- Email: [email protected]
Reactivity of heterogeneous F1 histones with glutaraldehyde and formaldehyde
380
Prelimitwry
notrs
60% of these sequences are present in the non-polysomal polyadenylated RNA (fig. 2). In addition, our results could not have resulted from the presence of mitochondrial polyadenylated RNA in our preparations of cytoplasmic polyadenylated RNA. This RNA, which has a very low sequence complexity [ 111, would account for less than 1 o/c of the observed difference in the hybridization plateaus of the two RNA populations. The possibility that all or part of the observed difference resulted from the leakage of nuclear polyadenylated RNA into the cytoplasm is more difficult to evaluate. However, several observations suggest that this is not the case. Nuclear polyadenylated RNA of Schneider cells has a sequence complexity 5-fold higher than cytopiasmic polyadenylated RNA (i.e., Roxt vatues are 5-fold higher) [12, 131. This is true for all frequency classes of RNA. Yet, we find that, while the frequent class of RNAs (those having a Roxt of 10-l moles/lxsec or less) react with essentially the same rates, the infrequent class reacts at significantly slower rates. Thus, either there has been preferential leakage of one RNA class and not of the other, or nuclear leakage is too small to influence our results. In conclusion, our data raise two questions about the role of the non-polysomal polyadenylated RNAs: are they mRNAs and if so, how is their translation controlled? We thank Dr Brian McCarthy for the use of his laboratory facilities and his helpful discussions. We also thank Dr Alan B. Blumenthal for critically reading this manuscript. This work was supported in part by a NIH Fellowship (CA 01060) from NC1 to A. Rizzino.
References 1. Ryffel, G U & McCarthy, B J. Biochemistry 14 (1975) 1379. 2. Bishop, J 0, Morton, J G, Rosbash, M & Richardson, M, Nature 250 (1974) 199.
ExpCell
Res 106 (1977)
3. Birnie, G D, MacPhail. E, Young, B D. Getz, M J & Paul, J, Cell differ 3 (1974) 221. 4. Levy, W, B & McCarthy, B J. Biochemistry 14 ( 1975) 2440. 5. LaTorre. J & Perry, R P, Biochim biophys acta 335 (1973) 93. 6. Rudland, P S, Proc natl acad sci US 71(1974) 750. 7. Schochetman. G & Perry, R P, J mol bio163 (1972) 577. a. MacLeod, M C, Biochemistry 14 (1975) 4011. 9. Lindquist McKenzie, S, Henikoff, S & Meselson, M, Proc natl acad sci US 72 (1975) I 117. 10. Levy, W, B, PhD Dissertation, University of California, San Francisco (1975). II. Hirsch, M, Spradling, A & Penman, S, Cell I (1974) 31. I?. Levy, W, B, Johnson, C B & McCarthy, B J, Nucleic acids res 3 (1976) 1777. 13. Levy, W, B, Johnson, C B & McCarthy, B J, Molecular biology of the mammalian genetic apparatus (ed P Tso) (1976). In press. Received August 3, 1976 Accepted November 24, 1976
Reactivity of heterogeneous Fl histones with glutaraldehyde and formaldehyde F. VARRICCHIO and G. JAMIESON, Jr, Memorial Sloan-Kettering Cancer Center, New York, NY looZl, USA Summary. The rates of glutaraldehyde and formaldehyde fixation of Fi histones have been investigated using chromatin from rat pancreas, chicken erythrocyte, and human spleen. These chromatins differ in number, type and relative proportion of Fl species present. In all cases the rates of fixation by glutaraldehyde and formaldehyde of the Fl components is much faster than for the other histones. The rates of fixation of Fl-type histones are similar in each case with the exception of one minor Fl histone from chicken which reacts slower than the rest of that Fl group. The results suggest that the interactions of all Fl type histones with DNA are similar.
Histones are now thought to be organized into an oligomeric complex, nu bodies [l]. The presence and regular distribution along the DNA helix of nu bodies was first suggested from results of an electron microscopic study by Olins & Olins [2]. More recent studies with a variety of chemical cross-linking agents have yielded results consistent with this hypothesis and have provided evidence that the nu body is com-
Preliminary
posed of a defined number of histone units [3,4,5-j. Thomas & Kornberg [3] have suggested that the nu body is an octamer composed of (F2a 1)2r (F3),, (F2u&, and Wa),. Fl histone reacts with glutaraldehyde and formaldehyde more rapidly than other histone classes [4, 51. When reacted with the bi-functional reagent glutaraldehyde, Fl histone has not been found to form polymers with other histones [4]. Instead Fl is thought to polymerize only with itself [4]. Bradbury et al. [6] used high field nuclear magnetic resonance (NMR) spectra of chromatin and the Fl-DNA complex to study the nature of these species. Their results suggest that Fl is complexed with DNA rather than with the other histones. The results of a study by Langmore & Wooley [7] using darkfield electron microscopy have shown that Fl histone may be involved in holding together adjacent DNA helices. Therefore the Fl histones are not considered to be a part of the nu body and may have a different role than the other histones. We have used chromatin from three different species which have histones with distinct and easily demonstrable differences in the Fl region, to determine the fixation rate of the minor Fl histones and Fl histone subfractions with glutaraldehyde and formaldehyde. Fl histones are by far the most heterogenous class of histones [8]. Panyim & Chalkley [9] have described a minor Fl-like histone called Flu whose occurrence appears to be in inverse relationship with the rate of cellular division. Fla histone is present in relatively high concentrations in the adult rat pancreas [lo]. In the chicken erythrocyte in addition to the well-known organ-specific histone F2C [ 111, the Fl histones can be separated by polyacrylamide gel electrophoresis into
