- Email: [email protected]
Structure, sequence and expression of the mouse Cx43 gene encoding connexin 43
Gene, 130 (1993) 191-199 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00
191
GENE 072 18
Structure, sequence and expression of the mouse Cx43 gene encoding connexin 43 (Gap junction; embryo; intron; trans~~ption initiation; promoter; APl; immediate early gene; membrane chann~is)
Ruth Sullivan, Christine Ruangvoravat, and Cecilia W. Lo
Daniel Joo, Judy Morgan, Baa Lin Wang, Xin Kang Wang
Biology Department, University of Pennsylvania, Philadelphia, PA 19104-6017, USA
Received by D.T. Denhardt: 27 November 1992; Revised/Accepted: 18 February/l9 February 1993; Received at publishers: 19 April 1993
SUMMARY
Gap junctions, membrane channels that mediate the diffusion of ions and small molecules between cells, are hypothesized to play a role in development and growth regulation. The Cx43 gene (encoding connexin 43) is one member of the gap junction gene family whose transcripts are expressed in a highly regionalized manner during mouse development. We cloned and sequenced Cx43 cDNAs from a 7.5-day mouse embryo cDNA library. These cDNA clones encode the authentic 43-kDa connexin. Analysis of RNA isolated from different regions of the 7.5day mouse embryo revealed that Cx43 transcripts are differentially expressed, with expression detected in the embryo proper, but not in the extraembryonic region containing the ectoplacental cone. Using one of the newly isolated mouse Cx43 cDNA probes, we screened a mouse genomic DNA library and cloned the Cx43 gene. Restriction mapping and sequencing of the cloned genomic inserts revealed that Cx43 contains two exons and a 10.5-kb intron located in the 5’ untranslated region (S-U’ZX). We mapped the Cx43 transcription start point (tsp) by RNase protection and primer extension analyses and showed that transcripts expressed in the 7.5-day mouse embryo and in adult tissues are initiated from the same tsp. The DNA sequence immediately upstream from the tsp contains a putative API-binding site and a degenerate TATA consensus sequence. A comparison of mouse, rat, human and bovine Cx43s showed that the 3’4JTR has an unexpectedly high degree of sequence homology. This includes conservation of four AUUUA motifs, a sequence associated with transcript instability in immediate early genes. FIowever, further analysis of Cx43 expression in NIH 3T3 and BALB/c 3T3 cells showed that Cx43 expression is not subject to serum induction, as has been found for the immediate early genes.
INTRODUCTION
Gap junctions are composed of membrane channels that allow the direct cell-to-cell transfer of small cytoCorrespondence
to: Dr. C.W. Lo, Biology Department, Goddard Laboratory, University of Pennsylv~i~ P~ladelphia, PA 191~~17, USA. Tel. (1-215) 898-8394; Fax (l-215) 898-8780; e-mail: [email protected]
Abbreviations: aa, amino acid(s); AMV, avian myeloblastosis virus; bp, base pair(s); CHX, cycloheximide; Cx, connexin(s); Cx, gene encoding Cx; FBS, fetal bovine serum; kb, kilobase or 1000 bp; nt, nucleotide(s); TPA, 12-0-tetradecanoylphorbol-13-acetate; tsp, transcription start point(s); UTR, untranslated region.
plasmic components such as ions, metabolic intermediates, and second messengers. The proteins that make up these junctions are encoded by a multigene family referred to as the connexins (Cx; for review see Beyer et al., 1990; Willecke et al., 1991). Analysis of various Cx polypeptides and the gap junctions they generate has revealed differences in their tissue distribution, permeability and gating properties, and sensitivity to modulation by factors such as pH and phosphorylation (for review see Bennett et al., 1991; Beyer et al., 1990). These obse~ations suggest that gap junctional communication and the differential expression of different Cx polypeptides may play an important role in many diverse physiological processes.
192 This may include the coordination of cell metabolism, growth regulation, and development. In the mouse embryo, Cx43 proteins and transcripts are both expressed in a highly regionalized manner (Ruangvoravat and Lo, 1992; Yancey et al., 1992). Thus, using in situ hybridization, we have shown that Cx43 transcripts are expressed in spatially restricted domains in many regions of the embryo undergoing tissue inductive events (Ruangvoravat and Lo, 1992). Additionally, in the limb bud and developing hindbrain, Cx43 transcripts were expressed in a graded pattern similar to the gradient of Hole gene transcripts found in these regions of the embryo. These in situ hybridization results, in conjunction with observations derived from Cx43 immunostaining studies (Yancey et al., 1992), suggest that Cx43mediated gap junctional communication may participate in many aspects of mammalian embryogenesis. More definitive insights into the possible role of Cx43 in mammalian development may be obtained with the use of molecular approaches to disrupt or otherwise manipulate the expression of Cx43 in a developmentally regulated manner. It is with this goal in mind that we cloned and characterized the mouse Cx43 gene. We began the present study with the cloning and characterization of a Cx43 cDNA from a 7.5day mouse embryo cDNA library. Using the mouse Cx43 cDNA probe, a Northern analysis was carried out to examine the expression of Cx43 transcripts in the mouse embryo. We also screened a genomic DNA library and cloned the mouse Cx43 gene gene. Its structure, sequence, and tsp were determined. As our analysis revealed that the 3’UTR of Cx43 contains four conserved AUUUA motifs, a sequence associated with transcript instability in immediate early genes (Shaw and Kamen, 1986; Jones et al., 1988; Schuler and Cole, 1988; Wilson and Treisman, 1988; Shyu et al., 1989; 1991), we carried out additional experiments to determine whether 043 may be inducible by serum stimulation, as is typical for immediate early genes.
