; (A-A) T984 mRNA x T233 cDNA.
Sequence Homology and Specificity between Myoblast and Embryonal Carcinoma Cells By way of reciprocal heterologous hybridization experiments, it is possible to examine the extent of homology between the myoblast and embryonal carcinoma cell polysomal poly(A)+ RNA populations. The final plateau difference between homologous and heterologous cDNA hybridization reflects the amount of RNA by mass absent in the heterologous RNA population. If this difference arises at low Rot values, then the missing sequences should be derived from an abundant class. Similarly, if the differences arise at high Rot values, then sequences should correspond to rare RNA species. This type of information can be obtained from heterologous hybridization provided
that complete hybridization of common sequences occurs and that this proceeds with first-order kinetics. If there are large differences in the relative frequency of common RNA sequences, then a much larger excess of RNA over cDNA (than in the homologous hybridization) will be required to ensure that these conditions are met. Titration experiments showed that PCC3 and myoblast RNA could hybridize, respectively, the same amount of myoblast and PCC3 cDNA at a given Rot value (where 80% of the cDNA had hybridized) in a range of RNA/cDNA ratios extending from 17OO:l to 25:l. Since the excess of RNA used was >I700 fold (by weight) over cDNA, then it is very probable that all reacting cDNA sequences were forming hybrids in heterologous hybridizations with firstorder kinetics.
Cell 512
Table
1. Analysis
of Homologous
Cell
Hybridization
Class of Abundance
Curve
Fraction of Hybridizable cDNA
Apparent Kinetic Constant
cl Pluripotent Teratocarcinoma Cell
Teratocarcinoma Derived Myoblast Cell
Friend Erythroleukaemic Cell
k
Base-Sequence Complexity Nucleotides
in
Number Messenger
of
Species
Number Copies Cell
RNA
of per
NTx~O-~
Abundant
0.309
3.5
0.111
56
601
Intermediate
0.33
0.48
0.8 74
437
82
Rare
0.358
0.03
14.550
7275
5.5
Abundant
0.30
3
0.126
63
476
Intermediate
0.34
0.35
1.224
612
5 5.5
Rare
0.36
0.018
25.199
12599
2.8
Abundant
0.36
5.6
0.081
40
1988
Intermediate
0.355
0.52
0.860
430
127
Rare
0.285
0.031
11.583
5791
8
7
The analysis of the Rot curves described in the text was performed as discussed in Experimental Procedures. LY is the proportion of hybridizable cDNA reacting in each abundance class, and K is the apparent first-order kinetic constant expressed as mole-‘-I-set+ (K = LnP/Rot,,, observed). The base-sequence complexity in nucleotides for each class is given by a. KS/K, where KS is the rate constant for 1 nucleotide (KS = 1.26 x IO6 mole-‘-I-set-‘), is deduced from the rate of hybridization between LY + p-globin mRNA and its cDNA, the data for which are given in Figure IA. K globin = 1050 mole-‘-I-set-’ for 1200 nucleotides. The number of copies per cell for each RNA sequence (of average length 2000 nucleotides) was estimated from the polysomal poly(A)+ RNA content per cell calculated according to the yield of RNA obtained from a known number of cells. This was found to be 1.2 x IO’, 1 .l x 10’ and 1.8 x 10’ pg per cell for PCCB, myoblast and Friend cells, respectively. The frequency of sequences in each class is given by: RNA content
per cell (pg)
= number of nucleotides in each class and divided
330 x IO6 pg (mole of nucleotide) (Avogadro’s number) 6 x IO=
per cell in polysomal poly(A)+ RNA. This number is then multiplied by 01to give the number of nucleotides present by NT (the base-sequence complexity in each class) to give the number of copies per cell of a given sequence.