notes
381
four bands. There is no band migrating in the position of Fla. Most human tissues contain Fla and all human tissues contain another Fl-like band, Flh [12] which migrates slightly slower than the principal Fl band. Materials
and Methods
Isolation of clrromafin and nuclei. Freshly excised rat pancreas, a normal human spleen removed at surgery (frozen at -20°C until use) and fresh whole chicken blood were processed essentially by methods described by de Pomerai et al. [13]. Nuclei were preDared bv homonenization in a Potter Elvehiem tube in ? vol of 0.24 fi sucrose, 10 mM Tris-HeI (pH 8.0), 5 mM M&I2 (solution A) to which was added NaHSO, to 0.05 M just prior to use. The homogenate was filtered through cotton gauze and then centrifuged at 1000 g for I5 min. The pellet was suspended in the same volume of solution A+NaHSO,, homogenized and centrifuged at 1000 g for IS min. This step was reoeated once more. The &let was susoended and hind homogenized in l/3 original volume df solution A made to 0.1% Triton X-100. This suspension was mixed with 4-5 vol of 2.2 M sucrose, 10 mM Tris-HCI (pH 8.0). 5 mM MgCI, (solution B) and layered over an equal volume of solution B prior to centrifugation at 100000 g for 1 h in an SW-27 rotor. The pellet of nuclei was hand homogenized in 3/4 original voiume of solution A without NaHS03 and centrifuged at i 000 g for 10 min. Nuclei were lvsed bv gentle hand homogenization of the pellet in 1 mG Tris-HCI (pH 8.0), 0.1 mM EDTA. 0.5 mM dithiothreitol. 12.5 % slvcerol and then centrifuged for 15 min at 1~000 g t;-pellet the chromatin. The chromatin was suspended in 10 vol of H,O, centrifuged again, and finally suspended in a small volume of HZ0 and stored at -20°C. Intact nuclei were prepared from the pellets from the ultracentrifugadon-step. Nuclei were hand homogenized in 3/4 original volume of 5~ IO-’ M triethanolamine (pH 7.2). 5 mM MgCI, (solution C) and centrifuged at I OCKIg for IO min. The pellet was suspended in a small volume of solution C 151. Isolation of the chicken erythrocyte chromatin was identical until the step involving the use of Triton X-100 which was replaced as follows. The pellet was hand homogenized next in 10 vol of a 0. I % aqueous solution of Triton X-100. Ultracentrifugation was omitted. Centrifugation at I OCGg for I5 min foilowed, before proceeding with the steps coming after ultracentrifugation in a standard preparation. Fixation. Fixation reaction conditions are based on those of Chalkley & Hunter 141. Chromatin was adjusted to an AZ60bf approx. Idaid made to 5X 1O-4 M triethanolamine @H 7.2). Glutaraldehyde was used at final concentrations of 0.014% and 0.035 %: formaldehyde at 0.6 % and 0.9 %. Reactions were done at 4°C for 0, 1, 2, 5, lo,20 min with glutaraldehyde and 0, 2. 5, 10, 20, 30 min with formaldehyde. In some fixation reactions 5 mM MgCI, was present. The reaction was terminated by addition of l/10 vol of 10 N H$O,. The acid-soluble fraction was dialysed for 4-6 h against
382
Preliminary
notes tion. Reaction rates were based on the amount of a species remaining at different time points compared to the zero time point.
Results Chromatin from three sources which are representative of the three different Fl profiles that we have found were selected for fixation with glutaraldehyde and formaldehyde. In many tissues, an Fl type histone which migrates slightly faster than FI occurs [9]. This histone, Fla, exists in un-
Fig. 1. Fl histones from different species. Histones were prepared and separated by polyacrylamide gel electrophoresis as described in Materials and Methods. The direction of migration is from top to bottom and the location of the Fl-types is noted at the left (A) Chicken erythrocyte; (B) adult rat pancreas; (C) human spleen.
0.4 N H,SO,. Histones were separated by electrophoresis using 6.4 M urea, 12 % polyacrylamide gels (OH 2.9) as described bv Panvim & Chalkley rl41. equal volumes of dialysed reaction mixtures were put on the gel so that in any one reaction series the amount of protein on each gel was derived from a constant amount of chromatin. The acid-insoluble material was prepared for SDS polyacrylamide gel electrophoresis bv addition of 0.1 to 0.2 ml of the following solution I-% SDS, 0.25 M Tris-HCI (pH 6.8). This m&ure was heated and 10 ~1 of ,3-mercaptoethanol was added. Samples were warmed just before being put on gels for electroohoresis. Ten oercent SDS oolvacrvlamide aels were piepared and run in the Tr&glycind buffer gystern as described by Laemmh [ 151. 3PC in .0.1%. Coomassie -. Gels were ._-_ stained 6 .h. at.^^_ -. Blue m 10% acettc actd, 10% methanol. Gels were destained for 3 days at 37°C in 10% acetic acid, 10% methanol. Gels were scanned at 600 nm in a Gilford model 2000 soectroohotometer eauiooed . . . with a linear transport attachment. Glutaraldehvde (EM-fixative grade) was obtained from Polysciences and 37% formaldehyde from Fisher Scientific Company. Areas under the curves were calculated by triangula-
-. L. - utsappearance -. rrg. of chicken erythrocyte histones during formaldehyde treatment. Reactions and other procedures were carried out as described in Materials and Methods. The direction of mieration is from left to right. (The top curve is the control.) The location of different histone arouns is noted. Each lower curve is the histone profile after 0.6% formaldehyde fixation for 2 and 5 min, respectively. The gels were scanned so that the F2b, F2n 2 peak=lOO%.