RESULTS AND DISCUSSION
(a) Cloning and characterization of Cx43 cDNA Using the rat Cx43 cDNA probe, pG2A (provided by David Paul; Beyer et al., 19871, we screened a mouse embryo cDNA library made from RNA isolated from the embryonic portion of the 7.5-day mouse conceptus (provided by Janet Rossant). Five positive plaques were obtained, and the two containing the largest EcoRI inserts (2.0 and 2.1 kb) were subcloned into plasmid vectors and sequenced in their entirety (data not shown; GenBank accession No. X61576). The sequences of the two clones were identical and showed extensive homol-
ogy to adult rat and mouse Cx43 cDNA, in both the coding (Beyer et al., 1987; Beyer and Steinberg, 1991; Lang et al., 1991; Henneman et al., 1992b) and noncoding regions (Lang et al., 1991). Both cDNAs encode the same polypeptide consisting of 382 aa (43 kDa; data not shown). The coding region of our Cx43 clones is identical to the mouse genomic Cx43 sequence reported by Henneman et al. (1992b). Although we observed three conservative nt changes when compared with the Cx43 cDNA sequence of Beyer and Steinberg (199 1) (positions 9, 15, and 1146 relative to the start codon at position I), these substitutions all lie within the primers used by Beyer and Steinberg (1991) to PCR amplify and clone the mouse Cx43 coding region. Our mouse Cx43 peptide sequence and also that of Beyer and Steinberg (1991) and Henneman et al. (199217)differ from adult rat Cx43 by a single aa (Lang et al., 1991); in mouse Cx43, Asn is found in place of Ser 341. It is interesting to note that another previously published mouse Cx43 sequence (Nishi et al., 1991) was reported to contain a Ser341 and, in addition, a second aa difference - a Thr in place of Met3** (data not shown). As the cDNA sequence of Nishi et al. (1991) showed numerous mismatches when compared with the mouse Cx43 sequence from this study and that of the other two groups (Beyer and Steinberg, 1991; Henneman et al., 1992b), it is not possible to evaluate the basis for the latter aa differences. The cDNA clones we have isolated do not represent full-length cDNAs, as Cx43 transcripts are over 3.0 kb in size (Beyer et al., 1987). Given that both cDNA inserts lacked a polyadenylation signal sequence and were terminated at the same position within the 3’-UTR (911 nt from the stop codon; data not shown), it is likely that the 3’ ends of these cDNA clones represent truncations at an endogenous EcoRI site (generated during cDNA library construction). This was subsequently confirmed in our genomic cloning studies (see below). (b) Northern analysis Northern blot analysis was carried out using riboprobes generated from the 2.1-kb cDNA clone (~132). RNA from adult mouse heart, a tissue known to express Cx43 abundantly (Beyer et al., 1987), exhibited a 3.2-kb band which is typical for Cx43 transcripts (lane H in Fig. 1B). This same band was observed in RNA from blastocyst embryos, embryos at 7.5-9.5 days of gestation, and decidual and placental tissue (Fig. 1B). A @actin probe (P-Act) was also included in these hybridizations to control for RNA integrity and loading, and as expected, all samples exhibited the 2.0-kb P-Act band (Giebelhaus et al., 1983). As our previous in situ hyb~dization studies suggested that Cx43 transcripts are expressed in a striking spatially
193
B. 4.5
8.5
7.5 F-z
IE
9.5 E
12.5 days PH
Fig. 1. Cx43 transcripts in mouse embryos and various adult tissues. (A) The 7.5-day embryo was manually dissected and separated into three parts for RNA isolation. This included the ectoplacental cone (C) (which gives rise to the placenta), the extraembryonic region (X) (which gives rise to the yolk sac), and the embryo proper (E). The decidua, a tissue which arises from the proliferation of maternal tissue at the implantation site, is not represented in this diagram. (B) RNA from various adult tissues and mouse embryos at different developmental stages (7.5, 8.5, and 9.5 days) were separated by electrophoresis in 2.2 M formaldehyde-1.3% agarose gels (Sambrook et al., 1989), blotted onto Nytran membranes (Stratagene, La Jolla, CA, USA), and then hybridized with a mouse Cx43 or mouse B-Act (encoding p-actin) riboprobe (made from plasmid pgAct, kindly provided by M. Goldrick, Ambion, Inc. Austin, TX, USA). For the 7.5-day mouse embryos, three fractions were obtained as described above. The 8.5-day conceptuses were separated into embryonic (E), placental (P), and decidual (D) tissues, while the 9.5-day embryo sample consisted of the embryo proper with the surrounding yolk sac membranes (E). Note that RNA from the 12.5-day placenta was also included in this analysis but exhibits no Cx43 transcripts. As expected, the 2.0-kb P-Act was observed in all samples.
