The expression of intracisternal A particle genes in the preimplantation mouse embryo

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The Expression of lntracisternal A Particle Genes in the Preimplantation Mouse Embryo ANN A. POZNANSKI~ AND PATRICIA G.

CALARCO

Intracisternal A particles (IAP), murine endogenous retrovirus, make up 0.3% of the mouse genome. They are expressed in some normal tissues, certain transformed cell lines, and show stage-specific patterns of expression in early embryos. We have used peptide-specific antisera and the polymerase chain reaction to explore type-specific expression of these IAP during preimplantation development. In this paper we show that the IAP core protein, ~‘73, characteristic of type I IAP, is present throughout preimplantation development while the grr(/-pol fusion protein ~120, characteristic of the variant type Ul, is synthesized and expressed only from the B-cell stage onward. Type I IAP RNA is present at all stages and appearance of ~120 at the 8-cell stage could represent new transcription or translation from a preexisting IAl message. The presence of type II IAP RNA varies according to stage, with t\vo sizes of type II transcripts present at all stages except the Z-cell stage at which time only the smaller of the two transcripts can be detected. The reappearance of the larger type II transcript subsequent to the 2-cell stage implies new transcription of this type II subspecies. The r: 1!1!n presence of type I, II, and ~‘73 in the unfertilized egg strongly suggests mattrnal inheritance from the oocyte. Academlr

Press. Inc

1982; Lueders ef al., 1982; Gattoni-Celli et al., 1983; Sheng-Ong and Cole, 1984; Burt et al., 1984; Greenberg et ah, 1985; Ymer et al., 1985; Kongsuwan et al., 1989). IAP elements have been grouped with Ty elements of yeast and copia elements of Drosophila as Class I retrotransposons (Fanning and Singer, 1987). The long terminal repeat of IAP genes has been shown to function as an efficient promoter in heterologous (Lueders et al., 1984) as well as homologous cells (Morgan et al., 1988). It is itself the target for transactivation by oncogene products (Luria and Horowitz, 1986), and has at least five distinct protein-binding domains (Falzon and Kuff, 1988). IAP form by budding an RNA nucleoid core into the endoplasmic reticulum (ER) of the cell, thereby acquiring an outer membrane. They remain in the ER and, when isolated, are not infectious (Hall et al., 1968; Kuff et al., 1968). IAP occur in large numbers in certain plasmacytomas (Dalton et nl., 1975) and diverse tumors from all three germ layers (Wivel and Smith, 1971) but are rarely seen in normal adult tissues. However, IAP sequences are transcribed in several normal tissues with the highest expression occurring in the thymus of young mice where the quantity and type expressed vary according to the strain of the mouse (Kuff and Fewell, 1985). There is also evidence for the selective activation of specific IAP sequences in the thymus (Grossman et

INTRODIJCTION

Intracisternal A particle (IAP) genes are a family of endogenous retroviral genes which are present in the mouse genome at approximately 1000 copies per haploid genome distributed among most, if not all, chromosomes (Lueders and Kuff, 1977). Biochemical and molecular analyses of isolated IAP reveal that they possess a high molecular weight polyadenylated RNA genome (Yang and Wivel, 1973; Wong-Staal et al., 1975; Lueders and Kuff, 1977), a core protein of 73,000 M, (Wivel et al., 1973), and a Mg 2+-dependent reverse transcriptase (Wilson and Kuff, 1972). IAP sequences do not share any significant homology with other classes of viruses (Lueders and Kuff, 1979; Yang and Wivel, 1974) except in the region encoding the polymerase where they share sequence homology with type B, type D, and avian but not mammalian type C viruses (Chiu et al., 1985). They also show some sequence homology with a unique endogenous virus of Asian murine species, Mus cerkolor and MUS caroli (Kuff et al., 1978; Callahan et ah, 1981), and show substantial sequence homology with segments of a related element of Syrian hamsters (Ono et al., 1985). IAP proviral elements can undergo transposition to new locations within the genome, thereby acting as insertional mutagens (Hawley ef al., 1982,1984; Rechavi ef al., 1 Present address and to whom at Department of Microbiology, cisco, CA 94143.

correspondence should IJniversity of California,

al.,

be addressed San Fran-

1987).

Two subfamilies guished and termed 271

of IAP genes have been distintype I and type II (Sheng-Ong and 0012.1606/91 Copyright All rights

$3.00

Y 1991 hy Academic Press. Inc. of reproduetm in an> form wwrwd.