not found in the spleen. Gel scans of the histone patterns after formaldehyde fixation of chromatin from chicken erythrocytes, rat pancreas and human spleen at different time points are shown in figs 2. 3, and 4. The scans showing the histone patterns after glutaraldehyde fixation are similar except that with increasing time of glutaraldehyde fixation, the Fl histone bands become less and less clear and therefore resolution of similarly migrating histones is lost. As had been previously reported [4, 51 the Fl histones react with formaldehyde and glutaraldehyde much faster than do the other histones. In the case of the chicken
Fig. 3. Disappearance of adult rat pancreas histones during formaldehyde treatment. Reaction conditions, cf caption to fig. 2.
usual abundance in adult rat pancreas (fig. la). The ratio Fl : Flu is about 0.33. In the chicken erythrocytes the Fl band can be resolved into four bands. One band is difftcult to resolve and since there is a loss of resolution as a result of the fixation reactions we will consider only three Fl bands here, noted Fl, Fl’ and Fl”. The organspecific histone F2c is a large band between the Fl group and the F241 (fig. la). In all human tissues examined [12] there is a prominent band which migrates slightly slower than Fl. We have noted this Fl type histone Flh (fig. lc). A histone with the electrophoretic mobility characteristics of Flu is present in most human tissues but is
Fig. 4. Disappearance of human spleen histones during formaldehyde treatment. Reaction conditions. cf caption to fig. 2.
384
Preliminary
notes
erythrocyte chromatin, the Fl reacts slightly faster than does the F2C histones. F5 histone in turn reacted faster than any of the remaining histones. One minor chicken erythrocyte Fl-type histone, Fl”, did react slower with formaldehyde than did the other two chicken erythrocyte histones. Newborn rat pancreas does not contain any detectable Fla histone as compared with the relatively large amount in adult rat pancreas. We reacted newborn rat pancreas which did not contain any Fla, with formaldehyde and glutaraldehyde and compared the rate of reaction of Fl histone from newborn rat pancreas with that in adult rat pancreas. No change in the rate of Fl histone fixation by either reagent was detected. When the nuclei were lysed to prepare chromatin, the nuclei and then the chromatin are exposed to EDTA. Since removal of all magnesium ions might have farreaching effects on chromatin structure, we repeated the reaction of adult rat pancreas chromatin with formaldehyde and glutaraldehyde in the presence of 5 mM magnesium. There was no difference in the reaction rate of Fl histones with glutaraldehyde and formaldehyde in the presence of added magnesium. The preparation of chromatin from nuclei involves extensive unfolding of chromatin and possible changes in the proximity of histones and other chromatin components. We performed the fixation reactions, therefore, on adult rat pancreas nuclei. Magnesium was present in the reactions to prevent lysis of nuclei. The glutaraldehyde and formaldehyde concentrations used for nuclei fixation were the same as those used with chromatin. Again, we did not discern any differences between the reaction rate of Fl histones in nuclei as compared with those in chromatin.
SDS electrophoresis of the 0.4 N H,SO, acid-insoluble residue of the fixation reactions after preparation for SDS gels by methods described in Materials and Methods, showed a difference between formaldehyde and glutaraldehyde fixation. Boiling dissociated the Fl bound to DNA by formaldehyde so that SDS gel patterns show an increasing amount of HI histone to be present with increased time of fixation which appears to be in direct proportion to the decrease of FI histone seen on the histone gel. This was not observed with glutaraldehyde-fixed preparations. Discussion Several groups have previously reported on the reaction rate of various histone types with formaldehyde and glutaraldehyde [4, 51. The FI histones have been reported to react much faster than any other histone type. The Fl histones are different from other histones in a number of ways, one of which is that the Fl histones have been shown to be more heterogeneous [8, II]. We have studied fixation of chromatin from three different sources which are representative of chromatin-containing specific and marked differences in the Fl histones which are present. The results presented in this paper show that all histones of the Fl type react at the same rate with the exception of one minor chicken Fl histone. The meaning of these results is greatly enhanced by the finding that the reaction rates of Fl histones with glutaraldehyde and formaldehyde are the same when additional magnesium is added to the reaction mixture and when nuclei rather than chromatin are used. The products of the fixation reaction with formaldehyde and glutaraldehyde are different. Formaldehyde is thought to fix histones to the DNA, whereas glutaraldehyde,
Prelitninary
a bifunctional reagent, fixes histones to each other. We have found that the acidinsoluble material from the formaldehyde fixation. when analysed by SDS polyacrylamide gel electrophoresis contained increasing amounts of histone. This indicates, then, that formaldehyde does bind histone to DNA and that the histone can then be released by boiling in 10 % SDS. The chemical basis for the differences in the rate of fixation by formaldehyde and glutaraldehyde of the histone groups probably lies in part in the differences in lysine content. Formaldehyde and glutaraldehyde are thought to react principally with the a-amino groups of lysine. The lysine content of calf thymus histones is 28.7, 16.7, 12.5, 9.8 and 10.1% for Fl, F2b, F2u2, F2a 1 and F3, respectively [ll]. The reaction rates of glutaraldehyde with the chicken erythrocyte histones are 0.04080, 0.00774,0.00590,0.00648, and 0.00582 K,,, (min-l) for Fl, F241, F2a 2, F2a 1, F3, respectively [S]. Comparison of the lysine content with reaction rates suggests that the lysine content per se would seem to be insufficient to account for the markedly more rapid fixation of the Fl histones. Possibly the proximity, accessibility and location of specific lysine residues are factors. The amino acid composition of the minor chicken erythrocyte Fl histones, the human Flh and rat Fla are not known but they are probably not greatly different from Fl . These minor histones all separated as a group when the Fl purification procedure of Oliver et al. [ 161 using 5 % perchloric acid is utilized. Specific roles for the minor Fl histones and F2c are indicated by the variations in their occurrence in specific situations. Since they are present in various ratios minor histones cannot be distributed uniformly in chromatin. Some interchange-
nof~s
385
ability of minor histones in terms of functional role is, of course, not excluded. The results of this study show that a number of minor Fl type histones are fixed at similar rates. This suggests that the interaction of most but not all Fi histones with DNA may be very similar and that they exist in a similar micro-environment. Possibly the portions of the histone molecule which interact with DNA are very simi!ar. A recent study by Chapman et al. [ 171 showed that the structure of Fl histone in solution varies along the length of the protein. They suggest that one end, N-terminal, of the Fl histone is a globular head which may have a specific binding site on the chromosome subunit structure, whiie the tail consists of a highly basic random coil which binds to DNA. Cole and coworkers [ 18, 191 have found that the areas of substantial variance of the primary structure of different rabbit thymus Fl histone fractions were restricted to certain region of the N-terminal N- bromosuccinimide fragment. Perhaps the amino acid sequences of the C-terminal half of the Fl molecule are highly conserved. These recent findings would reconcile the heterogeneity of the Fl-type histones with the homogeneity of reaction rates with formaldehyde and glutaraldehyde as reported here. This work was supported in part by grants from the NCI. CA-08748 and CA-19584.