Figures 2A and 2B present the results of crosshybridization between myoblast poly(A)+ RNA (from both T233 and T984 cell lines) versus PCC3 cDNA, and PCC3 RNA versus myoblast cDNA (of both T984 and T233 lines), respectively. The broken line indicates the homologous kinetics for the cDNA participating in the cross-hybridization. From Figure 2A, it can be seen that all hybridizable PCC3 cDNA can ,be converted into hybrid by RNA from either myoblast cell line, showing that all PCC3 polysomal poly(A)+ RNA sequences are present on myoblast polysomes. In addition, comparison between homologous and heterologous curves shows that myoblast RNA hybridizes less PCC3 cDNA in the intermediate class of sequences. The difference in this region is redressed at higher Rot values, suggesting that sequences situated in the intermediate class in PCC3 cells are possibly present at lower frequency in the rare class of myoblast polysomal poly(A)+ RNA. In view of this considerable homology, it is important to show that extensive heterologous hy-
bridization does not simply represent base mismatching. The melting points (Tm) of an homologous (PCC3 RNA x PCC3 cDNA) and an heterologous (PCCS RNA x myoblast cDNA) hybrid were therefore measured as described in Experimental Procedures, and the results are shown in Figure 3. It can be seen that the melting curves are identical, implying that the heterologous hybrid is as equally well matched as the homologous one. Furthermore, the high Tm value (92°C in 0.3 M NaCI) indicates that in both cases, cDNA and RNA are congruently paired along their length. This would argue that the extent of heterologous hybridization is a true measure of homology between the two different RNA populations. In the reciprocal cross-hybridization (Figure 2B), at Rot values characteristic of the abundant and intermediate frequency classes, PCC3 RNA hybridizes almost the same proportion of myoblast cDNA as in the homologous myoblast curve. It is not until higher Rot values, characteristic of the rare frequency class, that a clear difference arises between
Complexity 513
of Polysomal
Poly(A)+
RNA in Different
1001
Cell Lines
1
I
80
2 G
6o
I” .E
40
9 0 ” 20 s
-2
-1
0
1 LOG
Figure 2. Reciprocal and Myoblast cDNA
Heterologous and RNA
2
3
0
ROT Hybridization
60 between
PCC3
Polysomal poly(A)+ RNA and corresponding cDNA of myoblast and EC cell lines were used in reciprocal heterologous hybridizations, and the reaction kinetics were followed by digestion with Sl nuclease. The points represent the results from two independent experiments with each myoblast cell line. (A) Myoblast mRNA x PCC3 cDNA: (0-O) T 984 mRNA x PCC3 cDNA; (O-O) T233 mRNA x PCC3 cDNA; (- - -) homologous EC cell curve from Figure 1. (B) PCC3 mRNA x myoblast cDNA: (O-O) PCC3 mRNA x T233 cDNA; (A-A) PCC3 mRNA x T984 cDNA; (- - -) homologous myoblast curve from Figure 1.
the homologous and heterologous curves, 12% of cDNA failing to enter into hybrids. These results suggest that the sequences in the first two frequency classes are largely common to both cell types and present at similar frequencies, and that the differences lie predominantly in the rare frequency class. Presumably, the 12% of unhybridized cDNA mainly corresponds to the additional basesequence complexity of 10’ nucleotides expressed in the rare class of myoblast polysomal poly(A)+ RNA. Indeed, as a proportion of the cDNA representing the rare class, this fraction of nonhomologous cDNA corresponds closely to the disparity (1 .l x 10’ nucleotides) in base-sequence complexity between EC and myoblast cells. By using fractionated cDNA, it can be established more precisely whether the sequences specific to myoblast cells do in fact arise from the rare frequency class. To facilitate this, myoblast (T984) cDNA was fractionated into sequences hybridizing by a Rot value of 7 and into sequences hybridizing at Rot values greater than this [separation was effected, after homologous hybridization with T984 polysomal poly(A)+ RNA, by fractionation on hydroxyapatite as described in Experimental Proce-
70
80
Temperature Figure brids
3. Melting
Curves
for Homologous
90
100
’ C and Heterologous
Hy-
Melting curves for PCC3 mRNA/T233 cDNA and T233 mRNAI PCC3 cDNA hybrids were determined as described in Experimental Procedures. The proportion of cDNA participating in the melt has been normalized to 100% for the purpose of presentation. (O-O) PCC3 mRNA/PCC3 cDNA hybrid; (m-m) PCC3 mRNA/T233 cDNA hybrid.
dures]. At a Rot value of 7, 50% of cDNA was hybridized, this factor being reflected in the partition coefficient on hydroxyapatite of recovered cDNA in each fraction. Figure 4A shows that in homologous hybridization, up to 8590% of both the cDNA fractions enriched for abundant and rare sequences is capable of entering into hybrids. As expected, cDNA representing abundant sequences hybridizes faster than cDNA enriched in sequences arising from the rare frequency class. The derivative composite curve arising from these two curves had kinetics close to that of the analogous Rot curve with unfractionated cDNA (not shown). This indicates that the hybritiization characteristics of the cDNA had not been affected by the fractionation procedure. Figure 48 shows that in heterologous hybridization with EC cell polysomal poly(A)+ RNA, all reactive abundant cDNA sequences can be completely hybridized with very similar kinetics to the corresponding homologous curve. In contrast, cDNA enriched in sequences hybridizing at high Rot values cannot be completely hybridized by PCC3 RNA. As higher Rot values are approached, the difference between the heterologous and corresponding homologous curve increases, resulting in a plateau 1520% lower for the heterologous hybridization. This amplified plateau difference be-
Cell 514
-2
-1
0
1
2
3
Hybridization
of Frac-
LOG ROT Figure 4. Homologous and tionated Myoblast cDNA
Heterologous
Myoblast polysomal cDNA (of line T984) was fractionated into cDNA entering hybrid by a Rot of 7 and cDNA entering hybrid after a Rot of 7 during homologous reaction. These fractions represent, respectively, cDNA partially enriched in abundant and rare sequences. Each cDNA fraction was then hybridized homologously with T984 polysomal poly(A)+ RNA and heterologously with PCC3 polysomal poly(A)+ RNA, and analyzed in the same manner as for previous curves. The points represent the results from two independent experiments. (A) Homologous hybridization of fractionated T984 cDNA: (U-m) abundant cDNA; (A-A) rare cDNA. (6) Heterologous hybridization of fractionated T984 cDNA with PCC3 polysomal poly(A)+ RNA: (m---W) abundant cDNA; (A-A) rare cDNA; (- - -) abundant homologous curve; (....) rare homologous curve.
