VOL. 18, NO. 13, 1979
LATE EMBRYONIC HISTONE MRNAS
de Haas, G. H., Bonsen, P. P. M., Pieterson, W. A,, & van Deenen, L. L. M. (1971) Biochim. Biophys. Acta 239, 252-266. Klotz, I. M., & Hunston, D. L. (1971) Biochemistry 10, 3065-3069. Nieuwenhuizen, W., Kunze, H., & de Haas, G. H. (1974) Methods Enzymol. 32B, 147-154. Pattus, F., Slotboom, A. J., & de Haas, G. H. (1979a) Biochemistry (first paper of three in this issue). Pattus, F., Slotboom, A. J., & de Haas, G. H. (1979b) Biochemistry (second paper of three in this issue). Pieterson, W. A,, Vidal, J. C., Volwerk, J. J., & de Haas, G. H. (1974) Biochemistry 13, 1455-1460. Rietsch, J., Pattus, F., Desnuelle, P., & Verger, R. (1977) J . Biol. Chem. 252, 43 13-43 18. Slotboom, A. J., & de Haas, G. H. (1975) Biochemistry 14, 5394-5399. Slotboom, A. J., van Dam-Mieras, M. C. E., & de Haas, G. H. (1977) J . Biol. Chem. 252, 2948-2951.
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Slotboom, A. J., Jansen, E. H. J. M., Vlijm, H., Pattus, F., Soares de Araujo, P., & de Haas, G. H. (1978a) Biochemistry 17, 4593-4600. Slotboom, A. J., Verhey, H. M., Puijk, W. C., Dedieu, A. G. R., & de Haas, G. H. (1978b) FEBS Lett. 92, 361-364. Slotboom, A. J., Jansen, E. H. J . M., Pattus, F., & de Haas, G. H. ( 1 9 7 8 ~ )in Semisynthetic Peptides and Proteins (Offord, R. E., & DiBello, C., Eds.) pp 315-348, Academic Press, London. van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A+,& de Haas, G. H. (1975) Biochemistry 14,5387-5394. van Wezel, F. M., Slotboom, A. J., & de Haas, G. H. (1976) Biochim. Biophys. Acta 452, 101-1 11. Verger, R., & de Haas, G. H. (1973) Chem. Phys. Lipids 10, 127-1 36. Verger, R., & de Haas, G. H. (1976) Annu. Rev. Biophys. Bioeng. 5, 77-1 17. Verger, R., Mieras, M. C. E., & de Haas, G. H. (1973) J . Biol. Chem. 248, 4023-4034.
Histone Gene Switch in the Sea Urchin Embryo. Identification of Late Embryonic Histone Messenger Ribonucleic Acids and the Control of Their Synthesist Philip A. Hieter, Marvin B. Hendricks, Kari Hemminki, and Eric S . Weinberg*
ABSTRACT: During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these units. Four bands in the early mRNA sample are identified as H1, H3, H2A + H2B, and H4 m R N A while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both
H 3 and H2A mRNA. When a polysomal R N A preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A H 3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal R N A present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic R N A from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.
E o widely diverged classes of histone mRNA, each coding for all five histone types, are synthesized at particular times during embryogenesis in the sea urchin Strongylocentrotus purpuratus (Kunkel & Weinberg, 1978). In this organism, one class of histone gene transcripts (“early” RNA) is synthesized during cleavage and blastula stages. Another class (“late” R N A ) begins to be made during blastulation and represents the vast bulk of the histone mRNA synthesized at
the subsequent mesenchyme blastula. The two mRNA classes have been distinguished by properties of hybrids formed between cloned S . purpuratus histone DNA and each of the two transcript populations (Kunkel & Weinberg, 1978) and by the difference in histone subtypes which result from in vitro translation of mRNAs obtained from the two stages (Weinberg et al., 1977; Newrock et al., 1978). The sequences of the early mRNAs are very similar, if not identical, to the vast bulk of the several hundred histone gene repeats in the S.purpuratus genome (Kunkel & Weinberg, 1978). These genes consist of clustered 6-7 kilobase units, each of which contains genes coding for the five histone classes (see Figure 4) (Kedes et al., 1975; Weinberg et al., 1975; Cohn et al., 1976; Wu et al., 1976; Holmes et al., 1977; Overton & Weinberg, 1978). The late
From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. Received December 12, 1978. This investigation was supported by Grant GM22155 from the National Institutes of Health. P.A.H. and M.B.H. were supported by National Institutes of Health training grants and K.H. was a holder of a National Institutes of Health Fogarty International Fellowship.
+
0006-2960/79/0418-2707$01 .OO/O 0 1979 American Chemical Society
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BIOCHEMISTRY
histone mRNAs, on the other hand, are thought to be encoded in rare genes which may have a completely different organization (Kunkel & Weinberg, 1978; Overton & Weinberg, 1978). The shift in the form of histone mRNA synthesized in the embryo is correlated with the transition in the type of H2A, H2B, and H1 histones (Ruderman et al., 1974; Cohen et al., 1975; Newrock et al., 1977) which are made toward the end of the blastula stage. The synthesis of a single form of each of these histones is replaced by the synthesis of two or more other subtypes. The shift in the synthesis of the particular subtypes is a direct result of the replacement of one class of histone mRNA in the cell by another (Newrock et al., 1978). Although the early and late embryonic H 4 or H 3 histones cannot be distinguished by electrophoresis (Newrock et al., 1978), the early and late mRNAs coding for these histones are considerably diverged in sequence as are the other histone mRNAs (Kunkel & Weinberg, 1978). On the basis of hybrid stability studies, the late and early histone genes of S . purpuratus are found to be considerably more diverged from one another than are the early histone gene classes of two distantly related sea urchins, S. purpuratus and Lytechinus pictus (Kunkel & Weinberg, 1978). In L. pictus, the early and late histone mRNAs have recently been found to be different in size, and a divergence in sequence has been shown for the H4 m R N A by fingerprinting studies (Grunstein, 1978). Here we show that the newly synthesized late histone mRNAs in S.purpuratus differ considerably in electrophoretic mobility from the early mRNAs, and the various late R N A species are identified as mRNAs for particular histones. The mRNAs which hybridize to the histone DNA are shown to specifically translate particular histones in vitro. Finally, the accumulation of the late histone m R N A in the cell is shown to begin well before the mesenchyme blastula stage. Methods and Materials Preparation of RNA. Eggs were collected from S . purpuratus (Pacific Biomarine Supply), fertilized, and cultured at 17 “ C until the specified stage. Polysomal R N A was prepared from polysomal pellets and the R N A extracted as previously described (Weinberg & Overton, 1978). All glassware was soaked in 0.2% diethyl pyrocarbonate (DEP) and thoroughly rinsed with distilled water. The R N A was dissolved in 10 mM Tris-HC1, pH 7.5, and 0.5% sodium dodecyl sulfate (NaDodSO,), briefly heated to 60 “C, and fractionated on 15-30% sucrose gradients containing 10 mM Tris, pH 7.5, and 0.5% NaDodS04. A “9s” pool (7-12 S ) was taken and precipitated in 0.3 M sodium acetate and ethanol. The R N A pellet was dried under N 2 and stored in 0.1 mL of DEP treated sterile H 2 0 at -20 “C. Total cytoplasmic RNAs were prepared essentially as described by Ruderman & Pardue (1977). At the desired stage, embryos were washed twice with ice-cold artificial sea water and once with T N M (0.2 M NaCl, 5 mM Mg(OAc)2, and 20 mM Tris, pH 7.6) and then homogenized in 10 volumes of T N M containing 0.25% DEP in a Dounce homogenizer. The homogenate was centrifuged at 12OOOg for 15 min, and the supernatant was collected and precipitated with 2.0 volumes of absolute ethanol at -20 “C. The pellet was dissolved in 10 mM Tris-HC1, pH 7.5, and 0.5% NaDodS04 and extracted once with 1 volume of phenol and twice with 1 volume of chloroform-isoamyl alcohol (24: 1). Labeled R N A was prepared by culturing embryos in either 50 pCi/mL t3H]uridine (25-30 Ci/mmol; Amersham) or 100 pCi/mL [32P]orthophosphate(carrier free; ICN). Label was added at 7 h and the embryos were harvested at 9 h for late
HIETER ET A L .