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Cole, 1982). Type I genes include the full length gene of 7.2 kb, a 5.4-kb variant, and other deleted forms and comprise about 60% of all IAP genes (Lueders and Kuff, 1980; Lueders and Meitz, 1986). Type II genes contain major deletions involving the guy and pol genes, contain a unique inserted sequence of approximately 270 bp (Sheng-Ong and Cole, 1982; Lueders and Metiz, 1986), and comprise about 40% of a11 IAP genes (Lueders and Meitz, 1986). IAP-related proteins include a 73,000 M,. core protein (p’73), the main structural protein of IAP, which is equivalent to an uncleaved gag-precursor protein of a conventional retrovirus, and a larger protein of approximately 120k, (~120) equivalent to a gag-pal fusion protein (Kuff and Fewell, 1985). Both proteins have been shown to be in vitro translation products of IAP RNAs with ~73 translated from a type I RNA of 7.2 kb and ~120 from a type I variant RNA (Ial) of 5.4 kb (Paterson et ab, 1978; Kuff and Fewell, 1985). IAP are expressed as part of the normal program of preimplantation development (Calarco and Szollosi, 1973). Biochemical analyses of mouse embryos have described the synthesis of the ~73 core protein (Huang and Calarco, 1981a) and the presence of a 5.4-kb RNA in blastocysts (Piko et al, 1984). In this paper we show that the IAP core protein, ~73, is present throughout preimplantation development while ~120 is present only from the 8-cell stage onward. Although type I and type II genes are expressed at all stages, the expression of subtypes of these major classes varies according to stage. Evidence also suggests that type I and II transcripts and ~73 are inherited maternally. MATERIALS AND METHODS Embryo culture. Two-cell embryos were flushed from the oviducts of superovulated female ICR mice (12 weeks old; Harlan Sprague-Dawley, Inc.) and cultured as described previously (Calarco, 1975). Unfertilized eggs were obtained from superovulated, unmated ICR females; follicle cells were removed by hyaluronidase (75 units/ml, 5 min, Sigma). Antisera. Five different antisera were used in our comparison of IAP proteins: (1) Anti-IAP (A’IAP), a polyclonal rabbit antiserum made against a preparation of myeloma (MOPC-104E) IAP was prepared as previously described (Huang and Calarco, 1981b). (2) Anti-p73 (A1.3), a polyclonal rabbit antiserum, raised against electrophoretically purified IAP gag core protein (Kuff et ah, 1980). (3-5) Anti-peptide antisera (anti-6016, anti6020, and anti-6021) are polyclonal rabbit antisera made against ovalbumin-conjugated synthetic peptides (BIOSEARCH) whose amino acid sequences were derived from the gag region of a cDNA clone (8.3) of an IAP

element (Moore et ah, 1986). Peptide 6016 is: (C) ETVKAALPSMGKYM(nucleotides 910-957). Peptide 6020 is: (C) GRVHAPVEYLQIKEI (nucleotides 772 to 816 of clone 8.3). Peptide 6021 is: (C) GEGQFADWPQGSRLQ (nucleotides 562 to 606 of clone 8.3). Normal rabbit serum and IgG fractions were prepared as previously described (Huang and Calarco, 1981b). Morphology. Visualization of IAP was by techniques described previously: negative staining (Huang and Calarco, 1981a); transmission electron microscopy (Calarco and Szollosi, 1973); indirect immunofluorescence on sections (Sutherland et al, 1988). Protein kbeling and immunoprecipitation. Approximately 100 embryos were washed with methionine-free medium, labeled in a lo-p1 drop of methionine-free medium containing 100 PCi of [35S]methionine (spec. act. 1 mCi/mmol, Amersham) for 4 hr (37”C, 5% CO,), and rinsed. Extracts were prepared using a modification of the technique of Kuff and Fewell (1985). Labeled embryos were collected, washed, and lysed in 100 ~1 of lysis buffer (LB) (1% NP-40, 0.4% sodium deoxycholate, 30 mM Tris/HCl, pH 7.4,2, mM EDTA, 2 mM EGTA, 1.5 M NaCl, 0.5 mM PMSF) on ice for 5 min. The lysate was denatured by heating at 100°C for 3 min after the addition of SDS (final concentration 0.5%) and DTT (final concentration 1 mI@). After eentrifugation, iodoacetimide was added to the supernatant (final concentration 5 mM) in order to eliminate excess DTT and to alkylate proteins. Lysates were preabsorbed for 15 min on ice with 10 ~1 of normal rabbit serum (final concentration 1 mg/ml) and precipitated with Protein A Sepharose beads (Pharmacia). After addition of 10 ~1of normal or primary antisera (final concentration of 0.5 pg/ml) the samples were incubated at 4°C overnight, precipitated with Protein A Sepharose beads, washed twice with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.1 M sodium phosphate, pH 7.2,lO mM EDTA), and twice with a solution containing 0.05% NP40, 0.15 M NaCl, 50 mM Tris/HCl, pH 7.4, and 5 mM EDTA. The immunoprecipitated proteins were eluted with 40 ~1 of SDS sample buffer and analyzed on 7.5% polyacrylamide slab gels (Laemmli, 1970). Western blotting. Embryos were rinsed 3X in culture medium containing 3 mg/ml polyvinylpyrolidine in place of BSA. Lysates were prepared as described above and separated by 7.5% SDS-PAGE. Separated proteins were transferred to nitrocellulose by electroblotting (Towbin et al., 1979). Blots were incubated in BLOTTO (Johnson ef uZ., 1984) and 5% normal goat serum overnight at 4°C to block nonspecific reactivity. After incubation in primary antibody (1:lOO in BLOTTO, 1 hr, room temperature), the blots were washed with 0.1% Tween-20 in PBS and incubated in secondary antibody (1:500 BLOTTO, room temperature, 1 hr). Secondary an-