tween homologous and heterologous curves demonstrates that the qualitative sequence differences between EC and myoblast cells reside in sequences hybridizing at high Rot values. Polysomal Poly(A)+ RNA Sequences in Friend Erythroleukemic and Embryonal Carcinoma Cells Sequence Complexity The base-sequence complexity of polysomal poly(A)+ RNA from noninduced Friend cells (clone 70717e) was estimated in the same manner as for the EC and myoblast cell lines. The square symbols in Figure 5 illustrate the hybridization of Friend cell polysomal poly(A)+ RNA with its cDNA. In comparison to the PCC3 Rot curve (broken line), it is evident that Friend cell cDNA hybridizes with more rapid kinetics, implying that its poly(A)+ RNA population is less complex. The numerical analysis in Table 1 shows that the base-sequence complexity is 12.524 x IO6 nucleotides compared to 15.535 x IO6 for PCC3 cells. This represents about 1500 fewer sequences than in PCC3 cell polysomes.
Sequence Homology and Specificity between Friend Erythroleukemic and Embryonal Carcinoma Cells Also shown in Figure 5 are the results of reciprocal cross-hybridization experiments between Friend and PCC3 cell poly(A)+ RNAs and their corresponding cDNAs. 10% of Friend cell cDNA fails to react with PCC3 RNA. From comparison of the heterologous and the homologous curves, it appears that this 10% differential arises at low Rot values and is maintained until the respective plateaus are reached. This would imply that the cDNA sequences specific to Friend cells [with respect to PCC3 poly(A)+ RNA] originate from an abundant class of RNA. Friend cell polysomal poly(A)+ RNA is capable of converting all hybridizable PCC3 cDNA into hybrid except for a 7-10% fraction. In contrast to the preceding cross-hybridization, however, the difference between the homologous and heterologous curves lies at high Rot values, suggesting that the cDNA sequences specific to PCC3 cells are located in the rare class of RNA. It is conceivable that this fraction of cDNA represents the 1500 polysomal poly(A)+ RNA sequences expressed in PCC3 cells, but apparently lacking in Friend cell polysomes. These results imply that both PCC3 and Friend cells contain polysomal sequences which are either extremely dilute or absent in the polysomal compartment of the other. In the case of Friend cells, this appears to be a small number of abundant sequences, whereas in PCC3 cells, it appears to be a large number of sequences (about 1500) present in only a few copies per cell. In analogous fashion to the comparative studies with myoblast cells, fractionation of Friend cell cDNA was used to characterize more exactly the Friend-specific sequences. In this case, we were interested in sequences hybridizing at low Rot values. Friend cell cDNA was therefore hybridized to a Rot value of 1, after which unhybridized cDNA was separated from hybrid by hydroxyapatite fractionation. The cDNA fraction hybridizing at low Rot values was then heterologously hybridized with PCC3 poly(A)+ RNA to aRot value of 1. This was done to obtain abundant cDNA fractions enriched in sequences common to both cells and specific to Friend cells, providing that the sequence differences between these two cell types lie in the abundant class. After these fractionations, three Friend cell cDNA probes resulted: from the first fractionation, Friend cell cDNA sequences hybridizing at Rot values higher than 1; from the second fractionation, a partition of low Rot Friend cDNA (cDNA entering hybrid by a Rot of 1) into fractions enriched in sequences common (called Friend low Rot common) and specific (called Friend low Rot specific)
Complexity
of Polysomal
Poly(A)+
RNA in Different
Cell Lines
515
-. 2
0
-1
1
2
3
Log Rot Figure
5. Homologous
and Heterologous
Hybridization
of Friend
and PCC3
cDNA
Homologous hybridization between Friend cell polysomal poly(A)+ RNA and corresponding cDNA. together with reciprocal heterologous hybridization with PCC3 polysomal poly(A)+ RNA and cDNA. The points represent the results of two independent experiments in each case. (D-m) Friend mRNA x Friend cDNA; (A-A) Friend mRNA x PCC3 cDNA; (O-O) PCC3 mRNA x Friend cDNA; (- - -) homologous PCC3 curve from Figure 1.