cleavage stage preparations or was added at 24 h and the embryos were harvested at 26 h for mesenchyme blastula stage preparations. Electrophoresis of RNA. The 9 s histone RNA preparations were dissolved in electrophoresis buffer (50 mM Tris-borate, pH 8.3, and 1 mM Na2EDTA) containing 0.05% bromphenol blue and 7 M urea. This solution was briefly heated to 60 “ C before loading onto a 7.5% polyacrylamide slab gel (0.03 X 14 X 28 cm) containing 7 M urea. Electrophoresis was carried out at 300 V for 36-40 h. The locations of 32P-labeledR N A bands were determined by exposing the gel slab to Kodak XR-5 film at -20 “C. Densitometry was performed by scanning the autoradiographs at 546 nm by using a dual-beam scanner (Instrumentation Specialties Co.) linked to an Isco Model UA-5 absorbance monitor and a Model 1133 multiplexer expander. The locations of 3H-labeled RNA bands were determined by cutting a strip of gel lengthwise and slicing it into 1.18-mm sections. These slices were incubated in scintillation vials in 1 mL of 1 N N a O H at 37 “ C for 2 h, neutralized with 0.5 mL of 2 N HC1 containing 20 mM Tris-HC1, pH 7, and counted directly in scintillation fluor. For elution of R N A from the gel, a lane was sliced at 1.18-mm intervals, and appropriate slices were pooled into separate scintillation vials (for example, in Figure 3, 50 “pools” were made across a region that includes 83 slices). The gel slices were frozen, thawed, and macerated with a glass rod several times. RNA was eluted by incubating the macerated gel pieces for 2 h at 60 “ C in 1 mL of a sterile solution containing 0.5% NaDodSOl and 10 mM Tris-HC1, pH 7.5. The eluant was cleared of gel debris by centrifugation at 15OOOg for 10 min into a phenol pad, and the R N A precipitated from 2 volumes of ethanol and 0.1 volume of 3 M sodium acetate in the presence of 0.5 mg of Escherichia coli tRNA (Sigma; phenol-extracted). Recoveries of R N A from the gel by this method were around 90%. Preparation of DNA Hybridization Probes. Plasmid DNAs p C 0 2 , pSR1, pRC9, pRC39 were prepared as described previously (Overton & Weinberg, 1978) or by the acid-phenol extraction method of Zasloff et al. (1978). The plasmid pC02 consists of a complete histone repeat unit inserted at the Hind 111 site of pBR313 (Overton & Weinberg, 1978). The recombinant pSRl has the EcoRI fragment which contains the H 3 and H2A genes inserted at the EcoRI site of pMB9 (Overton & Weinberg, 1978). The plasmids pRC9 and pRC39 are subcloned fragments prepared by Cohn et al. (1976) and contain respectively a part of the H2A gene and some adjacent spacer inserted into pML21 and the complete H2B gene with adjacent spacers inserted into pGM15. Plasmid DNA was digested with the EcoRI restriction enzyme and, after denaturation, loaded onto Millipore nitrocellulose filter disks ( H A W P 0.45 pm) (Gillespie & Spiegelman, 1965) at 5 pg/filter. Digestion of 400 pg of pC02 D N A was performed with EcoRI and HhaI simultaneously in 50 mM NaCl, 6 mM Tris-HC1, pH 7.4, 6 mM MgC12, 5 mM 2-mercaptoethanol, and 100 pg/mL bovine serum albumin at 37 “C overnight. The resulting DNA fragments were electrophoresed on a 1.2% agarose slab gel for 17 h at 2 V/cm. The bands were visualized by staining in 1 pg/mL ethidium bromide (Sharp et al., 1973), and bands containing the histone genes (Overton & Weinberg, 1978) were excised from the slab. Each piece was macerated by extrusion through an 18-gauge syringe needle several times. DNA was eluted by incubating the gel material for 2 h in a solution containing 10 mM Tris-HC1, pH 8.0, and 1 mM EDTA. Gel debris was removed by centri-
VOL. 18, NO.
