[29] Two-dimensional gel analysis of protein synthesis

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[29] T w o - D i m e n s i o n a l G e l A n a l y s i s o f P r o t e i n S y n t h e s i s

By KEITH E. LATHAM, JAMES I. GARRELS, and DAVOR SOLTER Introduction Two-dimensional (2D) protein gel electrophoresis has been used extensively to investigate changes in gene expression during mouse embryogenesis. ~-~ The value of this technique lies in its ability to provide information about the synthesis of a large number of gene products with comparatively little starting material. Using high-resolution 2D gel electrophoresis, it is possible to detect over 2000 individual polypeptides in cultured fibroblast whole-cell lysates. 12 Quantitative gel image analysis combined with computer software for constructing protein databases has allowed more than 1200 individual polypeptides to be detected in mouse embryo lysates and their relative rates of synthesis to be quantified throughout preimplantation development. ~°'H Depending on the stage in question, between 3 and 10 preimplantation stage embryos and a single early postimplantation stage embryo can provide sufficient incorporated radiolabel for analysis. With the resolution that can be achieved with 2D gels, it is possible to address a variety of issues including transcriptional regulation, mRNA utilization and stability, posttranslational modification, and subcellular localization. 1-9 The combination of high-resolution gel formats and quantitative gel image analysis has increased the usefulness of this approach. This technology has been used to construct a 2D gel protein database for the mouse J O. Bensaude, C. Babinet, M. Morange, and F. Jacob, Nature (London) 305, 331 (1983). z p. Braude, H. Pelham, G. Flach, and R. Lobatto, Nature (London) 282, 102 (1979). 3 G. Flach, M. H. Johnson, P. R. Braude, R. A. S. Taylor, and V. N. Bolton, EMBO J. 1, 681 (1982). 4 C. C. Howe and D. Solter, J. Embryol. Exp. Morphol. 52, 209 (1979). 5 S. K. Howlett, Cell (Cambridge, Mass.) 45, 387 (1986). 6 S. K. Howlett and V. N. Bolton, J. Embryol. Exp. Morphol. 87, 175 (1985). 7 j. Levinson, P. Goodfellow, M. Vadeboncoeur, and H. McDevitt, Proc. Natl. Acad. Sci. U.S.A. 75, 3332 (1978). 8 j. Van Blerkom, Proc. Natl. Acad. Sci. U.S.A. 78, 7629 (1981). 9 j. Van Blerkom, in "Cellular and Molecular Aspects of Implantation" (S. R. Glasser and D. W. Bullock, eds.), p. 155. Plenum, New York, 1979. l0 K. E. Latham, J. I. Garrels, C. Chang, and D. Solter, Development (Cambridge, UK) 112, 921 (1991). II K. E. Latham, J. I. Garrels, C. Chang, and D. Solter, Appl. Theor. Elect. 2, 163 (1992). t2 j. I. Garrels and B. R. Franza, J. Biol. Chem. 264, 5283 (1989).

METHODS IN ENZYMOLOGY, VOL. 225

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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embryo. 1°'11 This chapter describes the techniques used to culture and label embryos for this analysis, and it offers practical suggestions for selecting an appropriate labeling and sampling strategy to detect changes in gene expression in genetically altered or experimentally manipulated mouse embryos. Methods

Embryo Isolation and Culture Embryos are isolated from superovulated mice at the 1-cell stage and cultured in vitro. Embryos can also be isolated at later stages to reduce the length of time spent in culture, although such embryos may differ subtly from in vitro cultured embryos. Adult female mice at least 6 weeks of age are superovulated with 5 IU of pregnant mare serum gonadotropin (PMSG) followed 46 hr later by 5 IU of human chorionic gonadotropin (hCG). Superovulated mice are placed in mating cages overnight and the embryos isolated approximately 20 hr post-hCG from the ampullae in HEPES-buffered Whitten's medium 13 supplemented with 100 /~M EDTA, 14 treated for 2 min with hyaluronidase to remove cumulus oophorous cells, and washed 4 times in Whitten's medium. Fertilized embryos bearing two pronuclei are selected and incubated at 37° in bicarbonate-buffered Whitten's medium supplemented with 100/xM EDTA 14 under an atmosphere of 5% COz and 5% 02. Alternatively, embryos may be cultured in CZB medium 15 until compacted and then switched to Whitten's medium.