between the two cell types. Figure 6 shows the hybridization kinetics of these Friend cell cDNA fractions with both homologous Friend cell and heterologous PCC3 cell polysomal poly(A)+ RNA. In Figure 6A, it can be seen that Friend cell RNA hybridizes at low Rot values a larger proportion of Friend low Rot-specific cDNA than does PCC3 RNA, finally reaching a plateau of 90% compared to 70% for PCC3 RNA at similar Rot values. The differences between the two curves clearly arise (as in the cross-hybridization with unfractionated cDNA) at low Rot values and are maintained until their respective plateaus are reached. The amplified plateau difference (20% versus 10% for unfractionated cDNA) indicates first, that at least some of the Friend-specific sequences are situated in the abundant class, and second, that there has been an enrichment for these sequences in this cDNA fraction. Contrary to this, Figure 6 shows that both Friend and PCC3 cell RNA can hybridize Friend low Rot common cDNA equally well, reaching plateau values of 80-85%. This demonstrates that the sequences in this fraction of cDNA are common to both cell types. Furthermore, in the homologous curve, a larger proportion of cDNA (66% of hybridiz-1 able cDNA) is hybridized at low Rot values characteristic of the abundant frequency class in Friend cell mRNA than in the heterologous hybridization curve, where 60% of hybridizable cDNA forms hy-
brid structures at Rot values characteristic of the intermediate class of PCC3 mRNA. This suggests that sequences present in abundance in Friend cells are found at lower frequency in PCC3 cells. In both the homologous and heterologous curves, very little of this cDNA fraction reacts at Rot values characterizing the rare frequency class. Also shown in Figure 66 are the homologous and heterologous hybridization curves with Friend cell cDNA hybridizing at Rot values >l. This cDNA represents mostly sequences drawn from the rare frequency class of Friend cell poly(A)+ mRNA, but also contains a proportion of the intermediate class. Both PCC3 and Friend cell RNA can hybridize this cDNA equally, reaching a plateau of 80%. This confirms that the sequences specific to the Friend cell are not situated in the rare class. As with the Friend low Rot common cDNA discussed above, PCC3 RNA drives this cDNA fraction into hybrid at higher Rot values than Friend RNA, showing that some of these sequences are also present at lower frequency in PCC3 cells. The main transition (containing 70% of the hybridizable cDNA), however, has a Rotliz value of about 20 mole-I-‘-set, giving this component a kinetic constant of 0.034 mole-l-‘set-‘. This is close to the value for the rare frequency class in PCC3 cells (0.031), thus indicating that the majority of rare Friend cell sequences are also present in the rare frequency class of PC63 cells. The remaining 30% of hybridizable cDNA
Cell 516
-2
-1
0
1
2
3
LOGROT Figure 6. Homologous tionated Friend cDNA
and
Heterologous
Hybridization
of Frac-
Friend cell cDNA was fractionated into three probes as described in the text and Experimental Procedures. Each fraction was then homologously and heterologously hybridized with Friend and PCC3 polysomal poly(A)+ RNA, respectively. The points represent the results from two independent experiments in each case. (A) (0-O) Friend mRNA x Friend low Rot specific cDNA; (A-A) PCC3 mRNA x Friend low Rot specific cDNA. (B) (0-U) Friend mRNA X Friend low Rot common cDNA; (A-A) PCC3 mRNA x Friend low Rot common cDNA; (W-W) Friend mRNA x Friend cDNA hybridizing at Rot values >I; (A-A) PCC3 mRNA x Friend cDNA hybridizing at Rot values >l.
reacts at Rot values characteristic of the intermediate frequency class of PCC3 mRNA. Discussion In this report, we have tried to define the extent of homology and specificity between mRNA sequences present in the polysomes (and therefore involved in specifying the cellular phenotype) of three quite distinct cell types, taking the polysomal poly(A)+ mRNA sequences in the EC cell as a reference population. There are several advantages in using the teratocarcinoma cell system for investigating the relation between differentiation and the extent of gene expression. With the exception of Galau et al. (1976), where the extent of genetic expression in polysomes was examined at different stages of sea urchin development and in adult structures, most studies have involved comparisons between fully differentiated tissues, the end products of terminal differentiation (Young et al., 1976; Axel et al., 1976; Hastie and Bishop, 1976). The use of a cloned pluripotent cell and committed
cell lines capable of terminal differentiation permits comparative studies not only between the multipotential and committed states, but also the terminally differentiated state. Furthermore, there is the added advantage that it is possible to work with homogeneous cell populations, which is difficult to control for when using tissues. Three qualifications, however, must be noted. First, although the committed cell lines exhibit the coordinate expression of tissue-specific markers, it is not clear that their patterns of gene expression are identical to the tissues they represent. Second, the absence of a direct precursor-product relationship between the EC cell and the Friend cell makes it necessary to interpret with caution whether any differences between messenger populations are a consequence of Friend cell commitment to the erythroid pathway or to some other processes arising from the different origins and life histories of the two cell lines. Third, our studies only refer to the poly(A)+ mRNA population and do not give any information on poly(A)) mRNA, reported to represent a substantial proportion of mRNA in HeLa cells (Milcarek, Price and Penman, 1974) and sea urchin embryos (Nemer, Graham and Dubroff, 1974). It is unclear, however, whether a large diversity is represented by this mRNA fraction. The results obtained indicate that the three polysomal poly(A)+ RNA populations each have basesequence complexities of similar magnitude (the largest difference being a factor of two), three analyzable frequency classes and comparable sequence abundance between the three cell types within these three frequency classes. The heterologous cross-hybridization experiments with both total and fractionated cDNA probes revealed several interesting points. First, all the PCC3 sequences are present on myoblast polysomes. Second, about 4500 sequences, primarily drawn from the rare frequency class, appear to be specific to myoblasts (with respect to the EC cell), those in the intermediate and abundant classes largely being found in common and present at similar levels. Third, for the Friend cell, the vast majority of the base-sequence complexity (close to the value reported by Birnie et al., 1974) is homologous to the EC cell, apart from a small group of sequences drawn from the abundance class and representing 10% of the total cDNA. As a proportion of the abundant class, this 10% cDNA fraction would represent about 12 average size mRNA sequences. Thus at least 6000 Friend polysomal poly(A)+ mRNA sequences are common to the EC cell. The additional 1500 sequences present in the EC cell polysomes appear to be lacking in the Friend cell polysomes. Fourth, the results showed that sequences abundant in one cell type can be found at lower frequency in another.