LATE EMBRYONIC HISTONE MRNAS
fugation at l5ooOg for 30 min into a phenol pad, and the DNA was precipitated from the eluant by the addition of 0.1 volume of 3 M sodium acetate and 2 volumes of ethanol. Recoveries of DNA from the gel slab were in the range of 3&40%. The D N A restriction fragments (A-E) corresponding to specific histone genes were loaded onto nitrocellulose filter disks at 0.5 pg/filter in the presence of 4.5 p g of E . coli DNA. Hybridization Reactions. Hybridization to the various histone D N A filter probes was performed as previously described (Kunkel & Weinberg, 1978). All solutions and vials were pretreated with 0.1% DEP. Various filters were stacked in a hybridization vessel containing labeled RNA, and the hybridization was generally carried out overnight in 3 X SSC and 0.1% NaDodSO, in Denbardt's buffer (1966) a t 62 "C. Filters were washed together with 3 X SSC and 0.1% NaDodSO. twice a t 55 "C and once at room temperature. The filters were dried and counted in a scintillation counter. In the case of preparative hybridization of 9s RNA to DNA filters, hybridization was performed in 50% formamide, 0.5 M NaCI, 0.2% NaDodSO,, 1 mM EDTA, and 40 mM Pipes, p H 6.5 (Casey & Davidson, 1978). The R N A sample was heated to 50 "C before hybridization. After 24-48 h at 37 "C, the filters were washed in two changes of 4 X SSC and 40% formamide at room temperature. Hybridized R N A was eluted in 99.8% formamide and 0.2% NaDodSO, at 40 "C for IO min and precipitated from 2 volumes of ethanol. Samples which were then to be electrophoresed were precipitated in 2 volumes of ethanol and 0.1 volume of 3 M sodium acetate, after adding 5 p g of phenol-extracted E. coli t R N A (Sigma). Samples prepared for translation were precipitated with 25 p g of E . coli 5s r R N A (Boehringer-Mannheim). Melting Profiles. Filters from hybridization reactions were recovered as previously described (Kunkel & Weinberg, 1978). Filters were incubated in I-mL aliquots of 0.1 X SSC added for 5 min and removed, at a series of increasing temperatures. The aliquots were counted directly in Triton X-toluene fluor. Cell-Free Translation of RNA. The wheat germ cell-free system was used for translation as previously described (Newrock et al., 1978). Either total cytoplasmic RNA, prepared from embryos at different stages, or polysomal RNA, hybridized to and eluted from histone DNA, was added as the template. A quantity of 5-7 p g of total cytoplasmic R N A or half (5 pL) of each filter-eluted sample was used in the translation reactions. The RNAs were washed once with 70% ethanol and once with absolute ethanol, dried under N z gas, resuspended in IO p L of distilled water, and stored at -70 OC until use. Incubations were carried out for 150 min at 25 "C with 5 pCi of [3H]leucine (105 C i / m M Amersham/Searle). After incubation, 3 pL of @-mercaptoethanol was added to each reaction tube which was then frozen at -20 "C until use. Triton X-100 Gel Electrophoresis. Electrophoresis in gels containing 0.9 M acetic acid, 6 mM Triton X-100, and 8.5 M urea was performed as previously described (Alfageme et al., 1974; Newrock et al., 1978). After the cysteamine and protamine scavenging steps, the samples (20 pL) were loaded onto a slab gel (30 X 18 X 0.15 cm) and electrophoresed for 19 h at 200 V. Immediately after electrophoresis, the gel was prepared for fluorography (Laskey & Mills, 1975). The samples from the wheat germ protein synthesis reaction were prepared as previously described (Newrock et al., 1978) with the following modification for translation of samples of filter-eluted RNA. After the centrifugation and washing steps, the proteins were extracted from the pellets with 20 pL of a solution containing 1 M urea, 5% mercaptoethanol, 5% sucrose, 5% acetic acid, and 4 pg/FL protamine (Sigma; Grade I). The
E L
IJ,
1979
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LH E
FIOURE I: Gel electrophoretic patterns of late cleavage (early) and mesenchyme blastula (late) polysomal 9s RNA. S. purpuratus embryos were labeled from 7-9 h (early) or 24-26 h (late) with ["P]orthophosphate, and a 7-12s polysomal fraction was isolated as described under Methods and Materials. Electrophoresis was performed on 7 M urea-7.5% polyacrylamide gels and the gel was then autoradiographed. Some material in the late samples did not enter the gel as is seen by the band at the origin at the top of the figure. Left: E and L are 7-12s polysomal RNA preparations labeled at the early and late stages, respectively. Right: another gel, in which the RNAs migrated more slowly, comparing unselected early pclysomal RNA (E) to late 7-12s polysomal RNA (L,) which was preparatively hybridized to pC02 DNA in 5wb formamide and 0.5 M NaCl and eluted before being electrophoresed.
samples were again centrifuged (IO000rpm for IO min), and the supernatants containing the histones were collected and used directly for electrophoresis. Results Cha8tge in Histone mRNA Electrophoretic Patterns. The early and late histone mRNA species can be shown to differ considerably in electrophoretic mobility on polyacrylamide gels. Polysomal R N A from embryos labeled with [JzP]orthophosphate at 7-9 or 24-26 h after fertilization was prepared by isolating a 7-12? fraction from sucrose gradients. The R N A was then electrophoresed on 7.5% polyacrylamide gels and subsequently analyzed by autoradiography. The patterns shown in Figure 1 compare the radioactive RNAs in the early and late R N A preparations. The slots marked E contain the 7-9-h sample and, as expected, contain major R N A species which correspond to previously studied histone mRNAs. The polysomal 24-26-h sample (L) also has prominent bands, all with different mobility from the early mRNAs. Since it had been shown that at least 0.5% of the newly made R N A in S . purpuratus mesenchyme blastula is histone transcript (Kunkel & Weinberg, 1978), the major R N A species resolved in lane L were candidates for the late histone mRNAs. The amount of label in these bands relative to the substantial background
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BIOCHEMISTRY
HIETER ET A L .
8 I
I
I
9 I
B
FIGURE2: Densitometric tracings of mesenchyme blastula (late) histone m R N A preparations prior to and after purification by preparative hybridization to pC02 DNA. Densitometry was performed on autoradiographs of lanes L and LH from Figure 1. Tracings are aligned with respect to the H4 mRNA peaks. For the determination of peak ratios, base lines were set by fitting smooth curves to the background. (A) Late R N A prior to purification by preparative hybridization. (B) Late RNA preparatively hybridized by pC02 DNA.