tibodies used were conjugated either to biotin or to horseradish peroxidase. Detection of biotinylated secondary antibody followed incubation in an avidin-conjugated alkaline phosphatase (Vector, 1:500 BLOTTO, for 30 min), rinsing, development (phosphatase substrate, 30 min, Vectastain ABC-AP), and a final rinse. Horseradish peroxidase-conjugated secondary antibody was detected by incubation in 0.05% 3,3’-diaminobenzadine in PBS, 0.02% cobalt chloride, 0.03% H,O,. Southern blotting and hybridization. Blotting was done according to Maniatis et al. (1982) and Church and Gilbert (1984). Hybridization of Southern blots is performed according to Church and Gilbert (1984). For the detection of both type I and type II sequences, an IAP plasmid pMIA1 (Lueders and Kuff, 1980) was used as a probe containing 5.2 kb of internal sequence from a genomic IAP sequence. For the detection of type I sequences a type I-specific probe, IAP 81 (Cole et al., 1981), was used. For the type II-specific IAP probe the plasmid pTAII.1 comprised of three copies of a 303-bp Hue111 fragment containing the type II-specific insertion AIIins (Sheng-Ong and Cole, 1984) and 19 bp of type I sequence was used. The mouse cDNA plasmid PAM91 (Minty et al., 1981) was used to detect actin. Plasmids were labeled by nick translation (BRL kit) to a specific activity of 2 X 10’ cpm/hg of DNA, using (LY-~‘P)dCTP (6000 Ci/mmole, NEN). RNA slot blotting. The procedure of RNA slot blotting was according to Piko et al. (1984). Embryo RNA slot blots were stripped, rehybridized with a ,&actin probe, and scanned with a laser-densitometer. The ratio of the amount of @actin RNA per embryo at the 8-cell stage to the blastocyst stage is 1 to 13.3. From the ratio of the densitometric signals from the 8-cell and blastocyst samples on the autoradiographs of the slot blots hybridized with the actin probe, the ratio of embryo RNA equivalents between these stages was deduced. This factor was used to normalize the densitometric signals from the same slot blots hybridized to the IAP probes and these values were used to compare relative transcription of type I IAP sequences at these stages. Reverse transcription and the po&merase chain reaction (PCR) (Saiki et al., 19&j’).RNA was isolated according to Rappolee et al., 1988 with the following modifications. The RNA was taken up in RQl-DNAse buffer (40 mM Tris/HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl,), digested with 2 pl(2 units) of RQl-DNAse (Promega Biotee) for 2 hours at 37°C and extracted once with phenol/ chloroform/isoamyl alcohol (24:24:1) and once with chloroform/isoamyl alcohol (24:l). The RNA was precipitated by a l/10 vol of 3 M NaOAc and a 2.5 vol of EtOH (20°C overnight), pelleted in a microfuge (15 min, 4”C), washed twice in cold 80% EtOH, dried in a speedvac, and finally resuspended in 20 ~1 of DEPC-treated dH,O.

First strand synthesis was according to Frohman et uh, 1988. Primers. The sequence of primers used for the IAP detection is as follows: APl, 5’-AGCAGGTGAAGCCACTG-3’; AP2, 5’-CAA TCCCTCTGCAGCTC-3’; AP3, 5’-CTTGCCACACTTAGAGC-3’. Primers APl and AP3 correspond to nucleotide positions 1652-1668 and 20512067 in MIA14, respectively (Mietz et ul., 1987), a full length type I IAP gene. Primer AP2 corresponds to nucleotide positions 164-180 within the type II-specific AIIins sequence in the cloned IAP gene 113(Lueders and Mietz, 1986). P-actin primers span an exoniintron border of the fl-actin gene such that the PCR amplification product of the cDNA is 243 bp and that of the genomic DNA is 373 bp. PCR. PCR was done according to Frohman et al., 1988. An annealing temperature of 50°C was used for 40 cycles on 1/20th of each embryo reverse-transcribed sample. Restriction endonuclease digestion of PCR products. The contents of the PCR tube are placed on parafilm and transferred repeatedly to a new spot until separated from the mineral oil. The products are then passed over a spun column of Sephadex G-50 in TE with the volume subsequently decreased by speedvac. The products are cut with internal enzymes (Promega, Madison, WI) for 1 hr at 37°C; SucI is used to cut the type I PCR products and AZuI is used to cut the type II PCR products. Both cut and uncut samples are analyzed on a 2% agarose gel containing ethidium bromide. RESULTS Mwpholopy