References 1. Felsenfeld, G, Nature 257 (1975) 177. 2. Olins, A L & Olins, D E, Science 183 (1974) 330. 3. Thomas, J 0 & Kornberg, R D, Proc natl acad xi US 72 (1975) 2626. 4. Chalkley, R & Hunter, C, Proc natl acad sci US 72 (1975) 1304. 5. Olins,DE&Wright,EB,Jcellbiol59(1973)304. 6. Bradbury, E M, Danby, S E, Rattle, H W E & Giancotti, V, Eur j biochem 57 (1975) 97. 7. Langmore, J P & Wooley, J C, Proc natl acad sci US 72 (1975) 2691. 8. Panyim, S, Bilek, D & Chalkley, R, J biol them 246 (1971) 4206. hp Cd/ RP.S!&‘I (IY77)
386
Preliminary
notes
9. Panyim, S & Chalkley, R, Biochemistry 8 (1969) 3972. 10. Marsh, W & Fitzgerald, P J, Fed proc 32 (1973) 2119. 11. Hnilica, L S, The structure and biological functions of histones. Chemical Rubber Co. Press, Cleveland, Ohio (1972). 12. Varricchio, F & Huvos, A. Unpublished results. 13. de Pomerai, D I, Chesterton, C J & Butterworth, P H W, Eur j biochem 46 (1974) 461. 14. Panyim, S & Chalkley, G R, Arch biochem biophys 130 (1969) 337. 15. Laemmli, U, Nature 227 (1970) 680. 16. Oliver, D, Sommer, R R, Panyim, S, Spikers, S & Chalkley, G R, Biochem j 129 (1972) 349. 17. Chapman, G E, Hartman. P G & Bradbury, E M, Eur j biochem 61 (1976) 69. 18. Jones, G M T, Rail, S C & Cole, R D, J biol them 249 (1974) 2548. 19. Rail, S C & Cole. D, J biol them 246 (1971) 7175. Received August 19, 1976 Accepted December 9, 1976
CeU surface changes on the mouse blastocyst at implantation E. J. JENKINSON
and R. F. SEARLE,
Reproductive
Immunology Group, Deportment of Pathology, The Medical School, University of Brisrol BSH 1 TD, UK
Su~nmar~~. Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.
To ensure its continued development the mammalian blastocyst needs to implant and establish an intimate association with the uterine tissues. In the mouse the initiation of implantation is apparently associated with functional changes in the outer surface of the trophectoderm which acquires the ability to adhere to the uterine wall. Similarly in vitro at the late blastocyst stage the trophectoderm undergoes a change in its
surface properties enabling it to attach and produce outgrowths on the bottom of the culture vessel [I]. Little is known, however, of the molecular basis of these events. It has been reported by Nilsson and his colleagues [2, 31 that the ability of blastocysts to bind positively charged colloidal iron particles is reduced at implantation indicating a decrease in surface negative groups, although the identity of these groups and the extent to which their reduction simply reflects alterations in the uterine milieu remains undetermined. In the present study the findings of Nilsson et al. have been confirmed and the nature of the surface groups investigated by examining their susceptibility to different enzyme treatments. In addition, changes in colloidal iron binding in the absence of maternal factors have been assessed using blastocyst outgrowth in vitro, a process considered to be analogous to implantation [4]. Materials and Methods Spontaneously ovulating random-bred female mice were caged with males overnight and examined for the presence of vaginal plugs the following morning. The date of vaginal plug was designated as day 0 post coitum (p.c.). Embryos. Pre-implantation blastocysts were obtained by flushing the uterine horns of pregnant females at 31 days p.c. with phosphate-buffered saline (PBS) containing 2 mglml bovine serum albumin (BSA). Peri-implantation embryos were studied in two ways. To obtain blastocysts in the immediate preimplantation and early implantation stages pregnant females were ovariectomized on day 2 p.c. and maintained in a state of experimentally-induced delay by sub-cutaneous injection of I mg of progesterone every second day until activated to implant by injeG tion of 0.1 pg 17p estradiol [SJ. Blastocysts were flushed from the uterus 14-18 h after estrogen administration. In order to examine changes in the trophectoderm at implantation in the absence of maternal influences blastocysts were recovered from the uterus at 31 days D.C. Followinn removal of the zona pellucida as described below these were cultured in vitro for three days in medium RPIMI (GIBCo-Biocult)+ 10% fetal calf serum in groups of 4-6 in 0.2 ml volumes in the wells of Cooke microtitre plates under 5 s CO* in air. During this time the aority of blastocysts attached to
Animals.