varied from preparation to preparation, the sample shown here being one of those richest in the visible bands. The background, even though we used a 7-12s fraction, is quite high. To find out whether the prominent bands were histone mRNAs, the polysomal R N A was first hybridized to D N A of the histone plasmid pC02 (a histone recombinant containing all 5 histone genes) and then eluted and electrophoresed in a similar gel. As is shown to the right in Figure 1, in lane LH, virtually all the major bands seen in lane L are purified by the hybridization procedure and the background becomes essentially nil. The major bands are therefore histone mRNAs. Newly synthesized early histone mRNA types are not present in the late preparation. There is evidently a complete shift in the form of newly synthesized mRNAs which are in polysomes in the 24-26-h embryo. The difference between the unhybridized and hybridized late R N A samples is seen dramatically in Figure 2 which compares densitometric tracings of lanes L and LH from the gels presented in Figure 1. It is clear that the purification of the late m R N A species by the preparative hybridization procedure is quite effective. At least 10 distinct bands, some
of which may contain multiple species, can be visualized from the late histone mRNAs (Figures 1 and 2B). This pattern is considerably more complex than the early histone m R N A pattern which contains only four major bands (Figure 1). This may be due to the fact that there are more different m R N A species needed to code for the increased number of subtypes in the late embryo. The relative efficiency of hybridization of the various bands may be estimated by comparing the densitometric profiles. The ratios of the area of each peak in the hybridized sample as compared to the corresponding peak in the unhybridized sample were 2.45, 3.33, 3.61, 4.18, and 3.01 for species 1, 2-4, 5-7, 8, and 9, respectively. The efficiency of hybridization of the various species is quite similar. A further indication of the difference between early and late histone mRNAs was demonstrated by electrophoresing early and late polysomal R N A preparations, each labeled with a different isotope, together in one slot. The gel was sliced, and R N A was eluted and then hybridized to pC02 DNA. In the top panel of Figure 3, it is shown that the locations of the early and late mRNAs do not coincide. Furthermore, the presence of all of the histone transcripts and their relative positions are the same as in the comparison made in Figure 1. Identification of the Late Histone mRNAs. Specific restriction enzyme-produced fragments of the cloned complete repeat (e.g., pC02) or D N A from cloned segments of the repeat can be used to probe for each m R N A type (Kunkel & Weinberg, 1978). For this purpose, we used recombinant DNA containing specifically the H 2 A and H 3 genes [pSRl : Overton & Weinberg (1978)], the H2B gene [pRC39: Cohn et al. (1976)], and the H2A gene [pRC9: Cohn et al. (1976)l. The various DNA probes are illustrated in Figure 4. The late and early mRNA fractions which specifically hybridize to the cloned DNA segments are indicated in Figure 3. In the case of the early R N A fraction, the H2A, H3, and H2B RNAs are limited to the middle two bands (2’ and 3’), the most prominent faster running one (3’) coding for H2A and H2B. By difference in hybridization with p S R l and pRC9, the slower running band (2’) appears to be H 3 mRNA, and this is confirmed by hybridization with a more specific H 3 probe (see below). The use of other specific probes (see below) was also necessary to identify bands 1 and 4 as H 1 and H 4 mRNAs, respectively. The late mRNA pattern is more complex and a t least 10 bands are shown to hybridize to pC02 D N A (Figures 1 and 3). The late mRNA bands 2-7 appear to contain H 3 and H2A sequences as shown by the hybridization with pSRl DNA. Hybridization with pRC9 DNA is weak, but indicates bands 2-4 as putative late H 2 A mRNAs. By implication, bands 5-7 therefore may contain H 3 mRNAs. It should be noted that the profile for the hybridization to the pSRl probe is not coincident with the profile obtained with the pC02 probe. This suggests that species other than H 3 and H 2 A m R N A s may be present in this region. Band 8 is clearly H2B mRNA, and there is also some hybridization to the H2B-specific pRC39 D N A in bands 2-5. Further indications of the mRNA identities are derived from restriction enzyme-produced fragments of pC02. There are eight histone DNA fragments which result from the digestion of this plasmid D N A with HhaI and EcoRI (Overton & Weinberg, 1978). The five largest fragments, A-E, diagramed in Figure 4 and a t the top of Figure 5, are specific for individual genes: fragment A contains only the H 1 gene; B contains only the H2B; C only the gene for H4; D is mostly H 2 A specific but also has a bit of H 3 gene; E has solely H 3 sequence. These fragments were prepared by electrophoresis
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VOL. 18, NO. 13, 1979 I
0
2
3
5
4
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50
60
70
80
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FIGURE 4: Restriction enzyme map of S.purpuratus histone genes. The uppermost map diagrams the repeating unit in native DNA. The lower maps indicate the genes and restriction enzyme sites in the plasmids used for hybridization. Solid bars indicate the position of coding regions. Diagonally marked bars represent the vector DNA.
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FIGURE 3: Hybridization of different DNA plasmid probes to late and early RNA fractionated by gel electro horesis. A mixture of 7-1 2s polysomal RNA from early embryos (PH-labeled; 400 000 cpm; 0) and from late embryos (32P-labeled; 14 X IO6 cpm, 0 ) was coelectrophoresed in a single lane on a 7.5% polyacrylamide gel as described under Methods and Materials. Autoradiography of the gel slab yielded a pattern as shown in lane L of Figure 1. A gel piece corresponding to the region of mRNA bands was sliced into 0.1 18-cm sections (84 sections in all) and pooled into 50 vials as indicated by the brackets (single gel slices were used when no brackets are shown). RNA was eluted and hybridized to the four plasmid DNA filter probes in 3 X SSC and 0.1% NaDodSO, in Denhardt's buffer at 62 O C overnight.
of the enzyme-digested plasmid D N A , and each D N A fragment was hybridized to the four early R N A fractions. Table I indicates the results of hybridization of early mRNA gel fractions to HhaI and HhaI + EcoRI fragments of pC02. Bands l', 2', 3', and 4' are unambiguously identified as H1, H3, H2A H2B, and H4, respectively. The results also demonstrate the high degree of specificity provided by the H h a I RI fragments to individual mRNAs. For example, the addition of EcoRI to the H h a I digestion of pC02 renders the A fragment absolutely HI specific. It is also evident that the efficiency of hybridization to the filter containing the D and E fragments is considerably lower than the efficiencies for fragments A, B, and C.
+
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FIGURE 5: Identification of histone mRNAs by hybridization of gel-purified fractions to gene-specific DNA restriction enzyme fragments. The same double-labeled RNA samples shown in Figure 3 were pooled somewhat differently into 11 fractions, as indicated by the brackets, and hybridized as in Figure 3 to nitrocellulose filters containing fragments A-C derived from HhaI + EcoRI digestion of pC02 DNA. The location of each fragment with respect to the total repeat as found in pC02 is diagramed in the insert. Panel A shows H1-specific hybridization, panel B is specific for H2B, and panel C is the probe for H4 mRNA. (0)3H-labeled early RNA; (m) 32P-labeledlate RNA. The insert shows a map of pC02 DNA. Regions coding for mRNAs are shown as solid blocks, and the spacer regions are thin lines. Sites of EcoRI ($)and HhaI (7) digestion are identified.