Complexity
of Polysomal
Poly(A)+
RNA in Different
Cell Lines
517
A Large Proportion of Sequence Diversity Is Common A number of interesting features arise from our findings. It is evident that there is a large subset of sequences, at least 6000 (of average size 2000 nucleotides), which is present in the three quite distinct cell types. The findings of other investigators are similar, where divergent cell types are found to exhibit a considerable homology between their messenger RNA populations (Ryffel and McCarthy, 1975; Galau et al., 1976; Axel et al., 1976; Young et al., 1976; Hastie and Bishop, 1976). For example, in comparing the polysomal mRNA sequences present in sea urchin oocytes, various embryonic stages and adult tissues, Galau et al. (1976) found that a group of sequences numbering 1500 was found to be common to all these stages of development and differentiation. It is interesting that in common with Galau and his colleagues, we find that an embryonic cell sharing the same antigenic determinants as the normal mouse cleavage embryo (Art2 et al., 1973; Nicolas et al., 1976) continues to exhibit a large homology with committed cell types. In other studies with mouse tissues, it has been found that of 12,000 sequences present in mouse kidney, lO,OOO-11,000 are shared with mouse brain and liver, tissues exhibiting similar total base-sequence complexities (Hastie and Bishop, 1976). Quantitatively, similar results have been reported for the homology between chick liver and oviduct (Axe1 et al., 1976) and mouse liver, embryo and brain (Young et al., 1976). It is therefore clear that much of the genetic information expressed as mature mRNA is common to different cell types and may be involved in specifying “housekeeping” functions probably concerned with the normal growth and maintenance of cells. Quantitative Variations of Common mRNAs The experiments using Friend low Rot common cDNA (largely representing sequences common with the EC cell drawn from the abundant Friend cell class) demonstrated that abundant sequences in Friend cells are found at lower frequency in the EC cell type, primarily situated in the intermediate frequency class. In addition, Figure 2A suggests that some intermediate EC cell sequences are situated in the rare frequency class of myoblast cells. Other investigators have also reported that common sequences are present at quantitatively different levels in tissues of differing phenotype (Young et al., 1976; Axel et al., 1976; Hastie and Bishop, 1976). Using isolated abundant cDNA of mouse liver and kidney, Hastie and Bishop (1976) showed that certain sequences present in abundance in liver are found in the rare frequency class in kidney, and that some abundant kidney sequences are
found in the intermediate class of brain and liver mRNA. Such alterations in the relative frequencies of common mRNA sequences imply some mechanism of quantitative modulation. This may well reflect differential rates of transcription of these genes in different cell types or a post-transcriptional modulation which appears to intervene between the relative abundance of sequences at the nuclear and cytoplasmic levels (Herman, Williams and Penman, 1976). It is conceivable that such differences in the abundance of common sequences may have a role in determining phenotypic diversity. In general, the slight differences between the kinetics of our homologous and heterologous reactions indicate that the majority of homologous sequences are present at similar concentrations in the three cell types. Specific Polysomal Sequences The extremely small number of sequences found to be specific for the Friend cell (with reference to the EC cell) suggests that in addition to quantitative modulation of common sequences, phenotypic differences may be specified by a small number of abundant sequences. Hastie and Bishop (1976) arrived at similar conclusions, where in mouse brain, liver and kidney, the most abundant class, representing a very small number of sequeces, was found to be characteristic of its tissue. It is interesting to note that reported levels for globin-specific RNA (the major specialized gene product of erythroid tissue) in the polysomes of this clone of Friend cells in the untreated state (Harrison et al., 1975) would put globin mRNA in the abundant class. The absence of detectable globin-specific RNA in EC cells (M. Jacquet, unpublished results) may mean that globin mRNA is represented in the fraction of specific cDNA. Nevertheless, the possibility cannot be ruled out that the specific