Prelimitwry
notrs
60% of these sequences are present in the non-polysomal polyadenylated RNA (fig. 2). In addition, our results could not have resulted from the presence of mitochondrial polyadenylated RNA in our preparations of cytoplasmic polyadenylated RNA. This RNA, which has a very low sequence complexity [ 111, would account for less than 1 o/c of the observed difference in the hybridization plateaus of the two RNA populations. The possibility that all or part of the observed difference resulted from the leakage of nuclear polyadenylated RNA into the cytoplasm is more difficult to evaluate. However, several observations suggest that this is not the case. Nuclear polyadenylated RNA of Schneider cells has a sequence complexity 5-fold higher than cytopiasmic polyadenylated RNA (i.e., Roxt vatues are 5-fold higher) [12, 131. This is true for all frequency classes of RNA. Yet, we find that, while the frequent class of RNAs (those having a Roxt of 10-l moles/lxsec or less) react with essentially the same rates, the infrequent class reacts at significantly slower rates. Thus, either there has been preferential leakage of one RNA class and not of the other, or nuclear leakage is too small to influence our results. In conclusion, our data raise two questions about the role of the non-polysomal polyadenylated RNAs: are they mRNAs and if so, how is their translation controlled? We thank Dr Brian McCarthy for the use of his laboratory facilities and his helpful discussions. We also thank Dr Alan B. Blumenthal for critically reading this manuscript. This work was supported in part by a NIH Fellowship (CA 01060) from NC1 to A. Rizzino.
References 1. Ryffel, G U & McCarthy, B J. Biochemistry 14 (1975) 1379. 2. Bishop, J 0, Morton, J G, Rosbash, M & Richardson, M, Nature 250 (1974) 199.
ExpCell
Res 106 (1977)
3. Birnie, G D, MacPhail. E, Young, B D. Getz, M J & Paul, J, Cell differ 3 (1974) 221. 4. Levy, W, B & McCarthy, B J. Biochemistry 14 ( 1975) 2440. 5. LaTorre. J & Perry, R P, Biochim biophys acta 335 (1973) 93. 6. Rudland, P S, Proc natl acad sci US 71(1974) 750. 7. Schochetman. G & Perry, R P, J mol bio163 (1972) 577. a. MacLeod, M C, Biochemistry 14 (1975) 4011. 9. Lindquist McKenzie, S, Henikoff, S & Meselson, M, Proc natl acad sci US 72 (1975) I 117. 10. Levy, W, B, PhD Dissertation, University of California, San Francisco (1975). II. Hirsch, M, Spradling, A & Penman, S, Cell I (1974) 31. I?. Levy, W, B, Johnson, C B & McCarthy, B J, Nucleic acids res 3 (1976) 1777. 13. Levy, W, B, Johnson, C B & McCarthy, B J, Molecular biology of the mammalian genetic apparatus (ed P Tso) (1976). In press. Received August 3, 1976 Accepted November 24, 1976
Reactivity of heterogeneous Fl histones with glutaraldehyde and formaldehyde F. VARRICCHIO and G. JAMIESON, Jr, Memorial Sloan-Kettering Cancer Center, New York, NY looZl, USA Summary. The rates of glutaraldehyde and formaldehyde fixation of Fi histones have been investigated using chromatin from rat pancreas, chicken erythrocyte, and human spleen. These chromatins differ in number, type and relative proportion of Fl species present. In all cases the rates of fixation by glutaraldehyde and formaldehyde of the Fl components is much faster than for the other histones. The rates of fixation of Fl-type histones are similar in each case with the exception of one minor Fl histone from chicken which reacts slower than the rest of that Fl group. The results suggest that the interactions of all Fl type histones with DNA are similar.
Histones are now thought to be organized into an oligomeric complex, nu bodies [l]. The presence and regular distribution along the DNA helix of nu bodies was first suggested from results of an electron microscopic study by Olins & Olins [2]. More recent studies with a variety of chemical cross-linking agents have yielded results consistent with this hypothesis and have provided evidence that the nu body is com-
Preliminary
posed of a defined number of histone units [3,4,5-j. Thomas & Kornberg [3] have suggested that the nu body is an octamer composed of (F2a 1)2r (F3),, (F2u&, and Wa),. Fl histone reacts with glutaraldehyde and formaldehyde more rapidly than other histone classes [4, 51. When reacted with the bi-functional reagent glutaraldehyde, Fl histone has not been found to form polymers with other histones [4]. Instead Fl is thought to polymerize only with itself [4]. Bradbury et al. [6] used high field nuclear magnetic resonance (NMR) spectra of chromatin and the Fl-DNA complex to study the nature of these species. Their results suggest that Fl is complexed with DNA rather than with the other histones. The results of a study by Langmore & Wooley [7] using darkfield electron microscopy have shown that Fl histone may be involved in holding together adjacent DNA helices. Therefore the Fl histones are not considered to be a part of the nu body and may have a different role than the other histones. We have used chromatin from three different species which have histones with distinct and easily demonstrable differences in the Fl region, to determine the fixation rate of the minor Fl histones and Fl histone subfractions with glutaraldehyde and formaldehyde. Fl histones are by far the most heterogenous class of histones [8]. Panyim & Chalkley [9] have described a minor Fl-like histone called Flu whose occurrence appears to be in inverse relationship with the rate of cellular division. Fla histone is present in relatively high concentrations in the adult rat pancreas [lo]. In the chicken erythrocyte in addition to the well-known organ-specific histone F2C [ 111, the Fl histones can be separated by polyacrylamide gel electrophoresis into