These restriction enzyme-produced fragments were then used to probe 11 pools of the mRNA species displayed in the gel shown in Figure 3. The results illustrated in Figure 5 confirm the identification of early and late H2B m R N A s described previously and identify the early H 1 and H4 mRNAs as bands 1' and 4' and the late H 1 and H 4 mRNAs as bands 1 and 9 and 10, respectively. As indicated above, the hybridizations with the various fragments is absolutely specific for these R N A fractions. Figure 6 further identifies the late mRNAs by using the restriction fragment probes to analyze the 10 late m R N A species (Figure 1) obtained by preparative hybridization, followed by gel electrophoresis. The results for H1, H2B, and H 4 (A, B, and C ) are the same as those in Figure 5, but also shown are results using the H 2 A and H 3 specific probes. The identification of bands 4-6 as H 3 m R N A agrees fairly well with the results derived from comparison of the hybridization of pSRl D N A and pRC9
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H I E T E R ET AL.
BIOCHEMISTRY
Table I: Identification of Early Histone mRNAsa cpm hybridized with specific gel fractions or cpm input
-
expt
probe
specificity
1'
2'
3'
4'
I
pc02 pSRl pRC9 pRC39
all H3 t H2A H2A-3' H2B input
518 39 21 39 4356
1047 48 1 33 80 4824
1982 469 175 349 5112
197 27 17 21 2808
I1
HhaI A B C+D E
H1 + H2A-3' H2B H4 t H2A-5' t H3-3' H3 input
98 4 7 7 3388
8 9 20 77 3752
75 158 50 20 3917
5 11 273 7 2184
I11
HhaI
H1 H2B H4 H2A-5' t H3-3' H3 inout
169 20 15 6 2 4636
4 5 0 17 59 437 1
3 106 7 35 2 4142
2 27 301 4 2 4322
+ EcoRI A B
c
D E
a RNA fractions were eluted from two gel slices containing the RNA species 1'-4' as in Figure 3 and hybridized to DNA immobilized on filters. The inputs listed were the counts per minute recovered from the gel and added to the hybridization vial. In each experiment, a particular RNA fraction was hybridized with a stack of filters containing the particular DNAs. Plasmid DNAs were present at 5 pglfilter, whereas restriction enzyme fragments were added at 0.5 pg/filter.
D N A in Figure 3. A surprising result was the lack of hybridization with fragment D in the light of the weak, but specific, hybridization shown with pRC9 DNA. As can be seen in Table I, however, the D fragment was an extremely inefficient probe for the early m R N A as well. The case of H2A m R N A is further discussed below. The conclusions reached from these experiments lead to the identification of the early and late mRNAs assigned in Figure 1. The identity of late H1, H2B, and H 4 mRNAs as bands 1, 8, and 9 and 10 is absolutely clear. Bands 4-6 clearly contain H 3 mRNAs. The identification of H2A mRNAs among bands 2-5 is reasonable but not completely proven. There may also be additional H2B mRNAs in bands 2-5 as shown by hybridization to pRC39 DNA. It is clear that the R N A species in the 2-7 region hybridize much more efficiently with the complete repeat DNA of pC02 or with the DNA of pSRl than with the smaller HhaI + EcoRI fragments D and E or the H2A subclone pRC9. It is not excluded, in any case, that a band may contain more than one species, especially for the bands 4 and 5 which might contain H2A and H3 mRNAs. Translation of Prehybridized RNA. The labeled R N A species identified above as particular late histone mRNAs are almost certainly the mRNAs for the late histone subtypes identified in various electrophoretic systems (Cohen et al., 1975; Weinberg et al., 1977; Newrock et al., 1977, 1978). The timing of appearance and disappearance of the two m R N A classes is found to be the same by translational and electrophoretic criteria. We show here that the R N A species isolated from 26-h embryos which hybridize to the early-type histone gene DNA do indeed code for the late histone subtypes. The specific identifications determined by monitoring the hybridization of the labeled R N A as described above also can be made by assaying the translational activity of hybridized RNA. A preparation of unlabeled 7-12s polysomal R N A from 26-h embryos (a time when little detectable early m R N A remains in the cell; see below) was hybridized to pC02, pSRl, and pRC9 DNA. In Figure 7 we present the Triton X-urea gel of translational products which are made when R N A eluted from each of these DNAs is used as a template in a wheat germ protein synthesis system. The late and early histone subtypes are easily differentiated on the Triton X-urea gel when unhybridized late and egg RNAs are used as templates
501
25L. 3 5 7
FRACTION FIGLRE 6: Identification of late R N A species by preparative hybridization and electrophoresis. Late hybridized and eluted 32P-labeled R N A species (the bands 1-10 shown in Figure 1, LH)were carved from the gel, eluted, and hybridized as in Figure 3 to restriction fragments A-E derived from an EcoRI HhaI digestion of pC02 D N A (see Figure 4 for the map). The spacings between the bars of the histogram are those of the gel shown in Figure 1.
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(lanes e and f). When the late R N A eluted from pC02 DNA was translated, all five m R N A classes were synthesized (lane b). The R N A eluted from the H 3 and H2A gene containing pSRl DNA translates only H 3 and H2A histones (lane c) and that from pRC9 D N A translates only H2A histones, mainly the y form (lane d). There is therefore a direct correspondence between the labeled species which hybridize specifically to particular probes and the translational activity attributed to
LATE EMBRYONIC HISTONE MRNAS
V O L . 18, N O . 1 3 , 1979
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m o w 7 Translation products of plasmid-hybridized RNA, displayed on a Trlton X-urea gel. Unlabeled 26-h 9s mRNA was hybridized to a stack of filters containing either pCOZ, pSRI, or pRC9 DNA. Hybridization was done at 37 "C, overnight, in 50% formamide, 0.6 M NaCI, 0.2% NaDodSO,, 1 mM EDTA, and 40 mM Pipes, pH 6.5. Melting of the RNA from the filters, translation in the wheat germ system, and electrophoresis on the Triton X-urea gel have been described under Methods and Materials. Additions to the wheat germ mixture were (a) no addition (endogenous control), (b) pC02-hybridized RNA, (c) pSR1-hybridized RNA, (d) pRC9-hybridized RNA, (e) 0.5 pg of unhybridized 26-h (late) polysomal RNA and (f) 0.5 pg of unhybridired total egg RNA. those RNAs. The case of H2A histone mRNA is particularly interesting since the results indicate that pRC9 is a specific probe for at least one late H2A histone mRNA. This reinforces the assignments made above of specifichands as H2A mRNAs. We will explain under Discussion possible reasons for the low level of hybridization of H2A mRNAs to pRC9 and the DHhI fragment. The band with fastest mobility on the gel, not previously identified as a histone, appears to be coded for by an R N A with the specificity of a histone mRNA. Time of Appearance of Late "As. In order to determine when the late histone mRNAs appear and when the early mRNAs disappear in the cell, we extracted total cytoplasmic R N A from embryos at different developmental stages and used it as templates in the wheat germ protein synthesis system. In Figure 8, a Triton X-urea gel of these translation products is shown. As expected, there is a replacement of the early H2A, HZB, and H 1 mRNAs in the cell by the late varieties as development proceeds (Newrock et al., 1978). The 21-h sample includes all early and late histone species. In addition to the histones identified in the legend and the fastest band mentioned above, a number of other polypeptides are synthesized. Starting at the top, there are a number of species above the H2A6, two prominent hands between the H2A and the H 1 region, another band just above the H1 proteins, and several proteins which have greater mobility than the H 4 histone. Most of these species are made on mRNAs which are not complementary to the histone DNA, the main ex-
HGURB 8: Translation products using total cytoplasmic R N A from different embryonic stages as a template in an in vitro wheat germ >)stem. Total cytoplasmic RNA irol3lcd and prcparcd for trawlation ac described under Methods and Matcrisls at 5 pg/50 pL of reaction volume uas used to prime a wheat germ system. Translation produn0 were prepared for electrophoresis and separated on a Triton X-urea gel and fluorographed as described under Methods and Materials.