Embryo Labeling and Lysis A summary of the procedure used for labeling and lysis of embryos is given (Table I). For labeling with L-[35S]methionine, embryos are washed through Whitten's or CZB medium containing 1 mCi/ml high specific activity (> 1100 Ci/mmol) L-[35S]methionine and then labeled in 40-/zl droplets of the same medium under oil. Incorporation of L-[35S]methionine is similar with the two media under these labeling conditions [e.g., 23,100 and 21,300 disintegrations/min (dpm)/embryo incorporated after 3 hr labeling in Whitten's and CZB medium, respectively, for the 2-cell stage]. For labeling of phosphoproteins, 2 mCi/ml of ortho[32P]phosphate (carrier13 W. K. Whitten, A c t a Biosci. 6, 129 (1971). i4 j. Abramczuk, D. Solter, and H. Koprowski, Dev. Biol. 61, 378 (1977). 15 C. L. Chatot, C. A. Ziomek, B. D. Bavister, J. L. Lewis, and I. Tortes, J. Reprod. Fertil. 116, 679 (1989).

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TABLE I EMBRYO LABELING AND LYSIS PROCEDURE FOR ANALYSIS OF L-[35S]METHIONINE- AND ORTHO[32p]PHOSPHATE-LABELED PROTEINS FROM PREIMPLANTATION STAGES 1. Isolate embryos at 1-cell stage and culture in vitro 2. Synchronize embryos by controlling time of fertilization or by using pick-off method 3. Wash embryos in medium containing 1 mCi/ml L-[35S]methionine (>1100 Ci/mmol) or 2 mCi/ml ortho[3ZP]phosphate (carrier-free) and label for 2-3 hr in same medium under oil 4. Wash embryos once in PBS containing 0.4% (w/v) PVP 5. Transfer embryos in a minimal volume to preheated (100°) SDS lysis buffer in a siliconized microcentrifuge tube 6. Heat for 30 sec in boiling water bath and cool on ice for 1 min 7. Digest with 1/10 volume of DNase/RNase on ice for 1 min 8. Freeze in liquid nitrogen and store at - 7 0 ° 9. Lyophilize, resuspend in an equal volume of 2D gel sample buffer, and quantitate incorporated radiolabel by TCA precipitation

free) in phosphate-free Whitten's medium is used. This labeling procedure yields significantly more incorporated radiolabel per embryo than was observed in some studies and permits the use of fewer embryos for analysis (Table II). Comparisons of several independent studies of protein synthesis in the mouse embryo (Table II) indicate that the use of concentrations of L-[35S]methionine greater than 1 mCi/ml or labeling times longer than 3 hr may be detrimental, as evidenced by reduced L-[35S]methionine incorporation. After labeling, embryos are washed in phosphate-buffered saline (PBS), pH 7.4, containing 0.4% (w/v) polyvinylpyrrolidone (PVP) to remove excess label and bovine serum albumin (BSA) and then lysed. The embryos must be lysed in such a way as to maximize solubilization and dissociation of proteins while preventing proteolysis. Lysis is performed in hot (100 °) sodium dodecyl sulfate (SDS) buffer containing 0.3% SDS, 1% (v/v) 2-mercaptoethanol, and 50 mM Tris-HCl (pH 8.0). 12This buffer is prepared in advance and stored in aliquots at - 7 0 °. Lysis in SDS efficiently solubilizes most proteins and dissociates most protein complexes, thereby improving separation during electrophoresis. For preimplantation stage embryos, a small volume of lysis buffer (13-30 /zl) is heated in a siliconized Eppendorf tube for 30 sec in a boiling water bath. The lysis volume is adjusted according to the number of embryos available and the number of gels (10/zl for each gel) to be run, allowing for an additional 2-3/zl for TCA (trichloroacetic acid) precipitation. The embryos are transferred to the hot buffer in a minimal volume and the lysis buffer heated for another 30 sec in the boiling water bath. If embryos are not lysed in hot SDS buffer, significant proteolysis can occur. After cooling

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T A B L E II COMPARISON OF Two-DIMENSIONAL GEL ANALYSES OF MOUSE EMBRYOS Latham et al. (1991)1° Disintegrations/min (dpm)/embryo ( x 104) 1-Cell 2.99 a 2-Cell 2.31 a 4-Cell 2.74 a 8-CeU 3.88 a Morula 8.0 a Blastocyst 15.7 a N u m b e r of 4-22 (12) d embryos/gel E x p o s u r e time 25-81 days (40) d N u m b e r of spots Up to 855 y resolved N u m b e r of regulated 502 spots (2-cell stage) Labeling Specific activity > 1100 Ci/mmol Concentration Time