notes
381
four bands. There is no band migrating in the position of Fla. Most human tissues contain Fla and all human tissues contain another Fl-like band, Flh [12] which migrates slightly slower than the principal Fl band. Materials
and Methods
Isolation of clrromafin and nuclei. Freshly excised rat pancreas, a normal human spleen removed at surgery (frozen at -20°C until use) and fresh whole chicken blood were processed essentially by methods described by de Pomerai et al. [13]. Nuclei were preDared bv homonenization in a Potter Elvehiem tube in ? vol of 0.24 fi sucrose, 10 mM Tris-HeI (pH 8.0), 5 mM M&I2 (solution A) to which was added NaHSO, to 0.05 M just prior to use. The homogenate was filtered through cotton gauze and then centrifuged at 1000 g for I5 min. The pellet was suspended in the same volume of solution A+NaHSO,, homogenized and centrifuged at 1000 g for IS min. This step was reoeated once more. The &let was susoended and hind homogenized in l/3 original volume df solution A made to 0.1% Triton X-100. This suspension was mixed with 4-5 vol of 2.2 M sucrose, 10 mM Tris-HCI (pH 8.0). 5 mM MgCI, (solution B) and layered over an equal volume of solution B prior to centrifugation at 100000 g for 1 h in an SW-27 rotor. The pellet of nuclei was hand homogenized in 3/4 original voiume of solution A without NaHS03 and centrifuged at i 000 g for 10 min. Nuclei were lvsed bv gentle hand homogenization of the pellet in 1 mG Tris-HCI (pH 8.0), 0.1 mM EDTA. 0.5 mM dithiothreitol. 12.5 % slvcerol and then centrifuged for 15 min at 1~000 g t;-pellet the chromatin. The chromatin was suspended in 10 vol of H,O, centrifuged again, and finally suspended in a small volume of HZ0 and stored at -20°C. Intact nuclei were prepared from the pellets from the ultracentrifugadon-step. Nuclei were hand homogenized in 3/4 original volume of 5~ IO-’ M triethanolamine (pH 7.2). 5 mM MgCI, (solution C) and centrifuged at I OCKIg for IO min. The pellet was suspended in a small volume of solution C 151. Isolation of the chicken erythrocyte chromatin was identical until the step involving the use of Triton X-100 which was replaced as follows. The pellet was hand homogenized next in 10 vol of a 0. I % aqueous solution of Triton X-100. Ultracentrifugation was omitted. Centrifugation at I OCGg for I5 min foilowed, before proceeding with the steps coming after ultracentrifugation in a standard preparation. Fixation. Fixation reaction conditions are based on those of Chalkley & Hunter 141. Chromatin was adjusted to an AZ60bf approx. Idaid made to 5X 1O-4 M triethanolamine @H 7.2). Glutaraldehyde was used at final concentrations of 0.014% and 0.035 %: formaldehyde at 0.6 % and 0.9 %. Reactions were done at 4°C for 0, 1, 2, 5, lo,20 min with glutaraldehyde and 0, 2. 5, 10, 20, 30 min with formaldehyde. In some fixation reactions 5 mM MgCI, was present. The reaction was terminated by addition of l/10 vol of 10 N H$O,. The acid-soluble fraction was dialysed for 4-6 h against
382
Preliminary
notes tion. Reaction rates were based on the amount of a species remaining at different time points compared to the zero time point.
Results Chromatin from three sources which are representative of the three different Fl profiles that we have found were selected for fixation with glutaraldehyde and formaldehyde. In many tissues, an Fl type histone which migrates slightly faster than FI occurs [9]. This histone, Fla, exists in un-
Fig. 1. Fl histones from different species. Histones were prepared and separated by polyacrylamide gel electrophoresis as described in Materials and Methods. The direction of migration is from top to bottom and the location of the Fl-types is noted at the left (A) Chicken erythrocyte; (B) adult rat pancreas; (C) human spleen.
0.4 N H,SO,. Histones were separated by electrophoresis using 6.4 M urea, 12 % polyacrylamide gels (OH 2.9) as described bv Panvim & Chalkley rl41. equal volumes of dialysed reaction mixtures were put on the gel so that in any one reaction series the amount of protein on each gel was derived from a constant amount of chromatin. The acid-insoluble material was prepared for SDS polyacrylamide gel electrophoresis bv addition of 0.1 to 0.2 ml of the following solution I-% SDS, 0.25 M Tris-HCI (pH 6.8). This m&ure was heated and 10 ~1 of ,3-mercaptoethanol was added. Samples were warmed just before being put on gels for electroohoresis. Ten oercent SDS oolvacrvlamide aels were piepared and run in the Tr&glycind buffer gystern as described by Laemmh [ 151. 3PC in .0.1%. Coomassie -. Gels were ._-_ stained 6 .h. at.^^_ -. Blue m 10% acettc actd, 10% methanol. Gels were destained for 3 days at 37°C in 10% acetic acid, 10% methanol. Gels were scanned at 600 nm in a Gilford model 2000 soectroohotometer eauiooed . . . with a linear transport attachment. Glutaraldehvde (EM-fixative grade) was obtained from Polysciences and 37% formaldehyde from Fisher Scientific Company. Areas under the curves were calculated by triangula-
-. L. - utsappearance -. rrg. of chicken erythrocyte histones during formaldehyde treatment. Reactions and other procedures were carried out as described in Materials and Methods. The direction of mieration is from left to right. (The top curve is the control.) The location of different histone arouns is noted. Each lower curve is the histone profile after 0.6% formaldehyde fixation for 2 and 5 min, respectively. The gels were scanned so that the F2b, F2n 2 peak=lOO%.