Friend cell cDNA may partially represent Friend virus-specific RNA, known to be present at fairly high levels in the untreated state (Pragnell et al., 1975). It should also be pointed out that these differences may also reflect alterations in gene expression due to transformation with Friend virus and not necessarily to commitment to erythroid differentiation. In this respect, the myoblast differs markedly, exhibiting a large group of rare sequences not present in the EC cell polysomes. As a fraction of the rare frequency class, the 12% of myoblast cDNA which is apparently specific would correspond to about 4500 sequences. This difference may reflect the relative state of development of the myoblast as compared to the Friend cell. The latter closely resembles the proerythroblast, an identifiable cell type in the terminal stages of erythropoiesis. In terms of developmental maturity, the myoblast may
Cell 518
represent an earlier precursor to the fully differentiated state, possibly requiring more genetic information to specify its function. Indeed, preliminary results show that polysomal base-sequence complexity in terminal cultures of fused myotubes is reduced to values close to the EC cell, almost all their sequences being homologous apart from a small group of abundant sequences (N. A. Affara, unpublished results). Terminal differentiation may perhaps be accompanied by an amplification of a small group of tissue-specific sequences. A cautionary note must be introduced here, however, since it is possible that the extra sequences present in the myoblast may represent aberrant gene expression caused by the abnormal nature of its karyotype (Boon et al., 1974), in contrast to the EC cells which appear to possess a diploid mouse karyotype (Jakob et al., 1973). It remains to be seen whether these apparently specific sequence differences reflect differential gene transcription (as appears to be the case with globin-specific sequences) or post-transcriptional controls resulting in qualitative differences at the polysomal level. It is important to point out, however, that failure to hybridize a fraction of heterologous cDNA may mean that these sequences are present in the driving RNA population, but at very low concentrations. Nevertheless, from the Rot values reached, this would mean that their levels are much lower than the rare frequency class, being present in less than one copy per cell. To demonstrate rigorously the absence of these sequences from an RNA population, it is necessary to purify their corresponding cDNA and hence increase the sensitivity for their detection Gene Number in Mammalian Genomes It can be seen that the total complexity between the three cell types does not exceed >13,000-14,000 sequences, thus representing a small fraction of the mouse genome (0.9% of duplex). It is not known what the total complexity is of mRNA utilized during the life cycle of the mouse; however, these figures are within the limits set on the coding potential of mammalian genomes (40,000-50,000 genes) based on various genetic arguments that a higher gene number would impose an unbearable genetic load, hence prejudicing the survival of a species (Haldane, 1957; Ohno, 1971; Ohta and Kimura, 1971; see review by Bishop, 1974). It therefore seems that commitment to a particular pathway of differentiation is not simply based on the selection of a subset of sequences already present in the EC cell polysomal compartment. Rather, it appears to involve the expression of new gene sequences (whose number can be large or small) in addition to a large population of common mRNAs, and quantitative modulation of certain shared sequences.
Experimental
Procedures
Cell Culture Embryonal carcinoma (strain PCC3) and myoblast cells (clones T233 and T984) were grown in large petri dishes (Falcon) in Oulbecco’s modified Eagle’s medium containing 15% fetal calf serum at 37°C and under 5% CO,. Friend erythroleukemic cells (clone 70717C; a gift from Dr. Paul Harrison at the Beatson Institute, Glasgow) were grown (in plastic Falcon flasks) in HAMS Flz medium containing 15% horse serum as previously described (Conkie et al., 1974). All cells were harvested during exponential growth. Cell lines PCC3 and T233 were derived from OTT6050, a strain 129 mouse teratocarcinoma. The Friend cell from DBA2 splenic tissue infected with Friend virus complex. Preparation of Polysomal RNA IO-15 min before harvesting, emitin (Boehringer) was added to cell cultures at a concentration of 50 rglml. Cells were then pelleted, washed at 4°C