1 mCi/ml 3 hr

H o w e and Solter (1979) 4

Levinson e t al. (1978) 7

Van Blerkom (1979) 9

0.09 b 0.084 b 0.476 b --0.57 b 170-420 e

0.13 0.18 -0.18 -0.47 10-119

----1.28 c -Single blastomere 8 months ___h

1-4 months ---g 105

>400 Ci/mmol 0.5 mCi/ml 5 hr

40-44 days Up to 600 34

600-1000 Ci/mmol 3-5 mCi/ml 1.5 hr

n.a. i

___h

h

a Average d p m / e m b r y o over entire stage. o Calculated by summing cytoplasmic and nuclear fractions. c Calculated from 800 dpm per 16-cell stage blastomere. d N u m b e r s in parentheses s h o w average values. e Divided into cytoplasmic and nuclear fractions. f High-quality spots only/5 g Samples were divided into cytoplasmic and nuclear fractions. h Not available. i n.a., Not applicable.

on ice for 1 min, 1/10 volume of a solution containing protease-free DNase I and RNase A (1.0 and 0.5 mg/ml, respectively; Worthington, Freehold, NJ), 1.5 M Tris-HC1 (pH 7.0), and 1.0 M MgC12 is added and the reaction incubated on ice for 1 min. The DNase/RNase solution is also prepared in advance and stored at - 7 0 °. After digestion, the sample is frozen in liquid nitrogen and stored at - 7 0 °. The entire lysis procedure should require less than 3 min to complete. The samples should be kept on ice after boiling to prevent proteolysis. Prior to electrophoresis, the sample is lyophilized, redissolved in an equal volume of 2D gel sample buffer,

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and an aliquot removed for quantitation of incorporated radiolabel by precipitation with TCA as described.16 Two-Dimensional Gel Electrophoresis

Selecting Gel Format Reproducibility and resolution are the primary concerns for selecting a gel format. No single gel format is capable of resolving every protein. The mouse embryo database used a pH 4-8 ampholine (British Drug House, Poole, UK) for isoelectric focusing and 10% acrylamide for the second dimension. 10,11This format gives reproducible spot resolution over the range of pI and Mr of approximately 4.4-6.7 and 20,000-127,000, respectively. A broader range ampholine (pH 3.5-10, LKB) can extend slightly the range of pl values resolved. 12,16 More acidic proteins (e.g., SPARC) have been resolved using the pH 3.5-10 ampholines and 0.1 M phosphoric acid as the anode solution. 17Additionally, nonequilibrium gels can be run as well as the standard equilibrium gels to visualize more basic and low-Mr proteins, 18 and altering the acrylamide concentration in the second dimension can reveal proteins of greater or lesser Mr.

Amount of Material Required for Analysis Gel electrophoresis and fluorography are performed as described.19'2° In general, analyses should be performed on duplicate samples in order to exclude the possibility that any observed differences are due to gel artifacts. To obtain sufficiently well-exposed gel images within a reasonable period of time, between 2 × 105 and 1 x 10 6 dpm ofL-[35S]methionine labeled protein should be applied to each gel in approximately 10/xl. For L-[35S]methionine-labeled samples, the best results are obtained with at least 4 × 105 dpm/gel which allows exposure times of approximately 4 weeks for the longest exposure (typically, 3-4 exposures of different lengths are obtained for each gel). For the 1-, 2-, and 4-cell stages, the mean incorporated radioactivity per embryo is relatively constant (29,900, 23, I00, and 27,400 dpm, respectively), 1° and between 16 and 20 embryos provide sufficient material for a single gel. This value increases to approxiJ6 j. I. Garrels, J. Biol. Chem. 264, 5269 (1989). 17 K. E. Latham and C. C. Howe, Roux's Arch. Dev. Biol. 199, 364 (1990). 18 R. Bravo, J. V. Small, S. J. Fey, P. M. Larsen, andJ. E. Celis, J. Mol. Biol. 154, 121 (1982). 19 j. I. Garrels, this series, Vol. 100, p. 411. 20 W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974).