and Distribution of IAP

IAP isolated from a myeloma cell line (MOPC-104E) and negatively stained exhibit the expected spherical structure of these ER-enclosed retroviruses (Fig. 1A). Transmission electron microscopy localized these viruses in ribosome-free ER cisternae of the 2-cell mouse embryo (Fig. 18). Of the three peptide-specific antisera made available to us, 6020 and 6021 readily detected cytoplasmic antigen immunocytochemically. Two-cell embryos had several positive immunoreactive regions in their cytoplasm while some antigen was seen on the cell surface (Figs. lC-1E). Older embryos, in addition, showed reactivity in the perinuclear cisternae (Fig. lD), as noted previously in IAP-expressing cell lines (Huang and Calarco, 1982). Detection of IAP Proteins Protein blot analysis using either an HRP-mediated or an alkaline phosphatase-mediated detection system shows that ~73, the IAP core protein, is present from the

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FIG. 2. Immunodetcction of IAP-related proteins on protein blots of mouse unfertilized eggs and embryos by various anti-IAP antisera. IAP-related proteins of unfertilized egos (IJE) and zygotes (Z) were detected with anti-6021 and A’IAP antisera. IAP-related proteins of 2-cell embryos were detected with anti-6016 anti-6020, anti-6021, and A’IAP antisera. IAP-related proteins of 8-cell emhrh-os were detected with anti-6020 and anti-6021 antisera. The 73%kDa IAP guy-related protein is recognized in all stages examined. Additional proteins are recognized by the various anti-IAP antisera: 60 kDa (anti-6020), X3 kDa and 100 kDa (A’IAP). Nonspecific proteins (approximately 160 and 90 kDa) are detected hy the hiotin/streptavidin-based detection systtrm regardless of the primary antiserum which is used. A normal rabbit serum (NRS) control immunoreaction is shown for the UE and 2-cell stages. NRS controls for the other stages were similarly negative. Molecular weight X 10m3 is indicated in the margins; markers are mgosin (200 kDa), phosphorylasc b (9’7.4 kDal. and bovine serum albumin (68 kDa).

unfertilized egg through the &cell stage (Figs. 2,3). The p73 protein is recognized prominently by the anti-peptide antisera, anti-6020 and anti-6021, and less so, but detectably, by A’IAP (Figs. 2,3). The core protein is recognized faintly, if at all, by anti-6016. Although all antisera have in common the recognition of ~73, additional proteins are also detected by the different antisera: a p43 by anti-6021, a p60 by anti-6020, and a ~83 as well as a ~100 by A’IAP. When the three anti-peptide antisera were used to immunoprecipitate IAP antigens, anti-6020 gave the most consistent results, so subsequent experiments used this antibody exclusively. Anti-6020 precipitates a prominent 120,000 M, gag-pal fusion protein (p120) from [35S]methionine-labeled embryos and from MOPC-315, an IAP-producing cell line (Fig. 4). Although ~120 is present in g-cell and blastocyst embryos, it is not obvi-

4

NRS

6016

6020

6021

97.4

68

43

A’IAP

FIG. 3. Comparison of immunodetection systems on protein blots of &cell mouse embryos. Anti-6016, anti-6020, anti-6021, A’IAP, and normal rahhit serum (NRS) were used with either a hiotin/streptavidinbawd detection system (ABC-AP, Vector) or with a secondary antibody directly conjugated to horseradish peroxidase (CAR-HRP). The 73kDa IAP f/tfg-related protein is recognized by antisera using both secondary detection systems, as the additional proteins are recognized hy specific anti-IAP antisera: 60 kDa (anti-6020), 43 kDa (anti-6021). Nonspecific proteins (160 and 90 kDa) are detected by the hiotin/ streptavidin-based detection system (ABC-AP) regardless of the primary antiserum which is used. These proteins are not detected when a goat anti-rabbit antiserum conjugated to horseradish peroxidase (CAR-HRP) is used as the secondary detection system. Molecular weight X10m3 is indicated in the margins; markers arc myosin (200 kDa), phosphorylase h (97.4 kDa), bovine serum alhumin (68 kDa), and ovalbumin (43 kDa).

ous in the unfertilized egg or the 2-cell stage. Previously it has been shown that anti-6020 antiserum is poor at precipitating the IAP core protein, ~73, from neoplastic cell lines (Kuff et (xl., 1986), and we have found this to be the case in embryos as well, i.e., p73 is only faintly detected by anti-6020 antiserum.