not found in the spleen. Gel scans of the histone patterns after formaldehyde fixation of chromatin from chicken erythrocytes, rat pancreas and human spleen at different time points are shown in figs 2. 3, and 4. The scans showing the histone patterns after glutaraldehyde fixation are similar except that with increasing time of glutaraldehyde fixation, the Fl histone bands become less and less clear and therefore resolution of similarly migrating histones is lost. As had been previously reported [4, 51 the Fl histones react with formaldehyde and glutaraldehyde much faster than do the other histones. In the case of the chicken
Fig. 3. Disappearance of adult rat pancreas histones during formaldehyde treatment. Reaction conditions, cf caption to fig. 2.
usual abundance in adult rat pancreas (fig. la). The ratio Fl : Flu is about 0.33. In the chicken erythrocytes the Fl band can be resolved into four bands. One band is difftcult to resolve and since there is a loss of resolution as a result of the fixation reactions we will consider only three Fl bands here, noted Fl, Fl’ and Fl”. The organspecific histone F2c is a large band between the Fl group and the F241 (fig. la). In all human tissues examined [12] there is a prominent band which migrates slightly slower than Fl. We have noted this Fl type histone Flh (fig. lc). A histone with the electrophoretic mobility characteristics of Flu is present in most human tissues but is
Fig. 4. Disappearance of human spleen histones during formaldehyde treatment. Reaction conditions. cf caption to fig. 2.
384
Preliminary
notes
erythrocyte chromatin, the Fl reacts slightly faster than does the F2C histones. F5 histone in turn reacted faster than any of the remaining histones. One minor chicken erythrocyte Fl-type histone, Fl”, did react slower with formaldehyde than did the other two chicken erythrocyte histones. Newborn rat pancreas does not contain any detectable Fla histone as compared with the relatively large amount in adult rat pancreas. We reacted newborn rat pancreas which did not contain any Fla, with formaldehyde and glutaraldehyde and compared the rate of reaction of Fl histone from newborn rat pancreas with that in adult rat pancreas. No change in the rate of Fl histone fixation by either reagent was detected. When the nuclei were lysed to prepare chromatin, the nuclei and then the chromatin are exposed to EDTA. Since removal of all magnesium ions might have farreaching effects on chromatin structure, we repeated the reaction of adult rat pancreas chromatin with formaldehyde and glutaraldehyde in the presence of 5 mM magnesium. There was no difference in the reaction rate of Fl histones with glutaraldehyde and formaldehyde in the presence of added magnesium. The preparation of chromatin from nuclei involves extensive unfolding of chromatin and possible changes in the proximity of histones and other chromatin components. We performed the fixation reactions, therefore, on adult rat pancreas nuclei. Magnesium was present in the reactions to prevent lysis of nuclei. The glutaraldehyde and formaldehyde concentrations used for nuclei fixation were the same as those used with chromatin. Again, we did not discern any differences between the reaction rate of Fl histones in nuclei as compared with those in chromatin.
SDS electrophoresis of the 0.4 N H,SO, acid-insoluble residue of the fixation reactions after preparation for SDS gels by methods described in Materials and Methods, showed a difference between formaldehyde and glutaraldehyde fixation. Boiling dissociated the Fl bound to DNA by formaldehyde so that SDS gel patterns show an increasing amount of HI histone to be present with increased time of fixation which appears to be in direct proportion to the decrease of FI histone seen on the histone gel. This was not observed with glutaraldehyde-fixed preparations. Discussion Several groups have previously reported on the reaction rate of various histone types with formaldehyde and glutaraldehyde [4, 51. The FI histones have been reported to react much faster than any other histone type. The Fl histones are different from other histones in a number of ways, one of which is that the Fl histones have been shown to be more heterogeneous [8, II]. We have studied fixation of chromatin from three different sources which are representative of chromatin-containing specific and marked differences in the Fl histones which are present. The results presented in this paper show that all histones of the Fl type react at the same rate with the exception of one minor chicken Fl histone. The meaning of these results is greatly enhanced by the finding that the reaction rates of Fl histones with glutaraldehyde and formaldehyde are the same when additional magnesium is added to the reaction mixture and when nuclei rather than chromatin are used. The products of the fixation reaction with formaldehyde and glutaraldehyde are different. Formaldehyde is thought to fix histones to the DNA, whereas glutaraldehyde,
Prelitninary
a bifunctional reagent, fixes histones to each other. We have found that the acidinsoluble material from the formaldehyde fixation. when analysed by SDS polyacrylamide gel electrophoresis contained increasing amounts of histone. This indicates, then, that formaldehyde does bind histone to DNA and that the histone can then be released by boiling in 10 % SDS. The chemical basis for the differences in the rate of fixation by formaldehyde and glutaraldehyde of the histone groups probably lies in part in the differences in lysine content. Formaldehyde and glutaraldehyde are thought to react principally with the a-amino groups of lysine. The lysine content of calf thymus histones is 28.7, 16.7, 12.5, 9.8 and 10.1% for Fl, F2b, F2u2, F2a 1 and F3, respectively [ll]. The reaction rates of glutaraldehyde with the chicken erythrocyte histones are 0.04080, 0.00774,0.00590,0.00648, and 0.00582 K,,, (min-l) for Fl, F241, F2a 2, F2a 1, F3, respectively [S]. Comparison of the lysine content with reaction rates suggests that the lysine content per se would seem to be insufficient to account for the markedly more rapid fixation of the Fl histones. Possibly the proximity, accessibility and location of specific lysine residues are factors. The amino acid composition of the minor chicken erythrocyte Fl histones, the human Flh and rat Fla are not known but they are probably not greatly different from Fl . These minor histones all separated as a group when the Fl purification procedure of Oliver et al. [ 161 using 5 % perchloric acid is utilized. Specific roles for the minor Fl histones and F2c are indicated by the variations in their occurrence in specific situations. Since they are present in various ratios minor histones cannot be distributed uniformly in chromatin. Some interchange-
nof~s
385
ability of minor histones in terms of functional role is, of course, not excluded. The results of this study show that a number of minor Fl type histones are fixed at similar rates. This suggests that the interaction of most but not all Fi histones with DNA may be very similar and that they exist in a similar micro-environment. Possibly the portions of the histone molecule which interact with DNA are very simi!ar. A recent study by Chapman et al. [ 171 showed that the structure of Fl histone in solution varies along the length of the protein. They suggest that one end, N-terminal, of the Fl histone is a globular head which may have a specific binding site on the chromosome subunit structure, whiie the tail consists of a highly basic random coil which binds to DNA. Cole and coworkers [ 18, 191 have found that the areas of substantial variance of the primary structure of different rabbit thymus Fl histone fractions were restricted to certain region of the N-terminal N- bromosuccinimide fragment. Perhaps the amino acid sequences of the C-terminal half of the Fl molecule are highly conserved. These recent findings would reconcile the heterogeneity of the Fl-type histones with the homogeneity of reaction rates with formaldehyde and glutaraldehyde as reported here. This work was supported in part by grants from the NCI. CA-08748 and CA-19584.