restricted manner in the early postimplantation mouse embryo (Ruangvoravat and Lo, 1992), we further examined the distribution of Cx43 transcripts by dissecting the 7.5day mouse embryo into three fractions (Fig. 1A) and analyzing the RNA isolated from each fraction by Northern blotting (Fig. 1B). This analysis showed no Cx43 transcripts in the ectoplacental cone region (lane
C in Fig. lB), while low transcript levels were observed in the extraembryonic fraction (lane X in Fig. lB), and higher levels were found in the embryo proper (7.5 days/ lane E in Fig. 1B). This pattern of Cx43 transcript localization is consistent with the results of the in situ hybridization studies, which showed abundant hybridization signal both in the embryo proper and in cells of the extraembryonic endoderm (hatched region in Fig. 1A). We also examined the expression of Cx43 transcripts in the decidua, a tissue derived from uterine stromal cells. In agreement with the in situ hybridization (Ruangvoravat and Lo, 1992) and immunolocalization studies (Yancey et al., 1992), a very high abundance of Cx43 transcripts was observed (see lane D in Fig. 1B). Note that in the decidual RNA, the intensity of the Cx43 band was actually greater than that of actin. An examination of RNA from 8.5 and 12.5day placenta revealed no transcripts in the 12.5-day placenta and only a very low level of transcripts in the 8.5-day placenta. Given the difficulty in removing all decidual tissue from the 8.5-day placenta, it is likely that Cx43 transcripts in the 8.5-day placental samples are derived from contaminating decidual cells. In contrast, with the 12.5-day placenta, as most of the decidua is resorbed, the placenta can be readily retrieved without adherent decidual cells. Thus, it is likely that the placenta, which is mostly derived from cells in the ectoplacental cone region, remains devoid of Cx43 transcripts. This result is in agreement with our in situ hybridization analysis which showed no Cx43 hybridization signal in the placenta proper (C.R. and C.W.L., unpublished observations). (c) Cloning and characterization of Cx43 We screened a mouse genomic DNA library with the ~132 cDNA probe and identified a number of positive clones, two of which were isolated and further characterized. Restriction mapping and DNA sequencing revealed that these two clones together contained the entire Cx43 gene (Fig. 2). Genomic Southern analysis was carried out using hybridization probes derived from the cDNA or the cloned genomic DNA, and in most restriction enzyme digests, only a single hybridizing band was released (data not shown). These results suggest that there may be only a single Cx43 gene in the mouse genome. The restriction patterns observed in the genomic Southern analyses are in complete agreement with the restriction map of the cloned genomic DNA, thereby further confirming that the cloned mouse genomic DNA contained the authentic Cx43 gene gene. We sequenced 7.5 kb of the cloned genomic DNA insert, and all coding sequences obtained were in complete agreement with those of the cDNAs. The sequencing carried out included the entire coding region, the 5’-UTR
194 13.2 kb 14 kb i I
f
I
Clone 1
1 Clone 2 pXbaiXmn4WTR
Fig. 2. Restriction map and structure of the mouse Cx43 gene, A mouse NIH 3T3 genomic library (Stratagene, La Jolla, CA) was screened with the mouse Cx43 cDNA probe (p132), and two overlapping h phage clones were isolated which in total contained the entire mouse CM3 gene. Cx43 is composed of two exons (open boxes denoted I and II) separated by a 10.Skb intron (thick line) in the 5’-UTR. The entire coding region (cross hatched) is found within exon II. Regions of the cloned inserts that were sequenced are indicated by the arrows. Dots next to arrows denote where sequencing was carried out using primers. In other regions, sequence info~at~on was obtained by sequencing deletion clones generated by exam&ease III digestion. The X&I-XmnI fragment indicated below the clone-2 insert as p~a/XrnnI*~-~T~ was subcloned and used for mapping tlte transcription initiation site in the RNase protection assay. E, EcoRI sites; X, Xhol sites.
and upstream sequences, 3.2 kb of in&on sequences, and 2.5 kb of sequences encompassing the entire 3’-UTR (Fig. 2). Analysis of the sequence data revealed the presence of two exons (exon I an& II in Fig. 2). Exon I contains most of the 5’-UTR, while the larger 3,5-kb exon II includes the remaining 13 nt of the S-UTR and all of the coding region and 3’-UTR. The coding region in exon II encodes a 43-kDa polyp~ptide which is identical to that of the mouse embryo-derived Cx43 cDNAs characterized above (data not shown). This result, in conjunction with the fact that our aa sequence is identical to the adult mouse Cx43 sequence reported by Beyer and Sternberg (1991), would suggest that Cx43 transcripts in the 7.5 day mouse embryo are likely the same as those expressed in adult tissues. As expected, the single intron which is 10.5 kb in size is bordered by typical splice junctions (see Fig. 4 for splice donor site; splice acceptor site and remaining sequences are in CenBank database, accession No. L10388). Overall, this gene structure appears to be conserved in the connexin gene family, as a single intron in the SUTR has also been reported for human and mouse Cx43 (Fishman et al., 1990; 1991; Hennemann et al., 1992b) and for various other connexin genes (Miller et al., 1988; Willecke et al., 1991; Hennemann et al., 1992a). The conservation of this gene structure suggests that sequences in the intron may be functionally important to connexin gene regulation. In some genes, enhancer like elements in the intron specify the developmental regulation of gene