ception being the fastest species which was mentioned in the previous section. Some of these proteins appear to be developmentally regulated, especially the band directly above the HI in 21- and 38-h samples. The early and late HI proteins themselves are not well resolved on this gel. Some mention must be made of the proteins which run in the H2A region in the 4- and 9-h lanes. Although there is a band at the H2Ad position, this species (called Y I ) has previously been shown to be different from the H2As which begins to appear at a later time (Newrock et al., 1978). It can be labeled with ["S]methionine and is oxidized by H202,whereas the H2A6 is not. As the Y I and H2As cannot be distinguished here, the exact time of onset of H2A mRNA accumulation cannot be determined. The faint bands in the HZA, and H2h6 positions in the 9-h sample may be small quantities of these late histones. but they may also be additional, as yet uncharacterized H2A variants. It is clear, however. that, between 9 and 15 h of development during the prehatching blastula period, an accumulation of late H2A mRNAs takes place. At I5 h there is also a significant amount of H2B, and H2B, mRNA. The presence of late HI protein is not obvious from this gel, but two bands are resolved in longer runs. The appearances of the three classes of late mRNA are reasonably coordinate, although we have not distinguished Y 1 and H2Ad here, so the time of appearance of HZA, is not certain. At 21 h, each species of the late and early types is synthesized to a similar extent. At 26 h, little detectable H2A. is present as shown by the translation products in Figure 7. In the 38-h gastrula. there is little if any HZA, and HI, mRNA, although a small amount of H2B. may still be present as seen in Figure 7. There is thus a detection of all late mRNA types in the cell at some point prior to the 15-h blastula. The early mRNAs persist until the mesenchyme blastula stages when they gradually disappear.
2714
BIOCHEMISTRY
HIETER ET AL.
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FIGURE 9: Stability of hybrids made with pC02 DNA and early and late polysomal RNA. Polysomal RNA prepared from embryos labeled with [3HJuridinefrom 7 to 9 h after fertilization and polysomal RNA prepared from embryos labeled with [32P]orthophosphatefrom 22 to 24 h were preparativelyhybridized as described in Figure 6 to pC02 DNA immobilized on filters. After elution, the two RNA preparations were mixed and rehybridized to $02 DNA under conditions described in Figure 3. Melting in 0.1 X SSC was done as described under Methods and Materials.
At times when the embryos are making late m R N A types exclusively (Figure l), there is still considerable early mRNA in the cell. The effect of the shift in synthesis of the two R N A classes which occurs is not immediately realized on the translational level. A degradation of the early m R N A most likely occurs before the cell solely synthesizes the late histone subtypes. Divergence of Polysomal RNAs. W e have previously demonstrated the large degree of sequence divergence in pulse-labeled histone R N A s from the early and late embryo (Kunkel & Weinberg, 1978; Kunkel et al., 1978). These new transcripts were demonstrated to be solely mRNA sequences by competition of hybridization by unlabeled polysomal mRNAs. Here we show that identical results are obtained when polysomal m R N A labeled for 2 h is used in hybrid formation. Figure 9 shows a difference of 11 O C in the T , of the two hybrid types. The R N A sample was a mixture of )H-labeled early and 32P-labeled late mRNA hybridized together to the same D N A filters. Similar results are found for hybrids made with each individual histone mRNA. The histone m R N A in polysomes is very much like the newly synthesized total cellular R N A in the presence and/or absence of early and late histone transcripts. Discussion Divergence of Early and Late “As. There is a dramatic shift in the presence of particular histone mRNAs in the cells of the sea urchin embryo during blastulation. W e have previously shown that the R N A s coding for each of the histones exist in a t least two classes which are quite diverged in sequence (Kunkel & Weinberg, 1978). Grunstein (1978) has also recently obtained this result for the H 4 mRNAs of L . pictus by comparing fingerprints of 32P-labeledearly and
late mRNAs. The sequences apparently differ in the third base positions of the codons with a minimum of 6% base substitution. Our values derived from the T , of hybrids (Kunkel & Weinberg, 1978; this report) using the relationship of 1 O C depression of T , for each 1% base substitution (Bonner et al., 1973) indicate a divergence of 9-14% for each of the mRNAs. The mRNAs for all the late histones, including the H 3 and H 4 proteins, are therefore quite different from the early embryonic histone mRNAs even though the proteins made a t the two stages are almost identical. The divergence of histone genes in different sea urchin species (Weinberg et al., 1972; Grunstein et al., 1976) is allowed by base substitution which does not change the coding specificity of the DNA (Birnstiel et al., 1973; Grunstein et al., 1976). A similar divergence takes place between the early and the late histone genes within a genome, probably also due to neutral base changes. The two gene sets, although obviously derived from an ancient precursor, apparently have been evolving independently for some 100 million years (Kunkel & Weinberg, 1978). Since the differences on the protein level appear to be minor, there must be extremely powerful selection for the particular amino acid sequence in the various histone subtypes. This property, and the fact that the shift of subtype synthesis occurs at a key developmental stage, reinforces the idea of the importance of the subtypes in chromatin patterning and gene expression (Cohen et al., 1975; Newrock et al., 1977). Differences in Electrophoretic Mobility. In addition to the sequence divergence and translational criteria, we have shown here that the 5’. purpuratus late and early histone mRNAs differ considerably in electrophoretic mobility. There appears to be great variation in mobilities of the H 4 and H2B mRNAs and somewhat less variation for the others. If the early H 4 m R N A is 400 nucleotides in length (Grunstein et al., 1976) and codes for a protein of 102 amino acids, then only 94 nucleotides are left in the nontranslated region. The late H 4 m R N A is