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mately 8 × 104 and 1-1.4 × 105 dpm/embryo for the morula and blastocyst stages, respectively. One study produced 2D gel data for a single, isolated 16-cell stage blastomere, although this required a prolonged exposure of 8 months. 9 Early postimplantation stage embryos incorporate enough activity so that single embryonic or extraembryonic regions or 3-4 isolated germ layers provide enough material for analysis. Using pH 4-8 ampholines (British Drug House) in the first dimension and 10% acrylamide in the second dimension, an average of approximately 700 high-quality spots (designated as high quality based on their shapes, intensities, and degree of overlap with neighboring spots 16) are reproducibly detected for samples of preimplantation stage embryos. A total of more than 1200 proteins have been detected as high-quality spots and monitored from fertilization through the blastocyst stage using this gel format. H Spots accounting for as little as 20 parts per million (ppm) of incorporated radiolabel can be detected and reliably quantified. For ortho[3Zp]phosphate-labeled samples, sufficiently dark exposures can be obtained within 1-3 weeks with approximately 300 embryos. With good reproducibility in the gel system, it is possible to align gels of embryos labeled with these different isotopes in order to study posttranslational modification.

Quantification of Gel Images In many cases, simple visual comparison of two gel images may be sufficient to meet experimental needs, particularly where one or a few particular proteins of known location are to be examined. In other cases, however, gel images must be compared in their entirety in order to define both qualitative and quantitative differences between cell or tissue types, between embryos of different stages, or between normal and genetically altered or experimentally manipulated embryos. The details of gel image analysis using the QUEST system have been described, j6 One method of quantitation utilizes gel calibration strips that contain known amounts of radiolabeled protein. These are processed for fluorography and exposed in parallel with each gel. Using these calibration strips, film densities are converted to disintegrations per minute per unit of area. Each detected spot is thus quantified in terms of dpm and, dividing by the total TCAprecipitable radioactivity applied to the gel, as a fraction of total incorporated activity (ppm).~6 One point that is worth considering for analysis of embryonic material, however, is the method used to detect radiolabeled proteins. Exposure of X-ray films by fluorography typically requires between a few weeks and several months. Recently, photostimulable phosphor imaging plates

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(e.g., Fuji Photo Film Co., Tokyo, Japan) have been developed that can significantly shorten the requisite exposure times. 2~ Application of this technology to the analysis of embryonic material may significantly reduce the exposure times required and possibly the amount of labeled material that is required as well. The resolution, format, and sensitivity obtained for embryonic samples was similar between several independent studies. Until recently, however, the technology to perform in-depth, quantitative analyses on gel images was not available, and only a relatively small number of developmentally regulated proteins were described (Table II). Quantitative gel image analysis combined with computer software for direct matching of multiple gel images greatly increases the efficiency and speed of gel image comparisons and has allowed many more developmentally regulated proteins to be revealed than was possible previously (Table II).l° Experimental Design

Reproducibility and Selection of Time Points for Analysis When devising a strategy for using 2D gels to analyze embryonic gene expression, care must be taken to select the appropriate time point(s) and controls. Protein synthesis patterns of mouse embryos can change a great deal. An extensive reprogramming occurs during the 2-cell stage, for example ~° (Figs. 1 and 2), and many proteins can undergo substantial quantitative changes in their rates of synthesis within as little as 3 hr.l° The amount of change that can occur within 3 hr of the 2-cell stage, for example, can exceed the difference observed between proliferating and quiescent fibroblasts.~°'22 Furthermore, proteins that are synthesized during the 2-cell stage include a set of proteins that are induced transiently during the mid 2-cell stage (Fig. 3). This set includes some proteins that are apparently synthesized almost exclusively during the 2-cell stage, appear and disappear within as little as 9-12 hr, and can change by 2-fold or more in synthesis within as little as 3 hr. Such transient expression illustrates a potential difficulty that can be encountered when analyzing embryonic protein synthesis patterns, namely, the selection of the proper time points for analysis. Meaningful interpretation of alterations that result from genetic differences between embryos or experimental manipulations may require that labeling be performed within a narrow period of time in order to visualize changes in the expression of such transiently expressed, 21 R. F. Johnston, S. C. Pickett, and D. L. Barker, Electrophoresis 11, 355 (1990). 2.' j. I. Garrels and B. R. Franza, J. Biol. Chem. 264, 5299 (1989).

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FIG. 1. Representative gels for 2-cell stage mouse embryos labeled for 3 hr beginning at (A) 3 hr, (B) 6 hr, and (C) 21 hr postcleavage. Note that even within as little as 3 hr (A versus B) the relative rates of synthesis of some proteins can change significantly and that the overall pattern of proteins synthesized changes a great deal over the course of the 2-cell stage.

stage-specific p r o t e i n s . A d d i t i o n a l l y , for stages w h e r e rapid c h a n g e s in e m b r y o n i c p r o t e i n s y n t h e s i s p a t t e r n s o c c u r , c o m p a r i s o n s s h o u l d b e perf o r m e d b e t w e e n s a m p l e s o f e m b r y o s that, to the g r e a t e s t e x t e n t p o s s i b l e , are c o n t e m p o r a n e o u s .