RNA slot blots of 2-cell- through blastocyst-stage embryos were hybridized with type I- and type II-specific 32P-labeled DNA probes. The blots were then stripped and rehybridized with an a-a&in cDNA probe (Minty et crl., 1981). On the basis of densitometric tracings of the

FIG. 1. Intracisternal A particles visualized by several techniques. (A) IAP isolated from MOPC104E in a negatively stained preparation using phosphotungstic acid. The particles are composed of two concentric shells, the outer ER-derived membrane of 100 nm in diameter and an inner core of 50 nm diameter. (B) Transmission electron micrograph illustrating a region of many IAP budding into tht: endoplasmic reticulum (er) in a 2.~11 mouse embryo. M, mitochondria; N, nucleus. (C) IAP visualized by anti-6020 and indirect immunofluorescence in a section of a 2-cell embryo. two positive regions arc denoted hy arrowheads. N, nucleus. (D) Section from an X-cell embryo prepared in the same manner as C. Arrowhead denotes visible surface fluorescence surrounding polar body, arrow indicates a rexion of perinuclear fluorescence. (E) Section from an X-cell embryo prepared in the same manner as C and visualized hy anti-6021 and indirect immunofluorescence. Surface fluorescence is visible as are positive cytoplasmic regions (arrowhead). (F) Control srrtion of a 2-cell embryo treated with normal rabbit serum and processed for indirect immunofluorescence.

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which proved free of genomic DNA contamination (see below) were examined for type-specific sequences by PCR. The results show that type I sequences are present at all stages, from the unfertilized egg through the blastocyst (Fig. ‘7, lanes 2-5), while the presence of type II sequences varies according to stage. Both type II PCR products, the 242 and the 292 bp, are present in the unA

FIG. 4. Immunoprecipitation of IAP-related antigens from cells and embryos. Anti-6020 antiserum was used to immunoprecipitate IAPrelated antigens from [%]methionine-labeled unfertilized eggs, 2-cell embryos, &cell embryos, blastocysts, and IAP-producing MOPC-315 cells. The 120-kDa IAP gag-pal fusion protein is prominent in immunoprecipitates of the g-cell and blastocyst stages and of the MOPC-315 plasmacytoma cell line. The 73-kDa IAP guy-related protein is recognized faintly by this particular antiserum. Molecular weight ~10~~ is indicated in the margins; markers are phosphorylase b (97.4 kDa) and bovine serum albumin (68 kDa).

autoradiographs the relative levels of transcription of type I and type II IAP genes were estimated at the g-cell and blastocyst stages relative to known transcript levels of a-actin from the data of Giebelhaus et al. (1983). The results demonstrate that both type I and type II IAP genes are present at all stages examined, although the signal from the 2-cell stage was too faint for densitometric analysis (Fig. 5A). By comparison with the hybridization to the a-actin probe the relative amounts of type I and type II IAP transcripts were determined. There appears to be a 40-fold increase in type I IAP transcripts and a 16-fold increase in type II transcripts per embryo seen between the 8-cell and blastocyst stages. Although IAP transcription increases on a per embryo basis from the 8-cell through the blastocyst stage for both type I (Fig. 5B) and type II (Fig. 5C) sequences, the amount of transcription per cell is roughly similar. In order to determine whether both types I and II genes are expressed during development DNA oligonucleotide primers were designed which, when used in PCR, amplified IAP sequences within cDNAs synthesized from the RNA of preimplantation mouse embryos. The strategy which we used to design primers for PCR was to choose sequences which were unique to either type I or type II sequences for the (3’) downstream primers and a primer complementary to a sequence common to both types I and II for the (5’) upstream primer. This allows the prediction of products which can be distinguished on the basis of size: 417 bp for type I IAP amplified sequences and 242 and 292 bp for type II IAP amplified sequences, the smaller product having a second deletion of 50 bp (Fig. 6). Embryo cDNA samples

B

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Type I IAP Transcripts '21

8 cell

Blastocyst embryo stage

n

Type II

8 cell

IAP Transcripts

Blastocyst embryo stage

FIG. 5. (A) RNA slot blots of 2-cell (20 through blastocyst (BL) stage embryos hybridized with type I- and type II-specific s2P-labeled IAP DNA probes. The slots represent 3000 2-cell embryos, 1060 S-cell (SC) embryos, and 609 blastocysts. The type I IAP probe was the 1.0.kb Sst to Bar~Hl sequence clone and the type II probe was pTAII.1. The autoradiograph shown here was overexposed to show the presence of 2-cell transcripts. (B, C) Laser densitometry scans were performed on autoradiographs exposed to give signals for the stages which were within a linear range of detection. Histograms of relative RNA levels were generated by comparison with the signal from the same blots after stripping and rehybridization with a fi-actin probe. (B) Relative transcription of type I IAP sequences at the 8-cell and blastocyst stages on a per embryo and a per cell basis. (C) Relative transcription of type II IAP sequences at the 8-cell and blastocyst stages on a per embryo and a per cell basis.