apparently considerably smaller, leaving even less in the nontranslated region. One must be careful in interpreting electrophoretic mobilities as size differences, as we do not believe that the 7 M urea used in the gels necessarily completely denatures the mRNAs. Histone mRNA mobilities on NaDodSO, gels have been shown to be extremely sensitive to urea concentration, acrylamide content, and temperature (Gross et al., 1976). It is difficult from this electrophoretic analysis alone, therefore, to determine the m R N A size. Identification of Late H2A mRNAs. The late histone mRNAs have been shown here, and previously (Kunkel & Weinberg, 1978), to hybridize to DNA of the type coding for the early mRNAs. The use of specific D N A probes has allowed us to unambiguously identify all the early histone mRNAs and the late histone mRNAs for H1, H4, and H2B (Table I and Figures 3-7). Also, several forms of late H 3 m R N A can be assigned to particular bands because of hybridization with pSRl D N A and fragment E of the HhaI + EcoRI digest of pC02 DNA. The identification of late H2A mRNAs presents more of a problem since the specific hybridization probes (DHhal+EcoRI fragment and pRC9) do not hybridize well to any labeled R N A species (although there is some specific hybridization in the region of bands 4-6 shown in Figure 3). The H 2 A mRNAs do hybridize to the D N A of a complete repeat or a segment of the repeat coding for H2A and H 3 (pSR1) since the R N A from such hybrids is an effective template for H2A proteins (Figure 7, lanes c and d). In addition, the complete repeat is shown to hybridize to d l the labeled bands evident in the late polysomal R N A (Figures
LATE EMBRYONIC HISTONE MRNAS
1-3). That pRC9 will specifically probe for late H 2 A sequences is shown convincingly by analysis of translation products by using prehybridized and eluted R N A as the in vitro template as seen in Figure 7. This very sensitive way of assaying for H 2 A m R N A demonstrates the presence of H2A,, and to a lesser extent H 2 A , , mRNAs. From Figure 3, it is evident that bands 3 and 4 hybridize greatly to pC02 (total) and pSRl (H3 H2A) D N A but do not hybridize to or are underrepresented in the more specific probes. By difference we assign late H 2 A m R N A s to bands 2, 3, and probably 4, with band 4 also containing H 3 mRNA. There is a plausible explanation of our poor efficiency in demonstrating labeled H 2 A mRNA with pRC9 or DHhaI+&&I DNA. As can be seen in Figure 4, both pC02 and pSRl DNA contain the complete H2A gene, whereas pRC9 contains only a portion of the 3’ end of the mRNA (distal to the 28th amino acid from the -COOH terminal end) and the DHhaI+&&IDNA contains only the mRNA proximal to a point near the beginning of the coding sequence, that portion up to amino acid 16 (Sures, Lowry, and Kedes, personal communication), Therefore, a large central portion of the coding sequence, 237 nucleotides, is not present in the two specific probes. Although the pRC9 shows specificity when it does hybridize to the late mRNAs (Figures 3 and 7), the amounts that do hybridize are low. The melts of the small amount of hybrids which do form with pRC9 and DHhaI+EcoRID N A are very similar to those of the other specific histone hybrids (Kunkel & Weinberg, 1978). Our results here indicate that the intact H 2 A DNA gene sequence hybridizes much more efficiently to the late R N A than D N A containing only a small part of the gene. The efficiency of hybridization of bands 3 and 4 to pC02 DNA is a t least as high as that for any other band (Figure 2). We therefore have assigned band 3, perhaps band 2, and a portion of band 4 as late H 2 A m R N A . The early histone D N A sequence a t the 3’ end of the H2A gene may be more like the y than the /3 and 8 form; the H2A DNA sequence as a whole, however, appears to hybridize with similar efficiency to all the late H2A species. Therefore, the 3’ end of the late genes may have evolved somewhat faster than the central portion of the coding sequence. The pRC9 is a reasonable probe for the early H 2 A m R N A (Table I), so the low efficiency of hybridization of the 3’ end of the early H 2 A gene to late H 2 A mRNAs is not solely due to the configuration of the probe itself. As was mentioned previously, this is not true for the DHhai+EcoR1 fragment which was a poor probe for both early (Table I) and late (Figure 6) R N A s in these experiments. The translation of hybrid-eluted mRNAs perhaps provides the most stringent criterion for the nature of the late mRNAs. The in vitro translation products are found to have the expected characteristics on Triton X-urea gels, and all late subtype mRNAs are present. The assay of mRNA type by translation allows a definitive assignment of a protein product as belonging to a particular histone class. The results of hybridization with the H2A-specific probe pRC9 and subsequent translation and electrophoresis confirm the assignment of particular late proteins as H2A histones. Another interesting species is the polypeptide with the fastest mobility in Figure 8. In Figure 6, this entity is present in the products of the m R N A eluted from the total repeat and from the H 3 H 2 A pr6be. It is absent in the products derived from the H 2 A probe (3’ end of H2A) alone. W e therefore consider that this polypeptide is perhaps a short H 3 or H 2 A fragment, the result cf premature termination of protein synthesis, or more unlikely, the translation product of a H2A-H3 spacer transcript. A polypeptide of this mobility is not found in the chromatin of the