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For certain periods of development, direct comparisons of embryos labeled in different experiments can be complicated by variability in the relative timing of protein synthetic changes. For example, comparisons between duplicate samples of mouse embryos, prepared in two independent experiments, at the mid 2-cell stage (9 and 12 hr postcleavage) when the rate of change in protein synthesis is greatest, revealed 2-fold or greater differences in 10-13% of the proteins analyzed, even though the patterns of proteins were essentially identical between duplicate samples (K. E. Latham, J. I. Garrels, and D. Solter, unpublished observations, 1992). Duplicate samples of cultured cell lines or mouse embryos at other stages (e.g., 4-cell stage) typically show approximately 2% 2-fold or greater differences. 2z Thus, for some stages of development, comparisons might produce potentially misleading results in the form of apparent quantitative differences in protein synthesis that are actually due to slight age differences between embryos. To minimize the chance of this occurring, comparisons should be made between samples of embryos that have been isolated at the same time and cultured identically.

Synchronization of Embryos In addition to the rapidity of changes in protein synthesis, asynchrony among mouse embryos can also complicate analyses. Asynchrony arises from variation in the time of ovulation, insemination, and fertilization. 23 As a consequence, the first cleavage division occurs in a population of eggs over a period of 9 hr or more. This asynchrony can potentially obscure the transient appearance of minor spots (like spots DF190 and EP60, Fig. 3) as well as the relative timing between different events. Thus, the use of synchronously developing embryos whenever possible is preferable. Synchronous cohorts can be collected by the pick-off method, z3 which consists of selecting at each cleavage division those embryos that cleave within a given time period (e.g., 1 hr). Typically, a group of approximately 500 embryos will produce 10-12 synchronous 1-hr cohorts of 25-40 embryos within a period of 4-5 hr. For experiments where synchronous 1-cell stage embryos are to be analyzed, either in vitro fertilization or delayed matings can be used. With the latter method, superovulated females are placed with male mice for 1 to 2 hr beginning at 13-14 hr after injection of hCG. A few mice can be sacrificed as early as 3 hr postmating to yield a few embryos bearing second polar bodies for immediate labeling. The remaining mice are sacrificed beginning at 6 hr postmating, and fertilized embryos are identified by the presence of two pronuclei. In this way, 23 V. N. Bolton, P. J. Oades, and M. H. Johnson, J. Embryol. Exp. Morphol. 79, 139 (1984).

482

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2 Cells

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4 Cells

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2D GEL ANALYSIS OF PROTEIN SYNTHESIS

483

embryos can be synchronized at fertilization to within 2 hr of each other. As many as 75% of the embryos obtained from delayed matings will cleave within a 2-hr period.

Frequency of Sampling A third consideration in designing a labeling strategy is the need to distinguish between discrete, qualitative alterations in the array of proteins synthesized and more subtle alterations in the temporal pattern of synthesis of a common set of proteins. Genetic alterations or experimental manipulations might, as their primary defect, lead to loss of expression or inappropriate expression of a particular gene. Alternatively, genetic or experimental manipulations of the embryo might affect the regulatory mechanisms that govern time- or stage-specific inductions and repressions, thereby altering the temporal pattern of gene expression. Mechanistically, these two types of effects are, of course, very different. The possibility that an alteration in the temporal pattern ofgene expression is involved can be at least partially addressed by examining the normal pattern of synthesis for a given protein. Androgenetic embryos (two paternal genomes) constructed with DBA/2 eggs by nuclear transplantation, for example, exhibit 11 proteins with quantitatively different rates of synthesis in comparison to unoperated or gynogenetic (two maternal genomes) embryos. 24 The normal patterns of synthesis of all 11 proteins reveal induction or repression between the 8-cell and blastocyst stages. Thus, a partial interruption or delay in the normal sequence of regulatory events occurred in the androgenones, possibly as a secondary consequence of some other effect of genomic imprinting. To discriminate between specific primary defects that alter the array of genes expressed and more subtle alterations in the time of expression, genetically altered or experimentally manipulated embryos should be sampled at as many time points as feasible 24 K. E. Latham and D. Solter, Development (Cambridge, UK) 113, 561 (1991).