References 1. Felsenfeld, G, Nature 257 (1975) 177. 2. Olins, A L & Olins, D E, Science 183 (1974) 330. 3. Thomas, J 0 & Kornberg, R D, Proc natl acad xi US 72 (1975) 2626. 4. Chalkley, R & Hunter, C, Proc natl acad sci US 72 (1975) 1304. 5. Olins,DE&Wright,EB,Jcellbiol59(1973)304. 6. Bradbury, E M, Danby, S E, Rattle, H W E & Giancotti, V, Eur j biochem 57 (1975) 97. 7. Langmore, J P & Wooley, J C, Proc natl acad sci US 72 (1975) 2691. 8. Panyim, S, Bilek, D & Chalkley, R, J biol them 246 (1971) 4206. hp Cd/ RP.S!&‘I (IY77)
386
Preliminary
notes
9. Panyim, S & Chalkley, R, Biochemistry 8 (1969) 3972. 10. Marsh, W & Fitzgerald, P J, Fed proc 32 (1973) 2119. 11. Hnilica, L S, The structure and biological functions of histones. Chemical Rubber Co. Press, Cleveland, Ohio (1972). 12. Varricchio, F & Huvos, A. Unpublished results. 13. de Pomerai, D I, Chesterton, C J & Butterworth, P H W, Eur j biochem 46 (1974) 461. 14. Panyim, S & Chalkley, G R, Arch biochem biophys 130 (1969) 337. 15. Laemmli, U, Nature 227 (1970) 680. 16. Oliver, D, Sommer, R R, Panyim, S, Spikers, S & Chalkley, G R, Biochem j 129 (1972) 349. 17. Chapman, G E, Hartman. P G & Bradbury, E M, Eur j biochem 61 (1976) 69. 18. Jones, G M T, Rail, S C & Cole, R D, J biol them 249 (1974) 2548. 19. Rail, S C & Cole. D, J biol them 246 (1971) 7175. Received August 19, 1976 Accepted December 9, 1976
CeU surface changes on the mouse blastocyst at implantation E. J. JENKINSON
and R. F. SEARLE,
Reproductive
Immunology Group, Deportment of Pathology, The Medical School, University of Brisrol BSH 1 TD, UK
Su~nmar~~. Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.
To ensure its continued development the mammalian blastocyst needs to implant and establish an intimate association with the uterine tissues. In the mouse the initiation of implantation is apparently associated with functional changes in the outer surface of the trophectoderm which acquires the ability to adhere to the uterine wall. Similarly in vitro at the late blastocyst stage the trophectoderm undergoes a change in its
surface properties enabling it to attach and produce outgrowths on the bottom of the culture vessel [I]. Little is known, however, of the molecular basis of these events. It has been reported by Nilsson and his colleagues [2, 31 that the ability of blastocysts to bind positively charged colloidal iron particles is reduced at implantation indicating a decrease in surface negative groups, although the identity of these groups and the extent to which their reduction simply reflects alterations in the uterine milieu remains undetermined. In the present study the findings of Nilsson et al. have been confirmed and the nature of the surface groups investigated by examining their susceptibility to different enzyme treatments. In addition, changes in colloidal iron binding in the absence of maternal factors have been assessed using blastocyst outgrowth in vitro, a process considered to be analogous to implantation [4]. Materials and Methods Spontaneously ovulating random-bred female mice were caged with males overnight and examined for the presence of vaginal plugs the following morning. The date of vaginal plug was designated as day 0 post coitum (p.c.). Embryos. Pre-implantation blastocysts were obtained by flushing the uterine horns of pregnant females at 31 days p.c. with phosphate-buffered saline (PBS) containing 2 mglml bovine serum albumin (BSA). Peri-implantation embryos were studied in two ways. To obtain blastocysts in the immediate preimplantation and early implantation stages pregnant females were ovariectomized on day 2 p.c. and maintained in a state of experimentally-induced delay by sub-cutaneous injection of I mg of progesterone every second day until activated to implant by injeG tion of 0.1 pg 17p estradiol [SJ. Blastocysts were flushed from the uterus 14-18 h after estrogen administration. In order to examine changes in the trophectoderm at implantation in the absence of maternal influences blastocysts were recovered from the uterus at 31 days D.C. Followinn removal of the zona pellucida as described below these were cultured in vitro for three days in medium RPIMI (GIBCo-Biocult)+ 10% fetal calf serum in groups of 4-6 in 0.2 ml volumes in the wells of Cooke microtitre plates under 5 s CO* in air. During this time the aority of blastocysts attached to
Animals.