in MSB buffer [O.Ol M Tris base (pH 7.5), 0.01 M MgCI,, 0.14 M NaCl and 40 pg/ml of heparin], repelleted and resuspended in 2-3 ml of the same buffer. Nonidet P40 (Shell) was added with mixing to a concentration of 0.5% (v/v), and nuclei were pelleted at 1000 X g for 4 min. After centrifugation at 8000 x g for IO min, the postmitochondrial supernatant was layered onto 1540% isokinetic sucrose gradients prepared in MSB buffer and centrifuged at 40,000 rpm for 40 min at 4°C in the Beckman SW41 rotor. Gradients were collected using the ISCO density gradient fractionator, reading the absorbance profile at 260 nm. Polysomes from disomes upwards were pooled and precipitated with 2 vol of 70% ethanol at -20°C during 4 hr. After centrifugation, the polysome pellet was deproteinized by centrifugation in CsCl (Glisin, Crkvenjakov and Byus, 1974; Young et al., 1976; Affara and Young, 1976). The pellet was dissolved in 5 ml of MSB containing 1% SDS, to which 7 g of CsCl and 5 ~1 of diethylpyrocarbonate per mg of polysomes were added. After dissolution of the CsCl and centrifugation at 7000 rpm (Beckman J13 rotor) at 25°C for 25 min, the aqueous phase containing the RNA was gently removed from beneath the protein/SDS pellicle and filtered through a sterile glass-fiber filter (GF82 Whatman) using a Swinex adaptor. The aqueous phase was then diluted with 4 vol of distilled water, and the RNA was precipitated overnight with 2 vol of 100% ethanol at -20°C. Protein contamination of RNA is 0.26% by this method (Affara and Young, 1976). Preparation of Poly(A)+ RNA Polysomal RNA dissolved in binding buffer [O.Ol M Tris base (pH 7.4) 0.2% SDS and 0.4 M NaCI] was passed over a 1 ml oligo(dT)cellulose column (Collaborative Research), equilibrated with the same buffer. The RNA was passed 3 times at room temperature over the oligo(dT)-cellulose to ensure binding of all poly(A)+ RNA, after which poly(A)RNA was eluted with 10 ml of binding buffer. Poly(A)+ RNA was then eluted from the column with 5 ml of elution buffer [O.Ol M Tris base (pH 7.4), 0.2% SDS] at room temperature and rebound a second time in the presence of binding buffer. After washing away unbound RNA, poly(A)+ RNA was recovered as described above, the RNA concentration was measured by ultraviolet abosrbance at 260 nm (1 OD/ml taken as 40 pg/ml of RNA), and poly(A) content was determined by titration with radioactive poly(U) (Bishop, Rosbash and Evans, 1974; Jacquet et al., 1977) and finally concentrated by precipitation in 0.2 M NaCl with E. coli ribosomal RNA (20 pg/ml) added as carrier. After dissolving in 20 ~1 of distilled H,O, the concentration of poly(A)+ RNA was then determined in the concentrated solution by titration with poly(U) on the basis of the poly(A) content determined above. The poly(A)+ RNA fraction represented 3-4% of the total polysomal RNA and showed a modal size distribution around 16s as ascertained by centrifugation on sucrose/formamide gradients. Synthesis and Fractionation Synthesis of cDNA PCC3, myoblast and Friend
of cDNA cell
polysomal
cDNAs
were
synthe-
Complexity
of Polysomal
Poly(A)+
RNA in Different
Cell Lines
519
sized under the same conditions. The reaction mixture (loo-250 ~1) contained 40 mM Tris-HCI (pH 7.9) at 37°C. 100 mM KCI, 5 mM MgCI,, 10 mM &mercaptoethanol, 0.4 mM each of dATP, dGTP and dTTP, 0.08 mM 3H-dCTP (4000 cpmlmole; Amersham), 200 pg/ml actinomycin D, 0.6 pg oligo(dT)Iz,,, 1-5 pg poly(A)+ RNA and 50 units of reverse transcriptase. The reaction was carried out at 3PC for 45 min. 3H-cDNA was then purified as described previously (Jacquet et al., 1974,1977). The mean size of cDNA on alkaline/sucrose gradients (calculated according to McEwen, 1967) was found to be between 5.5-6S, representing a length of 400-500 deoxynucleotides. This is approximately 20% of the mean size distribution of the poly(A)+ mRNA template.
Measurement of Hybrid Tm Hybridization reactions (0.8 ~1) were permitted to reach completion (one per point to the melting curve) under the hybridization conditions described above. While still sealed in capillaries, the hybrids were heated at the appropriate temperature for 5 min, flash-cooled in an ice/salt mixture and immediately expelled with 200 ~1 of the Si nuclease assay buffer, and the level of Slresistant cDNA was determined as described above. The amount of hybrid resistant to Sl nuclease was then plotted against increasing temperature. The Tm is taken to be the point at which 50% of hybridizable cDNA has been rendered sensitive to Sl nuclease by melting the hybrid structure.