FIG. 2. Enlarged regions of gels from the 2-cell and 4-cell stage. Each panel shows the same region of gels obtained for L-[35S]methionine-labeled 2-cell (A-C) and 4-cell (D, E) stage embryos labeled at the indicated times after the first and second cleavage divisions, respectively. For the 2-cell stage, proteins that decline (downward pointing arrows), appear transiently (ovals), or increase (upward pointing arrows) during the 2-cell stage are indicated. For the 4-cell stage, proteins that are synthesized at a constant rate (ovals) or increase or decrease (arrows) during the 4-cell stage are indicated. Horizontal chevrons mark four proteins that are present in all five gels to facilitate alignment. Note that most of the major proteins of the 2-cell stage change significantly in rates of synthesis, whereas most of the major 4-cell stage proteins are synthesized at relatively constant rates.

484

GENE EXPRESSION: PROTEINS

ppm

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1 1Cell 0 02Cells~ 4Cells 4 20 0. 80-

DF190

0

ppm EE60 ppm EP60

400

.......

I. .......

I 80OOj~L~

~

60. 40200

ppm 5OOOO 70kDa Complex

,oooo4

I

_o-Ot rl r 1000O o

m

I. li

I

! liin . . . . _

I

L

time (h)

FzG. 3. Transiently induced proteins of the 2-cell stage mouse embryo. Graphs show the rates of synthesis of four representative proteins that are synthesized at elevated rates during the mid 2-cell stage. Individual bars show the rate of synthesis expressed as the fraction (parts per million) of incorporated radiolabel at sequential 3-hr time intervals beginning at the times indicated. Standard spot numbers, assigned to each protein using the protein database, I° are given to the left of each graph. Arrows positioned at 20 ppm denote the minimum intensity (20 ppm) that could be reliably quantitated. Note that these proteins are essentially stage-specific.

a n d c o m p a r e d to a c a r e f u l l y s t a g e d s e r i e s o f n o r m a l e m b r y o s . L a b e l i n g at m u l t i p l e t i m e p o i n t s will a l s o f a c i l i t a t e t h e o b s e r v a t i o n o f t r a n s i e n t c h a n g e s in p r o t e i n s y n t h e s i s as w e l l as c o m p a r i s o n s b e t w e e n i n d e p e n d e n t experimental series.

Analysis of Postimplantation Stages T w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s h a s a l s o b e e n a p p l i e d to t h e a n a l y sis o f p o s t i m p l a n t a t i o n s t a g e s o f m o u s e e m b r y o g e n e s i s . 25 A n a l y s i s o f m o r e a d v a n c e d s t a g e s b y 2D gel e l e c t r o p h o r e s i s b e c o m e s m o r e difficult a s m u l t i 25 K.-F. Murach, M. Frei, D. Gerhauser, and K. Iilmensee, J. Cell. Biochem. 44, 19 (1990).

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pie cell and tissue types become established. Characterization of protein synthetic patterns must account for both stage and cell or tissue specificity. The analysis of protein synthesis patterns of isolated tissues or embryonic regions offers a useful means of identifying tissue- or region-specific differences in protein synthesis as well as time-dependent changes in gene expression. Additionally, proteins that are selectively synthesized at enhanced rates in specific tissues or regions should be more easily visualized in lysates of the isolated tissue fragments than in lysates of whole embryos. Indeed, several proteins are more easily detected in lysates of isolated germ layers from 6.5 and 7.5 day embryos than in lysates of whole embryonic or extraembryonic regions (K. E. Latham, J. I. Garrels, and D. Solter, unpublished observations, 1993). Detection and accurate quantitation of such proteins will be facilitated by isolation or enrichment of that cell or tissue type. Thus, 2D gel analysis of isolated tissues or spatially distinct regions (e.g., anterior versus posterior) offers a sensitive means of detecting developmentally significant differences in gene expression. Protein Identification Two potentially very useful applications of 2D gel electrophoresis in embryology are to identify developmentally regulated genes and to monitor the expression of a particular known protein in different tissues or at different stages. The former requires the identification and/or characterization of a specific protein spot that exhibits a tissue- or stage-specific pattern of expression. The latter application requires the localization of a particular known protein of interest within the 2D gel pattern so that the synthesis of that protein can be followed. Limitations in the amount of embryonic material available may preclude the application of methods such as direct protein microsequencing and immunoblotting, particularly for proteins that are synthesized transiently and do not accumulate to high abundances. An indirect method of spot identification, based on the construction of the mouse database, is to align a 2D gel image containing spots to be identified with a 2D image containing previously identified spots. Twelve proteins have been identified in mouse embryo gels by alignment with 2D gels of rat fibroblast in which these proteins had been previously located. 22 This approach offers the advantage that tentative identifications can be assigned to embryonic proteins based on identifications made using more readily available material. For all but the most easily located, well-characterized proteins (e.g., actin), identifications made in this manner must be confirmed directly. Immunoprecipitation provides a convenient means for this purpose, and for locating spots that correspond to particular known proteins in gels of embryonic lysates. For example,