r--

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of the amplified sequences to 32P-labeled IAP probes confirmed their identities as IAP cDNA sequences (data not shown). Additional confirmation of the identity of type I IAP sequences was provided by restriction endonuclease digestion with SucI to predicted size fragments of 361 and 54 bp (Fig. 9). The identity of type II IAP amplified sequences was confirmed by digestion with AluI to predicted size fragments of 78 bp and either 214 or 164 bp (Fig. 9). The question of genomic contamination was addressed in two ways. First, a control amplification used primers which span an exon/intron border of the @-actin gene. These /j-actin primers give products which can be distinguished as amplifications of cDNA templates or contaminating genomic DNA on the basis of size (data not shown). However, due to the high copy number of IAP genes (1000 copies per haploid genome) a negative result for genomic DNA contamination at the level D-actin represents was felt to be inadequate. A second control was performed using IAP primers to amplify any IAP gene sequences present as contaminating genomic DNA in our RNA samples. This was done by subjecting half of each RNA sample, after treatment with RNase-free DNase, to alkaline hydrolysis which removes all RNA. Thus, the presence of a signal would indicate genomic contamination. Only samples negative by this test were used (Fig. 7). DISUJSSION

AND

292

BP

FIG. 6. Polgmerase chain reaction primer design. Sequences unique to type I (.4P3) and type II (AP2) IAP sequences were used as the downstream primers. A sequence common to type I and type II IAP sequences was used as the upstream primer (API 1. All primers are 17 nucleotides in length. The amplification products predicted hg this strategy are 117 hp for type I sequences and 242 and 292 bp for type II sequences (50 bp are deleted in some type II sequences resulting in the UO-hp amplification product). Primer AP2 is located within the type II-specific rlIIins sequence (indicated by the hatched box).

fertilized egg, the &cell embryo, and the blastocyst (Fig. 7, lanes 12, 14, and 15). At the 2-cell stage type II sequences are undetectable by ethidium bromide staining (Fig. 7, lane 13). However, when Southern transfers of the amplified sequences are hybridized with 32P-labeled type II DNA probes, it can be seen that the smaller of the two type II products (242 bp) is present at the 2-cell stage while the larger (292 bp) is not (Fig. 8). The identity of the amplified IAP sequences was confirmed in two ways; by hybridization of type I- and type II-specific IAP DNA probes to Southern transfers of the amplified sequences and by restriction digestion of the amplified sequences to predicted sizes, based on their restriction maps. Hybridization of Southern transfers

IAP Protei?a The results from protein-blotting experiments indicate that throughout the course of preimplantation development, from the unfertilized egg through the blastocyst stage, IAP-related proteins are present in the mouse embryo. both the ~73 gag protein and the ~120 gag-p01 fusion protein are detected by the anti-IAP antisera used, although the different antisera vary in their ability to detect these proteins. The titers of the antipeptide antisera against electrophoretically purified ~73 in an enzyme-linked solid-phase immunoassay (Moore et (I(., 1986) correspond in general to their activity against ~73 on Western blots of preimplantation embryos seen here, with the weakest reactivity seen with the anti-6016. The lack of detection of ~120 by any of the anti-IAP antisera on protein blots suggests that this protein is present in very small amounts, or it fails to blot, or it is unrecognizable by these antisera in this particular assay. All antisera have in common the recognition of ~73, but additional proteins are recognized by specific antisera. The ~100 and p43 may correspond to proteins found in minor amounts in IAP cores (~100, ~45, and ~30). The p45 shares tryptic peptides of the core protein

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FIG. 7. Detection of IAP mRNA by amplification of mouse unfertilized egg (UE) and embryo cDNAs by the polymerase chain reaction (PCR) using type I- and type II-specific IAP primers. The PCR products are shown on ethidium bromide-stained agarose gels. Type I amplification product (417 bp) from UE (lane Z), 2-cell (lane 3), g-cell (lane 4), and blastocyst (lane 5). Type II amplification products (242,292 bp) from UE (lane 12),2-cell (lane 13), g-cell (lane 14), and blastocyst (lane 15). Amplification of samples treated by alkaline hydrolysis to control for genomic contamination (see text). UE (lane 7), 2-cell (lane 8), 8-cell (lane 9), and blastocyst (lane IO).