Fractionation of cDNA Myoblast cDNA (4 x lo5 cpm) was homologously hybridized to an 100 fold excess of polysomal poly(A)+ RNA to a Rot value of 7, at which point 50% of the cDNA was in hybrids. Hybridized and unhybridized cDNA were then bound at room temperature to hydroxyapatite (0.5 g) in 0.05 M sodium phosphate (pH 6.8), followed by elution of unhybridized cDNA with 6 x 3 ml washes of 0.16 M sodium phosphate at 60°C. Hybrid was then eluted with 0.4 M sodium phosphate at the same temperature. Both fractions were then treated with 0.3 M NaOH at 37°C for 3 hr. filtered through G50”Sephadex [developed in 10 mM NaCl, 10 mM Tris base (pH 7.5), 1 mM EDTA] and precipitated in 0.2 M NaCl with 2 vol of ethanol at -20°C in the presence of E. coli ribosomal RNA (20 pglml) added as carrier. The pelleted cDNA fractions-enriched, respectively, in abundant and rare sequences-were dissolved in distilled H,O. Friend cell cDNA was fractionated and purified under identical conditions. Three Friend cell cDNA probes were prepared. First, cDNA (7.5 x IO* cpm) was homologously hybridized to a Rot of 1 with a 50 fold excess of Friend polysomal poly(A)+ RNA. Hybridized (50%) and unhybridized cDNA were then separated on hydroxyapatite. Unhybridized cDNA formed the high Rot cDNA fraction enriched in rare sequences. Second, hybridized cDNA from above was recovered (as described above) and heterologously hybridized with an 100 fold excess of PCC3 polysomal poly(A)+ RNA to a Rot value of 1. Hybridized cDNA (45% of cDNA called low Rot common) and unhybridized cDNA (55% of cDNA called Friend low Rot specific) were once again separated on hydroxyapatite and purified as described above.
Numerical Analysis of Multicomponent Hybridization The hybridization reaction of a mixture of different RNA species present at different relative concentrations can be described as the sum of each individual reaction. For n components, the concentration of DNA or RNA in hybrid (H) as a function of Rot will be:
Conditions for RNA/DNA Hybridization and DNAIDNA Reassociation RNA/DNA hybridization and DNA/DNA reassociation were performed under the same ionic conditions.The reaction mixture contained 0.1 M Tris base (pH 8.0) at 37°C (pH drops to 7.2 at 7O”C), 0.3 M NaCl and 1 mM EDTA. The driver component excess for RNA/DNA hybridization was >1700 fold. The concentration of the driver and the time of incubation were adjusted to obtain the required Rot values. Reactions were carried out in sealed, siliconized capillaries containing between 0.8-5 ~1 vol (measured using Hamilton micro-syringes), and incubation was at 70°C after heating at 100°C for 3 min. Reactions were stopped by plunging the capillaries into an ice/salt mixture followed by immediate expulsion of the reaction mixture with 200 ~1 of the buffer for Sl nuclease digestion. RNA/DNA Hybrid Analysis The amount of RNA/cDNA hybrid structure was determined by resistance to single-strand-specific Sl nuclease. Sl nuclease from Aspergillus orizae was purified from oramylase powder (Sigma) by chromatograpy on DEAE-cellulose and filtration through Sephadex GlOO as described by Vogt (1973). The expelled reaction was split into two 100 ~1 aliquots. 36 units of Sl nuclease were added to one aliquot, while the other was precipitated with 5% TCA. Reaction conditions and measurement of TCA-precipitable material were as previously described (Jacquet et al., 1974, 1977).
H = 2 Hi = c qDo(1 1-D 1-n
- e-KiRot);
or if 4 = 1 (~yi is the fraction
of cDNA
(1) in the ith component),
H - = 1 - IZ (aie-KiRot), Do l--n
we get:
(2)
where Do and Ro are the initial DNA and RNA concentations, H/ Do the fraction of DNA in hybrid structure, Ki the apparent firstorder rate constant of the ith component and t the time in seconds. Provided that the complementary DNA probe reflects quantitatively the frequency components in the RNA population, then Ki for a given component can be related to the base-sequence complexity of the component by the factor 01,, the fraction of the total RNA comprising this component, and the ratio of Ki of different components gives the relative frequency of the RNA sequences in these components. Thus by defining Ki and a, for each component in an heterogeneous RNA population, it is possible to estimate the base-sequence complexity and frequency distributions of the RNA sequences present. In this paper, we have used a linear transformation of the curves which allows a direct determination of oli and Ki. A single-component first-order reaction can be linearized by plotting -Ln [l - (H/ Do)] as a function of Rot. The following equation can be derived from equation (2): Y=-Ln
(
I-&
>
=KRot
When plotted in the same way {-Ln [I - (H/Do)] versus rnulticomponent reaction gives a multiphasic curve which described by the following equation: Y=-Ln = -Ln
1-2 i 1 (Lyie-KiROt +
But at high components
Rot}, a can be
+ c+emKn -,‘)
Rot values (>LnP/Kn), the contribution of the n - 1 is neglectable, and the equation becomes:
Y = Kn Rot - Ln an.
(5)
Kn is therefore given by the slope of the asymptote at high Rot and with the Y axis. Thus having a” = emYo, where Yo is the intercept defined both 01and K for the last component, its contribution to H (the amount of hybrid structure) can be substraced. H/Do is then normalized for n - 1 components, and the (n - 1)‘” component is defined in a similar way to the nth component. By this regressive procedure, (Y and K can be determined for each component in the RNA population. It should be noticed from equation (4) that at low Rot values (