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FIG. 4. Identification of tropomyosin isoforms by immunoprecipitation. Blastocysts were labeled and subjected to immunoprecipitation as described in the text using a rabbit polyclonal antiserum to tropomyosin. 22(A) Immunoprecipitate from labeled mouse 3T3 cells, (B) immunoprecipitate of labeled mouse blastocysts, and (C) control blastocyst whole-cell lysate. The positions of tropomyosins 1-6 are indicated.

a rabbit polyclonal antitropomyosin serum 22 was used to verify the identification of tropomyosin isoforms in blastocyst stage lysates prepared from approximately 450 embryos (Fig. 4). An effective immunoprecipitation protocol for mouse embryos is as follows. Embryos are labeled as described above and lysed in 100/.d of immunoprecipitation buffer [20 mM Tris-HC1, pH 8.0, 1% (v/v) Triton X-100, 0.5% sodium deoxycholate, 0.15 M NaC1, 5 mM MgC12, and 1 mM freshly added phenylmethylsulfonyl fluoride (PMSF)]. To this lysate are added 5/~1 of 10% SDS and 10 t~l of RNase/DNase (see above). After a

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1-min incubation on ice, 10 tzl are frozen immediately as a whole-cell lysate. An additional 100/~1 lysis buffer is added and the lysate centrifuged (13,000 g) for 5 min at 4°. The lysate is precleared by incubation with 10/xl of normal serum and 50/xl protein A-conjugated Sepharose (25% suspension in lysis buffer) for 30 min on ice with agitation every 5 min. The supernatant is incubated with 1-5/xl of primary antiserum on ice for 1 hr. The final incubation is for 30 min on ice after addition of 50/xl protein A-conjugated Sepharose with agitation every 5 min. The Sepharose beads are centrifuged for 30 sec at 13,000 g. If an antibody for which protein A has low affinity is to be used, an additional incubation with the appropriate secondary antibody can be included. An aliquot of the supernatant is frozen in liquid nitrogen and stored at - 7 0 °. The precleared (from above) and antigen-containing pellets are washed 6 times with 0.5 ml lysis buffer. During the final wash, the material is transferred to a new tube. After washing, the pellets are boiled for 1 min in 30/xl of SDS buffer (above) and centrifuged. The supernatants are frozen in liquid nitrogen and stored at - 70 °. The whole-cell lysate, precleared and antigen-containing pellet fractions, and supernatants are lyophilized, resuspended, and processed for 2D gel electrophoresis. Gels are obtained for the whole-cell lysate, immunoprecipitate alone, mixed whole-cell lysate and immunoprecipitate, precleared fraction, and supernatant fraction. Immunoprecipitated proteins are visualized on the gel receiving immunoprecipitate alone (Fig. 4) and on the gel receiving the mixture of whole-cell lysate and immunoprecipitate (not shown). The latter allows location and identification of antigen(s) within a gel of a whole-cell lysate as a spot(s) that is significantly enhanced in intensity relative to the whole-cell lysate alone. Proteins that coprecipitate with the antigen will also be enhanced. The supernatant gel should exhibit a specific reduction in the relative intensity of the same spot(s). The precleared sample provides a control for specificity. This procedure should be generally applicable to identification of specific proteins in 2D gels of preimplantation stage mouse embryos. It is also possible to use purified proteins obtained from alternate sources to identify spots in 2D gel patterns.17 Purified proteins are coelectrophoresed with labeled whole-cell lysates and later visualized either by staining (e.g., with silver or Coomassie blue) or by autoradiography (e.g., iodinated proteins). The position of the purified protein within the pattern of spots obtained from the whole-cell lysate is thus determined by comigration. If, however, different posttranslational modifications or alternatively spliced isoforms are expressed between the embryo and the source from which the purified protein was prepared, then confirmation by immunoprecipita-