expression (Kassis et al., 1986; Konieczny and Emerson, 1987; Renucci et al,, 1992), while in a gene such as the ribosomal gene rpL32, DNA in the first intron actually contributes to promoter function (Atchison et al., 1989; Chung and Perry, 1989; Nakano et al., 1991). In future studies, it will be interesting to determine if the intron of the Cx gene family harbors such regulatory elements. (d) Mapping the tsp To determine the tsp for Cx43, we carried out a RNase protection assay using a riboprobe generated from the plasmid pXba~Xmn~5’-~~R (see Fig. 2). A single major protected band was obtained (Fig. 3A), and this same band was observed with RNA isolated from the decidua, ovary, heart, and 7.5”day mouse embryo (Fig. 3A). Yeast tRNA was used as a negative control and, when incubated with the riboprobe, did not produce protected bands as obtained with the mouse RNA samples (data not shown). Compa~son with the posi~on of a 186-nt RNA marker (position denoted by arrowhead in Fig. 34, data not shown) indicates that the protected RNA fragment is 193 nt in length. This would suggest that the tsp is 192 nt upstream from the splice donor site in exon 1. However, as RNA is known to migrate anomalously in sequencing gels, we further used primer extension analysis to map the tsp more precisely. For this analysis, deciduaI RNA was subjected to primer extension analysis using a 40-mer primer derived from exon 1 (nt 350-389 in Fig. 4). The primer extension products obtained consisted of a
195
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B. ACGT
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193
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Pig. 3. Determination of the tsp for Cx43. (A) Identification of tsp by RNase protection. RNase protection assays were carried out using total RNA isolated from the 75day embryo (E), decidua (Dl,D2,D3), adult ovary (0), or adult heart (H). These assays were performed with the RNase protection kit from Ambion, Inc. (Austin, TX), using a riboprobe generated from the pXba/Xmn-S-UTR plasmid. Note that the same protected band was obtained in all samples. Negative control samples did not display this band. The slight difference in mobility of sample D2 is due to a distortion of gel migration during electrophoresis. The migration position of a 186-m RNA marker (not shown; position denoted by arrowhead) in conjunction with a DNA sequencing ladder, both of which were run in parallel with the RNA samples, suggest that the protected fragment is 193 nt in length. The sequencing ladder was generated using a primer in the S-UTR (nt 372-389 in Fig. 4). Samples Dl, D2, and D3 contain I, 3, and 5 pg of decidua RNA, respectively. Methods: Total RNA was hyb~d~~d with the riboprobe at 45°C for 14 h prior to RNase treatment. Following RNase treatment, the protected products were loaded on 8 M urea/6% acrylamide sequencing gels. DNA sequencing ladders and RNA markers were electrophoresed in parallel to allow the sizing of the protected fragments. The amount of RNA and the RNase concentration used were titrated to optimize for the detection of the protected fragment. Yeast tRNA was used as a negative control and, when incubated with the riboprobe, did not generate the protected band obtained with the mouse RNA samples. (B) Identification of the Cx43 tsp by primer extension. Primer extension performed with decidual (D) RNA using a 40-mer primer (complementary to nt 350-389; Fig. 4) resulted in a doublet which, when compared with the sequencing ladder, was determined to be 172/171 nt in length. The sequencing ladder spans DNA in exon I and was obtained using primer to nt 372-389 in Fig. 4, Methods: ‘*P-radiolabeled primer was hybridized with total RNA from the decidua for 12 h at 40°C in hybridization buffer containing 80% formamide (Sambrook et al., 1989). Following hybridization, primer extension was carried out for 2 h at 40°C using AMV reverse transcriptase (Promega, Madison, WI, USA) under standard conditions (except 50 mM TrisI-ICl pH 8.5 was used to optimize enzyme activity). The samples were then ethanol-precipitated and run on 8 M urea/6% polyacrylamide sequencing gels. Yeast tRNA was used as a negative control.
closely spaced doublet of 172/173 nt (Fig. 3B). Note that the finding of a doublet probably results from the fact that the primer extension assay entailed first generating cDNAs using AMV reverse transcriptase, an enzyme previously shown to transcribe the cap structure of mRNA (Gupta and Kingsbury, 1984). Consequently, both the authentic cDNA product and one that is longer by one nt are expected (Gupta and Kingsbury, 1984). Thus, the primer extension data place the tsp at 189/188 nt 5’ of the exon 1 splice donor site, i.e., 4 or 5 nt downstream from the tsp suggested by the RNase protection assay (see asterisks in Fig. 4). The near identity of the tsp indicated by these RNase protection and primer extension assays would suggest that there are probably no addi-
tional exons upstream from exon I. Hence, we assign the tsp for Cx43 to nt 218 (Fig. 4), i.e., 188 nt 5’ of the exon 1 splice donor site. However, it should be noted that a mouse ovary Cx43 cDNA sequence characterized by Nishi et al. (GenBank accession No. M63801) contained 12 additional nt 5’ of this tsp. As these additional nt are identical to immediately adjacent sequences found in the corresponding position of the Cx43 genomic DNA, it is possible that Cx43 may have more than one tsp. Consistent with this possibility is our finding of a slightly larger protected fragment upon greatly overexposing gels containing products of the RNase protection assays (data not shown). This minor band likely accounts for less than 5% of the total products. Thus, the tsp site we have
CCGcclTllmccrcCcrcCCcrrrclcclAticcccTccl-rc
GGClTGAM~MMGCTCTGTGf
,tGCTmAtC*CGF*iC*CUtT 240
498
GWATCCGT
Fig. 4. The nt sequence from exon 1 and flanking regions of the mouse Cx43 gene. Exon 1 (underlined) contains only the first 188 nt from UTR of the Cx43 transcript. The fsp corresponds to the C residue denoted by the asterisk. The region of the 5’-UTR included in the Cx43 clone is indicated by a horizontal bracket. Note the presence of a palindromic sequence (divergent arrows) just upstream from the end of the sequence. The small vertical arrow denotes the position where premature te~ination was observed in primer extensions carried out with a complementary to nt 363-402 (see section g). (Sequence submitted to GenBank, accession No. L10387).