and may represent specific or nonspecific proteolytic fragments of p73 (Marciani and Kuff, 1974). A ~47 has product of the been shown to be the in vitro translation integrase region of a type II IAP gene (Lueders et al., 1989), but we would not expect to recognize this protein with our antisera made against peptides of the gag region. Some of these additional proteins also may correspond to antigens detected by two peptide-specific antisera (anti-6020, anti-6021) on the surface of embryos, a finding that corroborates the earlier detection of surface antigen by A’IAP (Huang and Calarco, 1981b). When IAP proteins are immunoprecipitated from [35S]methionine-labeled embryos the data show that ~73 is synthesized from the 2-cell through the blastocyst stage with little to no synthesis in the unfertilized egg (these data; Huang and Calarco 1981a), while the 120kDa protein is synthesized only from the S-cell through the blastocyst stage. Again, the different peptide-spe-

cific antisera differ in their ability to immunoprecipate both antigens. For example, by immunoprecipitation anti-6020 does not strongly recognize the IAP core protein, ~73, of embryos (our data) nor of N4 or MOPC-104E cells (Kuff et al., 1986), but does recognize ~73 in an enzyme-linked solid-phase immunoassay (Moore et al., 1986), suggesting that access of the anti-6020 to ~73 may be different. However, the ability of anti-6020 to im-

Type

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49

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bp bp

50

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3-C

FIG. 8. Southern transfer of type II IAP products amplified from 2-cell (Z-C) and &cell (8-C) embryo cDNA and hybridized with a type II-specific IAP probe, pTAII.1. Two PCR products of 292 and 242 bp are detected in the &cell amplification while only the 242-bp PCR product is detected in the 2-cell amplification.

FIG. 9. Restriction endonuclease digestion of PCR products. The identity of amplified IAP sequences is confirmed by restriction digestion to predicted sizes based on their restriction maps. Type I IAP products are digested with Sac1 to fragments of 366 and 49 bp. Type II IAP products are digested with AluI to fragments of 78 bp and either 214 or 164 bp.

POZNANSKIANDCALARCO

IAP i?l thr Earl!/ Mouse Enllwgo

munoprecipitate ~120 in embryos of preimplantation stages is greater than that of the other two anti-peptide antisera, and greater than that of A’IAP.

IAP RNA The data from the embryo RNA slot blots represent the first evidence for the presence of both IAP type I and II sequences in preimplantation mouse development and suggest that although total levels of transcript increase during development, the transcript level is relatively constant on a per cell basis. Our analysis of relative transcription corroborates and extends the data of Piko et al. (1984) in which the amount of 3H-labeled IAP-related RNA was assayed by hybridization to filter-bound cloned IAP genes. They found that the rate of synthesis of IAP RNA per embryo increases about 30-fold during preimplantation development but that the rate of synthesis per cell remains nearly the same, about 4 fg per cell over a 5-hr period. Our results from PCR argue that IAP type-specific gene transcription varies during preimplantation development in the mouse. Sequence data predict a PCR product of 41’7 bp for transcripts derived from type I IAP genes and this product is present at all stages examined, from the unfertilized egg through the blastocyst. The detection of ~120 was particularly interesting since it represents a gag-pal fusion protein translated from a variant type I IAP gene (type IA1 transcript of 5.4 kb) (Paterson et al., 1978; Kuff and Fewell, 1985) and has never before been reported in cleavage-stage embryos. The probes we used detect both the full length 7.2-kb and the 5.4-kb type I IAP transcripts, but it seems likely that transcription of IA1 IAP elements begins at approximately the g-cell stage, although the presence of ~120 could represent new transcription or translation from a preexisting IA1 message. The presence of an IAP RNA of 5.4 kb has previously been reported in blastocysts (Piko et al., 1984) and in situ hybridization with a type I IAP probe also detects RNA at this time (Moshier et al., 1985). Sequence data predict PCR products of 242 and 292 bp for transcripts derived from type II IAP genes. Both these products are present at all stages other than the Z-cell stage. At the two-cell stage, however, only the smaller product of 242 bp is present, as determined by the more sensitive Southern analysis. Since maternal transcripts decrease dramatically at the 2-cell stage (Bachvarova and De Leon, 1980), the absence of the larger type II transcript may represent such a loss prior to embryonic transcriptional activation. The reappearance of the larger type II transcript subsequent to the 2-cell stage implies new transcription of this type II

279

subspecies. Our analysis of unfertilized eggs strongly suggests that ~73, the 7.2-kb type I RNA, and both type II RNA subspecies are inherited maternally with zygotic transcription of type I and II genes beginning at least subsequent to the 2-cell stage. This is consistent with reports of IAP expression in polar bodies (Yang et al., 1975), parthenogenetically activated eggs (Biczysko et al., 1973), and n-amanitin-treated embryos (Calarco, 1975).