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[29]

tion of labeled embryonic proteins is necessary. Immunoprecipitation can also reveal additional isoforms of some proteins that are not easily detected in gels of whole-cell lysates (Fig. 4C). Protein Databases Numerous 2D gel studies have been described for the mouse embryo. 1-9 Variability in gel conditions, resolution, and embryo staging and insufficient quantitation can limit the amount of information that can be gained from 2D gels. These problems can largely be overcome by adopting a protein database approach. 1° This approach combines standardized gel electrophoresis with a system for quantifying and matching gel images. 12,22,26-30The advantage of this approach is that each detected protein spot can be quantified and followed through a series of gel images representing samples collected in a number of independent experiments. Thus, the data are cumulative, and most of the data contained within the gel images can be extracted. The database approach also allows for spot annotation to record such information as protein names, known modifications, subcellular localization, and regulatory patterns. 16 The details of constructing a protein database are described elsewhere 12'16'22and will not be duplicated here. Conclusion The ability of high-resolution 2D gel electrophoresis to resolve so many gene products for analysis makes this technique an especially valuable one for the embryologist. Continued improvement in spot resolution, detection, and quantification should increase the value of this technique further, particularly when combined with computer software for accumulating and managing diverse types of data in the form of a 2D gel database. Additionally, it should be possible to integrate 2D gel data with data obtained through other approaches such as molecular cloning. Immuno26 j. E. Celis, K. Dejgaard, P. Madsen, H. Leffers, B. Gesser, B. Honore, H. H. Rasmussen, E. Olsen, J. B. Lauridsen, G. Ratz, S. Mouritzen, B. Basse, M. Hellerup, A. Celis, M. Puype, J. Van Damme, and J. Vandekerckhove, Electrophoresis 11, 1072 (1990). 27 j. E. Celis, B. Gesser, H. H. Rasmussen, P. Madsen, H. Leffers, K. Dejgaard, B. Honore, E. Olsen, G. Ratz, J. B. Lauridsen, B. Basse, S. Mouritzen, M. Hellerup, A. Andersen, E. Walbum, A. Celis, G. Bauw, M. Puype, J. Van Damme, and J. Vandekerckhove, Electrophoresis 11, 989 (1990). 28 R. A. VanBogelen, M. E. Hutton, and F. C. Niedhardt, Electrophoresis 11, 1131 (1990). z9 R. D. Appel, D. F. Hochstrasser, M. Funk, J. R. Vargas, C. Pelligrini, A. F. Muller, and J.-R. Scherrer, Electrophoresis 12, 722 (1991). 3o I. Ali, Y. Chan, R. Kuick, D. Teichrow, and S. M. Hanash, Electrophoresis 12, 747 (1991).

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precipitation offers a good means of identifying spots on 2D gels that correspond to the products of specific cloned genes. Antisera can be generated from cloned DNA sequences using synthetic oligopeptides or polypeptides generated with expression vectors as immunogens. The combination of molecular cloning and 2D gel protein database approaches offers an excellent means of identifying developmentally regulated genes, characterizing in detail their patterns of expression, and examining the mechanisms that regulate these patterns of expression. Acknowledgments This work was supported in part by U.S. Public Health Grants HD-17720, HD-23291, and HD-21355 from the National Institute of Child Health and Human Development and CA-10815 from the National Cancer Institute. The QUEST Center at Cold Spring Harbor Laboratory was supported by a grant (P41RR02188) from the National Institutes of Health Biomedical Research Technology Program. K.E.L. was supported in part by a training grant from the NCI (CA 09171-14).

[30] O n e - D i m e n s i o n a l G e l A n a l y s i s o f H i s t o n e S y n t h e s i s

By

MARIA WIEKOWSKI a n d MELVIN L . DEPAMPHILIS

Introduction All eukaryotic DNA is organized into a nucleoprotein complex referred to as chromatin, The basic unit of chromatin is the nucleosome, consisting of approximately 200 bp of DNA wrapped around a histone octamer. The nucleosome consists of 146 bp of " c o r e " DNA tightly associated with two copies each of histones H2A, H2B, H3, and H4 and about 60 bp of loosely associated "linker" DNA. Histone H 1 associates with linker DNA and thereby pulls nucleosomes together into a regular repeating array called the 30-nm fiber. Histone composition can change during development. For example, in Drosophila, the core histones are always present, but histone H1 does not appear until the blastula stage) In other animals, such as the sea urchin, sea worm, mud snail, and annuran amphibians (e.g., Xenopus), embryo-specific H1 variants are present, z-6 In Xenopus, histone H1 first t S. 2 R. 3 A. 4 D.

C. R. Elgin and L. E. Hodd, Biochemistry 12, 4985 (1973). R. Franks and F. C. Davis, Deo. Biol. 98, 101 (1983). M. Flenniken and K. M. Newrock, Dev. Biol. 124, 457 (1987). Poccia, J. Salik, and G. Krystal, Dev. Biol. 82, 287 (1981).

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