mapped likely represents the major tsp for Cx43, while a minor population of Cx43 transcripts may be derived from another tsp slightly further upstream. (e) Analysis of sequences at the 5’ end An examination of sequences upstream from the tsp (Fig. 4) for promoter/enhancer elements revealed a degenerate TATA box positioned 22 bp upstream from the tsp (Fig. 4) (Bucher, 1990). In addition, a consensus APlbinding site was found (41 bp upstream from the tsp; Fig. 4) (Angel et al., 1987; Lee et al., 1987). The presence of an API -binding site in Cx43 is particularly interesting, as these elements exhibit enhancer activity that can specify inducibility by the tumor promoter TPA (Angel et al., 1987; Lee et al., 1987), and gap junctional communication and the expression of Cx43 transcripts are observed to be modulated by TPA (see for example Brissette et al., 1991). Other members of the connexin gene family, specifically genes encoding Cx26 and Cx32, exhibit TATA-less promoter regions (Hennemann et al., 1992a). For Cx26, the promoter region is observed to be G + C-rich and to contain several GC boxes that are potential SPl binding sites (Hennemann et al., 1992a). In contrast, Cx32 exhibits only 49% G + C and has no GC boxes in its 5’ upstream sequences (Hennemann et al., 1992a). Both Cx26 and Cx32 have numerous putative regulatory motifs in the regions 5’ to tsp. Such motifs include consensus recognition sequences for NFKB and CAMP-response elements. Although such consensus sequences were not found in our 2 16 bp of 5’ flanking sequences, it will be interesting to determine if such sites exist in more distal 5’ sequences, especially in light of the complex physiological regulation of Cx43 expression reported by others (see, for example regulation by cyclic nucleotides; Mehta et al., 1992). However, functional assays, either in vitro or in uiuo, will be needed to demonstrate which of these various sequence elements in the different Cx genes are truly of physiological importance. Analysis of the nt sequence within the Cx43 5’ UTR revealed that it is highly A+T-rich, being composed of
the ScDNA cDNA primer
only 30% G + C residues (Fig. 4). This would suggest that this region is likely relatively unstructured and therefore could provide for the efficient translation of the Cx43 message (Kozak, 1991a,b). Moreover, nt surrounding the AUG closely match the Kozak (1991a) consensus sequence for translation initiation. This includes the presence of a purine at position -3 (relative to the start codon) and a G at position +4. This further suggests that the Cx43 transcript is likely to be translated efficiently. Interestingly, we noted that embedded within this A+T-rich S-UTR is a 30-nt palindrome (Fig. 4). Although the theoretical AG for this hairpin structure is only -9 kcal, we observed a primer extension block that maps precisely to the beginning of this palindrome (nt 336, see arrow in Fig. 4; data not shown) in the course of our attempts to map the tsp using a 40-mer primer (nt 363-402 in Fig. 4). This extension block was not observed with primers derived from sequences situated further upstream from this 40-mer (such as that used for the experiment shown in Fig. 3). It is also interesting to note that the cDNA insert in the Cx43 cDNA clone, ~132, terminates downstream from this palindrome (see the horizontally bracketed region in Fig. 4), thereby further suggesting that a secondary structure may be formed in this region at least under some conditions. (f) Analysis of the 3’-UTR At the 3’ end of Cx43, we observed two polyadenylation signal sequences 1696 and I71 8 bp 3’ of the stop codon (data not shown). This positioning of the poly(A)addition sites is consistent with the 3.2-kb size of the Cx43 transcript. Note the presence of two closely spaced EcoRI sites in the 3’-UTR (Fig. 2). The more 5’ of these two EcoRI sites is positioned precisely where our cDNAs terminated, thereby confirming that our cDNA clones were truncated at an endogenous EcoRI site as discussed above. A comparison of the 3’4JTR of the mouse Cx43 with that of rat (Lang et al., 1991), human (Fishman et al., 1990), and cow (Lash et al., 1990) revealed a surprisingly
197
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Fig. 5. Analysis of c-fos, c-myc, and Cx43 transcripts in tissue culture cells after serum stimulation. (A) Total cellular RNA isolated from quiescent (-FBS) or serum stimulated BALB/c 3T3 cells (+FBS) in the presence or absence of CHX were resolved by gel electrophoresis, transferred to a nylon membrane, and hybridized with c-fos, c-myc, C&3, or ribosomal protein L32-encoding cDNA (rpL32) probes. To monitor for possible induction typical of the immediate early-type genes, this analysis included RNA isolated at various time points after serum addition. Methods: BALB/c 3T3 cells (Mohn et al., 1991; a gift of R. Taub, University of Pennsylv~ia, Philadelphia, PA) were grown in Dulbecco’s modified Eagle’s medium supplements with 10% fetal bovine serum/2 mM ~ut~ine/lO units ~~cillin~i0 pg str~tomycjn per ml. To obtain quiescent cells, the cultures were grown to su~nfluen~ in the above medium, then changed to a medium containing 0.5% serum (Lau and Nathans, 1987). After culturing for another 48-72 h, the quiescent cells were stimulated with a medium containing 20% serum in the presence or absence of 10 pg CHX/ml, then harvested at various time points for RNA isolation (Chomczynski and Sacchi, 1987) and Northern blotting. Hybridization was carried out sequentially with c-myc (encoding exons 2 and 3; see Stone et al., 1987; gift of W. Lee, University of Pennsylvania, Philadelphia, PA), c-fos (Mohn et al., 1991; gift of R. Taub, University of Pennsylvania, Philadelphia, PA), rpL32 (Meyuhas and Perry, 1980; gift of R. Perry, Fox Chase Cancer Institute, Philadelphia, PA), and the mouse Cx43 cDNA probe. Prior to each rehybridization, the blot was stripped by boiling in 10 mM TrisHCl pH 7.5/l mM EDTA/l% SDS for 20 min. Hybridization and wash conditions used for detecting the c-myc, c-fos, and ribosomal protein L32 transcripts have been described previously (Wang et al., 1992). Hybridization for Cx43 detection was carried out using riboprobes derived from ~132 at 1 x lo6 cpm/ml at 65°C overnight, and then the blot was washed in four changes of 0.12 x SSC/O.l% SDS at 65°C (30 min each). After washing, blots were autoradiographed at -70°C for l-3 days on Kodak X-Omat film with a DuPont Cronex Lightning Plus intensifying screen. SSC is 0.15 M NaCl/O.OlS M Nascitrate pH 7.6. (B). The relative transcript abundance detected by the Northern analysis was quantitated densitometrically using Image QuantTM software ~3.0 (Molecular Dynamics, Sunnyville, CA) and plotted graphically. The amount of RNA loaded in each lane was normalized with the rpL32 transcript. The Northern blot and the quantitation data for c-fos and c-myc were adopted from Wang et al. (1992).