IAP and Develqment Two types of IAP have been reported in mouse embryos; a particle with a small core, abundant at the 2cell stage, designated “epsilon” (Yotsuyanagi and Szollosi, 1981) or “small A” (Chase and Piko, 1973), and a particle with a larger core present in much lower numbers in the oocyte and later cleavage stages, designated “large A” (Chase and Piko, 1973). Several hypotheses could explain the appearance of abundant epsilon particles exclusively at the 2-cell stage: (1) The absence of the larger type II transcript, reported here, favors the production of epsilon particles; (2) Maternally inherited transcripts form the epsilon particles while zygotic transcripts form the large A particles; (3) The presence of ~120, and by inference the type IA1 variant, from the 8-cell stage onward, reported here, favors production of large A particles; (4) Maturation of the ER subsequent to the 2-cell stage may interfere with epsilon particle formation; (5) The epsilon particles seen at the 2-cell stage may be a different virus. Our results do not distinguish between these hypotheses. A resolution of the relationship between these two types of particles awaits isolation of the two particles from embryos and comparison of their RNA genomes. IAP-related genetic information makes up approximately 0.3% of the total genome in Mus musculus. Further, a low level of IAP transcription is constitutive for many normal mouse tissues but enhanced IAP expression is characteristic of only three situations: (1) certain normal proliferating cells, such as immature thymocytes, (2) neoplastic mouse cell lines, and (3) early embryonic development. What can the expression of these repetitive IAP genes tell us about development? At least four possible reasons for IAP gene expression during early development can be envisioned. First, the location of particular IAP genes with respect to actively transcribed cellular genes may bring the IAP sequences under the control of cellular regulatory sequences. Second, particular IAP genes could be located in regions that were favored for transcription due to some local conformation or opening of the chromatin (possibly due to hypomethylation). Third, the regulatory sequences of

280

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the IAP genes themselves may be responding to the presence of trans-acting factors present during this time. Comparison of factors acting upon IAP genes with those that act upon other early transcribed genes in the mouse might lend clues about the initial activation of the embryonic genome. The presumption in this case is that the embryonic cell produces these factors to ensure the expression of cellular genes. Fourth, the IAP transcripts or related proteins may actually have a function. In summary, IAP expression brings to our attention that these vertebrate retrotransposons are present during early developmental stages. Expression as part of the normal program of early development may explain the dispersion of these sequences throughout the murine genome and suggests how they may play a role in contributing to genetic variability during murine evolution. Although the information encoded by IAP genes may be only tolerated by the embryo, their expression during the earliest stages of development may be useful in designing strategies to examine other genes active during this same period. This work was made possible by generous gifts from our colleagues. We would like to thank Ed Kuff and Kira Lueders (NCI) for providing antisera A 1.3 and plasmids PMIAl and pTAII.1, Kevin Moore (DNAX) for providing antisera to peptides 6016, 6020, and 6021, Ru Chih Huang (Johns Hopkins) for providing plasmid IAP 81, Randall Moon (University of Seattle) for providing mouse n-actin cDNA plasmid pAM91, Dan Rappolee (UCSF) for providing p-actin primers, and Kira Lueders (NCI) and Mike Frohman (UCSF) for their advice and comments on the manuscript. REFERENCES BACHVAROVA, R., and DE LEON, V. (1980). Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev. Bid. 74, 1-8. BICZYSKO,W., PIENKOWSKI, M., SOLTER, D., and KOPROWSKI, H. (1973). Synthesis of endogenous type A virus particles in parthenogenetically stimulated mouse eggs. J. Nutl. Colccer h/.sl-.52, 483-489. BURT, D. W., REITH, A. D., and BRAMMER, W. J. (1984). A retroviral provirus closely associated with the Ren-2 gene of DBA/B mice. Nucleic Acids Res. 12, 85798593. CALARCO, P. G. (1975). Intracisternal A particles formation and inhibition in preimplantation mouse embryos. Biol. Reprod. 12, 448-454. CALARCO, P. G., and SZOLLOSI,D. (1973). Intracisternal A particles in ova and preimplantation stages of the mouse. Nuturc (London) 243, 91-93. CALLAHAN, R., KUFF, E. L., LUEDERS, K. K., and BIRKENMEIER (1981). Genetic relationship between the Mus cervicolor M432 retrovirus and the Mus musculus intracisternal type A particle. J. Viral. 40, 901-911. CHASE, D. G., and PIKO, L. (1973). Expression of A- and C-type particles in early mouse embryos. J. Notl. Cuvcer Znsf. 51, 1971-1975. CHIU, I. M., HUANG, R. C., and AARONSON, S. A. (1985). Genetic relatedness between intracisternal A particles and other major oncovirus genera. Virus Res. 3, l-11. CHURCH, G. M., and GILBERT, W. (1984). Genomic sequencing. PNAS 81,1991-1995. COLE, M. D., ONO, M., and HUANG, R. C. C. (1981). Terminally redun-

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