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sea urchin embryo (Cohen et al., 1975; Weinberg et al., 1977; Newrock et al., 1977, 1978). Leuel of Control. The shift in the m R N A and total cytoplasmic R N A coding for histones has been followed by assaying the translational potential of RNAs extracted from embryos at different embryonic stages. There is a substitution on the polysomes of late m R N A to replace an early H 1 mRNA (Ruderman et al., 1974) and H 2 A and H2B mRNAs (Weinberg et al., 1977; Newrock et al., 1978). The shift in the R N A population not only occurs in the polysomes but also in the total cytoplasmic R N A of the embryo (Newrock et al., 1978; this report). The transition therefore appears to be the result of a disappearance of early histone mRNAs and a new synthesis of late mRNAs. The shift in histone synthesis therefore seems to be a t a pretranslational level. However, we cannot rule out solely with our in vitro translational assay the possibility that the various mRNAs are always present but can exist a t times in untranslatable form, both in vivo and in vitro. This possibility is now excluded by filter hybridization blot experiments which show no detectable late mRNA in the early embryo and no early mRNA in the late embryo (M. B. Hendricks and E. S . Weinberg, unpublished experiments). The appearance of late subtypes may occur as early as 9 h as there are traces of P,y,G-like products of H 2 A and y,6 of H2B translated in vitro as seen in Figure 8. However, a t this stage we already know that the 8-like H2A protein is not really 6, but another species (Newrock et al., 1978), and small amounts of other early variants may exist which electrophorese in the late H2A region of the gel. By 15 h there are large amounts of late forms of the histones in the translation products. At the beginning of mesenchyme blastula, at 21 h, the a and various late m R N A species appear to be present in roughly equal amounts. By 26 h, there is no detectable early m R N A as assayed by the presence of translational products of prehybridized R N A (Figure 7) although small amounts of H2B,,6 are seen in the gastrula products in Figure 8. Within the period of 5 h, there seems to be a degradation of large amounts of histone mRNAs in the cell. The distribution of mRNAs on the polysomes during these stages is very similar to their relative presence in the total cytoplasmic R N A (Newrock et al., 1978; Hendricks, Weinberg, Newrock, and Cohen, unpublished experiments). There does not appear to be any control in both the appearance and disappearance of mRNAs from polysomes a t the level of mRNA selection for formation of polysomes. Although there may be small amounts of late m R N A synthesized as early as 9 h as detected in our translation assay (Figure 8), we find no labeled late m R N A in the polysomal 7-9-h samples. Neither the electrophoretic criterion (Figure 1) nor the melting profiles (Figure 9) give any evidence of labeled late m R N A a t this stage. The level of synthesis of the late mRNAs rises dramatically in subsequent stages. Our previous experiments with pulse-labeled RNAs from early and late embryos (Kunkel & Weinberg, 1978) also did not detect the presence of late-type R N A in the 9-h sample. The melting profiles of hybrids made with early R N A show no low-melting components, indicative of late sequences. The level of transcription, therefore, of the late R N A sequences is very low or shut off completely in the early embryo. Similarly, the transcription of early sequences in the late embryo is low or nonexistent. Modulation of the expression of these genes, therefore, is most probably at, or instantaneously after, transcription. As explained elsewhere (Kunkel & Weinberg, 1978; Overton & Weinberg, 1978), the genes coding for late mRNAs
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are thought to be organized apart from the main class of repetitive histone DNA. The early genes, such as those encoded in pC02 and in other D N A probes used here, crosshybridize with the late mRNAs but are considerably diverged in sequence. The genes coding for the late mRNAs have yet to be isolated or characterized. The late genes are probably present in few copies, yet support the synthesis of considerable m R N A over a long period of embryonic development (Kunkel & Weinberg, 1978). The initial transcripts of the late genes may be large as opposed to the mRNA-sized transcripts of the early genes (Kunkel et al., 1978). The two histone gene systems are utilized a t different embryonic stages and may show quite different modes of transcription and gene organization. Acknowledgments We thank Patricia Kuwabara and Vicki Murtif for their assistance in the work, Robert Donnelly for preparation of the HhaI fragments of pC02, and L. Kedes and R. Cohn for sending us pRC9 and pRC39 plasmids. References Alfageme, C. R., Zweidler, A., Mahowald, A., & Cohen, L. H. (1974) J . Biol. Chem. 249, 3729-3736. Birnstiel, M. L., Weinberg, E. S., & Pardue, M. L. (1973) in Molecular Cytogenetics (Hamkalo, B. A,, & Papacanstantinou, J., Eds.) pp 77-93, Plenum Press, New York and London. Bonner, T. I., Brenner, D. J., Neufeld, B. R., & Britten, R . J. (1973) J . Mol. Biol. 81, 123-135. Casey, J., & Davidson, N. (1978) Nucleic Acids Res. 4, 1539-1552. Cohen, L. H., Newrock, K. M., & Zweidler, A. (1975) Science 150, 994-997. Cohn, R. H., Lowry, J. C., & Kedes, L. H. (1976) Cell 9, 147-1 61. Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 23, 64 1-646. Gillespie, E., & Spiegelman, S. (1965) J . Mol. Biol. 12, 829-842.
HIETER ET A L .
Gross, K., Probst, E., Schaffner, W., & Birnstiel, M. (1976) Cell 8, 455-469. Grunstein, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4 135-4 139. Grunstein, M., Schedl, P., & Kedes, L. (1976) J . Mol. Biol. 104, 351-369. Holmes, D. S., Cohn, R. H., Kedes, L. H., & Davidson, N . (1977) Biochemistry 16, 1504-1512. Kedes, L. H., Cohn, R. H., Lowry, J. D., Chang, A. C. Y., & Cohen, S. N. (1975) Cell 6 , 359-369. Kunkel, N . S., & Weinberg, E. S. (1978) Cell 14, 313-326. Kunkel, N. S., Hemminki, K., & Weinberg, E. S. (1978) Biochemistry 17, 2591-2598. Laskey, R. A., & Mills, A. D. (1975) Eur. J . Biochem. 56, 3 35-34 1. Newrock, K. M., Alfageme, C. R., Nardi, R. V., & Cohen, L. H. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 421-431. Newrock, K. M., Cohen, L. H., Hendricks, M. B., Donnelly, R. J., & Weinberg, E. S. (1978) Cell 14, 327-336. Overton, G. C., & Weinberg, E. S. (1978) Cell 14, 247-257. Ruderman, J. V., & Pardue, M. L. (1977) Del;. Biol. 60, 48-68. Ruderman, J. V., Baglioni, C., & Gross, P. R. (1974) Nature (London) 247, 36-38. Sharp, P. A., Sugden, W., & Sambrook, .I. (1973) Biochemistry 12, 3055-3063. Weinberg, E. S., & Overton, G. C. (1978) Methods Cell Biol. 19, 273-286. Weinberg, E. S., Purdom, I. F., Birnstiel, M. L., & Williamson, R. E. (1972) Nature (London) 240, 225-228. Weinberg, E. S., Overton, G. C., Shutt, R. H., & Reeder, R. H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4815-4819. Weinberg, E. S., Hendricks, M. B., Donnelly, R. J., Newrock, K. M., & Cohen, L. H. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 1093. Wu, M., Holmes, D. S., Davidson, N., Cohn, R. H., & Kedes, L. H. (1976) Cell 9, 163-169. Zasloff, M., Cinder, G. D., & Felsenfeld, G. (1978) Nucleic Acids Res. 5 , 1139-1 152.