high degree of sequence conservation (data not shown). This would suggest that this region is likely to be of functional importance. Included in this region are five AUUUA motifs, four of which are conserved across all four species (data not shown). The one AUUUA motif in mouse not conserved in the other species is actually located downstream from the two polyadenylation consensus sites. This A + U-rich sequence motif is suggested to play a role in message instability in immediate early genes such as c-fos (Wilson and Treisman, 1988; Shyu et al., 1989; 1991). This class of genes is rapidly induced upon growth factor or serum stimulation and is likely to
play an important role in the alteration of gene expression required for cell cycle progression (Bravo, 1990; Herschman, 1991). With regard to the possible role of gap junctions in growth control and cell cycle regulation, it is interesting to note that a cDNA encoding the gap junction gene Cx26 was isolated in a subtraction library screen designed to identify tumor suppressor genes (Lee et al., 1991). To determine whether or not the mouse Cx43 belongs to the class of immediate early genes, we examined quiescent (serum-starved) NIH 3T3 and BALBfc 3T3 cells for the transient elevation of Cx43 transcripts following
198 serum stimulation. Typical immediate-early genes, such as c-myc and c-fos, exhibit a rapid but transient rise of transcript abundance upon serum or growth factor stimulation (Bravo, 1990; Herschman, 1991). This effect is greatly accentuated in the presence of CHX (Lau and Nathans, 1987). Thus, we isolated RNA from NIH 3T3 or BALB/c 3T3 cells that were serum starved, then subsequently serum stimulated either in the presence or absence of CHX. Northern analysis of RNA harvested from these cells at various time points after the initiation of serum stimulation revealed no obvious change in Cx43 transcript abundance upon serum stimulation, even in the presence of CHX (Fig. 5; also data not shown). In contrast, c-myc and c--0s showed the expected transient sharp rise in transcript abundance, and this increase was accentuated in the presence of CHX (Fig. 5). These results demonstrate that at least in NIH and BALB/c 3T3 cells, Cx43 is not regulated in a manner characte~stic of immediate-early genes. (g) Conclusions We cloned and characterized mouse Cx43 cDNA and genomic DNA. Using the cloned mouse Cx43 probes, we further characterized the expression of Cx43 transcripts in mouse embryos and in tissue culture cells subjected to serum stimulation. These studies provide the foundation for future research in which molecular approaches will be used to manipulate the expression of Cx43 for further examining its role in development and growth regulation. The data we have presented demonstrate that : (1) Cx43 transcripts are expressed in a spatially restricted manner in the 7.5-day mouse embryo. This is indicated by the complete absence of Cx43 transcripts in the 7.5day ectoplacental cone and the 12.5-day placenta. (2) The Cx43 gene isolated from the genomic library screen encodes the same transc~pt~protein as the cloned cDNA. This finding, in conjunction with other data, suggests that Cx43 transcripts expressed in the embryo are likely identical to those in adult tissues. (3) Similar to other connexin-encoding genes, Cx43 is composed of two exons with a single large intron positioned in the S-LJTR. (4) The transcription initiation site is mapped to a C residue situated 188 nt upstream from the splice donor site of exon 1. This same initiation site is observed for Cx43 transcripts expressed in the mouse embryo and in various adult tissues. (5) Cx43 contains an APl-binding site and a degenerate TATA box. (6) The 3’-UTR of Cx43 is highly conserved. This includes four AUUUA motifs, a sequence associated with message instability in immediate early genes. (7) Cx43 expression in NIH and BALB/c 3T3 cells does
not exhibit the serum inducibility which is characteristic of immediate early genes.
ACKNOWLEDGEMENTS
We would like to thank David Paul for the generous gift of the rat Cx43 probe and Janet Rossant for the mouse embryo cDNA library. This work was supported by N.I.H. grant HD21355 and NSF grant DCB-06886. C.R. was partially supported by NRSA postdoctoral fellowship GM-141 16, and R.S. was supported by predoctoral fellowships from the March of Dimes and NIH VMSTP, 5-T32GM07 170.
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199 tity of histone and actin messenger RNA during the development of preimplantation mouse embryos. Dev. Biol. 98 (1983) 148-154. Gupta, KC. and Kingsbury, D.W.: Complete sequences of the intergenic and mRNA start signals in the Sendai virus genome: homologies with the genome of vesicular stomatitis virus. Nucleic Acids Res. 12 (1984) 3829-3841. Hennemann, H., Kozek, G., Dahl, E., Nicholson, B. and Willecke, K.: Molecular cloning of mouse connexins 26 and 32: similar genomic organization but distinct promoter sequences of two gap junction genes. Eur. J. Ceil Bioi. 58 (1992a) 81-89. Hennemann, H., Suchyna, T., Lichten~rg-Frate, H., Jungbluth, S., Dahl, E., Schwarz, J., Nicholson, B-J. and Willecke, K.: Molecular cloning and functional expression of mouse connexin 40, a second gap junction gene preferentially expressed in lung. J. Cell Biol. 117 (1992b) 1299-1310. Herschman, H.R.: Primary response genes induced by growth factors and tumor promoters. Annu. Rev. Biochem. 60 (1991) 281-319. Jones, NC., Rigby, P.W.J. and Ziff, E.B.: Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor virus. Genes Dev. 2 (1988) 267-281. Kassis, J.A., Poole, S.J., Wright, D.K. and G’Farrell, P.H. : Sequence conservation in the protein coding and intron regions of the engruiled transcription unit. EMBO J. 5 (1986) 3583-3589. Konieczny, SF. and Emerson, C.P.: Complex regulation of the muscle specific contractile protein (troponin I) gene. Mol. Cell. Biol. 7 (1987) 3065-3075. Kozak, M.: An analysis of vertebrate mRNA sequence: intimations of translational control. J. Cell Biol. 115 (1991a) 887-903. Kozak, M.: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. BiolChem. 266 (1991b) 19867-19870. Lang, L.M., Beyer, E.C., Schwartz, A.L. and Bitlin, J.D.: Molecular cloning of a rat uterine gap junction protein (connexin 43) and analysis of gene expression during gestation. Am. J. Physiol. 260 (1991) E787-E793. Lash, J.A., Critser, E.A. and Pressler, M.L.: Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos. J. Biol. Chem. 265 (1990) 13113-13117. Lau, L.F. and Nathans, D.: Expression of a set of growth related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc. Natl. Acad. Sci. USA 84 (1987) 1182-I 186. Lee, SW., Tomasetto, C. and Sager, R.: Positive selection of candidate tumor-suppressor genes by subtractive hybridization. Proc. Nat]. Acad. Sci. USA 88 (1991) 2825-2829. Lee, W., Mitchell, P. and Tjian, R.: Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell 49 (1987) 741-752. Mehta, P.P., Yamamoto, M. and Rose, B.: Transcription of the gene for the gap junctional protein connexin 43 and expression of functional cell-to-cell channels are regulated by CAMP. Mol. Biol. Cell 3 (1992) 839-850. Meyuhas, 0. and Perry, R.P.: Construction and identification of cDNA clones for mouse ribosomal proteins: applications for the study of protein gene expression. Gene 10 (1980) 113-129.
Miller, T., Dahl, G. and Werner, R.: Structure of a gap junction gene: connexin 32. Biosci. Rep. 8 (1988) 455-464. Mohn, K.L., Laz, T.M., Hsu, J.C., Melby, A.E., Bravo, R. and Taub, R.: The immediate-early growth response in regenerating liver and insulin stimulated H-35 cells: comparison with serum stimulated 3T3 cells and identification of 41 novel immediate-early genes. Mol. Ceil. Biol. 11 (1991) 381-390. Nakano, Y., Nishihara, T., Sasayama, S., Miwa, T., Kamada, S. and Kakunaga, T. : Transcriptional regulatory elements in the 5 upstream and first intron regions of the human smooth muscle (aortic type) ~-actin-encoding gene. Gene 99 (1991) 285-289. Nishi, M., Kumar, N.M. and G&la, N.B.: Developmental regulation of gap junction gene expression during mouse embryonic develop ment. Dev. Bio1.146 (1991) 117-130. Renucci, A., Zappavigna, V., Zakanay, J., Izpsiua-Belmonte, J.-C., Burki, K. and Duboule, D.: Comparison of mouse and human Hox4 complexes defines conserved sequences involved in the regulation of Hex-4.4. EMBO J. 11 (1992) 145991468. Ruangvoravat, C.P. and Lo, C.W.: Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains. Dev. Dynam. 194 (1992) 261-281. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Schuler, G.D. and Cole, M.D.: GM-CSF and oncogene mRNA stabiiities are independently regulated in truns in a mouse monocytic tumor. Cell 5.5 (1988) Ii 15-l 122. Shaw, G. and Kamen, R. : A conserved AU sequence from the 3 untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46 (1986) 659-667. Shyu, A.-B., Greenberg, M.E. and Belasco, J.G.: The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev. 3 (1989) 60-72. Shyu, A.-B., Belasco, J.G. and Greenberg, M.E.: Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 5 (1991) 221-231. Stone, J., De Lang, T., Ramsay, G., Jakobovits, E., Bishop, J.M., Varmus, H. and Lee, W.: Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol. Cell. Biol. 7 (1987) 1697-1709. Wang, X.K., Lee, G., Liebhaber, S.A. and Cooke, N.E.: Human cysteinerich protein: a member of the LIM/double finger family displaying coordinate serum induction with c-myc. J. Biol. Chem. 267 (1992) 9176-9184. Willecke, K., Hennemann, H., Dahl, E., Jungbluth, S. and Heynkes, R.: The diversity of connexin genes encoding gap junctional proteins. Eur. J. Cell Biol. 56 (1991) l-7. Wilson, T. and Treisman, R.: Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3’ AU-rich sequences. Nature 336 (1988) 396-399. Yancey, S.B., Biswal, S. and Revel, J.-P.: Spatial and temporal patterns of distribution of the gap junction protein connexin 43 during mouse gastrulation and organogenesis. Development 114 (1992) 203-212.
191
GENE 072 18
Structure, sequence and expression of the mouse Cx43 gene encoding connexin 43 (Gap junction; embryo; intron; trans~~ption initiation; promoter; APl; immediate early gene; membrane chann~is)
Ruth Sullivan, Christine Ruangvoravat, and Cecilia W. Lo
Daniel Joo, Judy Morgan, Baa Lin Wang, Xin Kang Wang
Biology Department, University of Pennsylvania, Philadelphia, PA 19104-6017, USA
Received by D.T. Denhardt: 27 November 1992; Revised/Accepted: 18 February/l9 February 1993; Received at publishers: 19 April 1993
SUMMARY
Gap junctions, membrane channels that mediate the diffusion of ions and small molecules between cells, are hypothesized to play a role in development and growth regulation. The Cx43 gene (encoding connexin 43) is one member of the gap junction gene family whose transcripts are expressed in a highly regionalized manner during mouse development. We cloned and sequenced Cx43 cDNAs from a 7.5-day mouse embryo cDNA library. These cDNA clones encode the authentic 43-kDa connexin. Analysis of RNA isolated from different regions of the 7.5day mouse embryo revealed that Cx43 transcripts are differentially expressed, with expression detected in the embryo proper, but not in the extraembryonic region containing the ectoplacental cone. Using one of the newly isolated mouse Cx43 cDNA probes, we screened a mouse genomic DNA library and cloned the Cx43 gene. Restriction mapping and sequencing of the cloned genomic inserts revealed that Cx43 contains two exons and a 10.5-kb intron located in the 5’ untranslated region (S-U’ZX). We mapped the Cx43 transcription start point (tsp) by RNase protection and primer extension analyses and showed that transcripts expressed in the 7.5-day mouse embryo and in adult tissues are initiated from the same tsp. The DNA sequence immediately upstream from the tsp contains a putative API-binding site and a degenerate TATA consensus sequence. A comparison of mouse, rat, human and bovine Cx43s showed that the 3’4JTR has an unexpectedly high degree of sequence homology. This includes conservation of four AUUUA motifs, a sequence associated with transcript instability in immediate early genes. FIowever, further analysis of Cx43 expression in NIH 3T3 and BALB/c 3T3 cells showed that Cx43 expression is not subject to serum induction, as has been found for the immediate early genes.
INTRODUCTION
Gap junctions are composed of membrane channels that allow the direct cell-to-cell transfer of small cytoCorrespondence
to: Dr. C.W. Lo, Biology Department, Goddard Laboratory, University of Pennsylv~i~ P~ladelphia, PA 191~~17, USA. Tel. (1-215) 898-8394; Fax (l-215) 898-8780; e-mail: [email protected]
Abbreviations: aa, amino acid(s); AMV, avian myeloblastosis virus; bp, base pair(s); CHX, cycloheximide; Cx, connexin(s); Cx, gene encoding Cx; FBS, fetal bovine serum; kb, kilobase or 1000 bp; nt, nucleotide(s); TPA, 12-0-tetradecanoylphorbol-13-acetate; tsp, transcription start point(s); UTR, untranslated region.
plasmic components such as ions, metabolic intermediates, and second messengers. The proteins that make up these junctions are encoded by a multigene family referred to as the connexins (Cx; for review see Beyer et al., 1990; Willecke et al., 1991). Analysis of various Cx polypeptides and the gap junctions they generate has revealed differences in their tissue distribution, permeability and gating properties, and sensitivity to modulation by factors such as pH and phosphorylation (for review see Bennett et al., 1991; Beyer et al., 1990). These obse~ations suggest that gap junctional communication and the differential expression of different Cx polypeptides may play an important role in many diverse physiological processes.
192 This may include the coordination of cell metabolism, growth regulation, and development. In the mouse embryo, Cx43 proteins and transcripts are both expressed in a highly regionalized manner (Ruangvoravat and Lo, 1992; Yancey et al., 1992). Thus, using in situ hybridization, we have shown that Cx43 transcripts are expressed in spatially restricted domains in many regions of the embryo undergoing tissue inductive events (Ruangvoravat and Lo, 1992). Additionally, in the limb bud and developing hindbrain, Cx43 transcripts were expressed in a graded pattern similar to the gradient of Hole gene transcripts found in these regions of the embryo. These in situ hybridization results, in conjunction with observations derived from Cx43 immunostaining studies (Yancey et al., 1992), suggest that Cx43mediated gap junctional communication may participate in many aspects of mammalian embryogenesis. More definitive insights into the possible role of Cx43 in mammalian development may be obtained with the use of molecular approaches to disrupt or otherwise manipulate the expression of Cx43 in a developmentally regulated manner. It is with this goal in mind that we cloned and characterized the mouse Cx43 gene. We began the present study with the cloning and characterization of a Cx43 cDNA from a 7.5day mouse embryo cDNA library. Using the mouse Cx43 cDNA probe, a Northern analysis was carried out to examine the expression of Cx43 transcripts in the mouse embryo. We also screened a genomic DNA library and cloned the mouse Cx43 gene gene. Its structure, sequence, and tsp were determined. As our analysis revealed that the 3’UTR of Cx43 contains four conserved AUUUA motifs, a sequence associated with transcript instability in immediate early genes (Shaw and Kamen, 1986; Jones et al., 1988; Schuler and Cole, 1988; Wilson and Treisman, 1988; Shyu et al., 1989; 1991), we carried out additional experiments to determine whether 043 may be inducible by serum stimulation, as is typical for immediate early genes.
RESULTS AND DISCUSSION
(a) Cloning and characterization of Cx43 cDNA Using the rat Cx43 cDNA probe, pG2A (provided by David Paul; Beyer et al., 19871, we screened a mouse embryo cDNA library made from RNA isolated from the embryonic portion of the 7.5-day mouse conceptus (provided by Janet Rossant). Five positive plaques were obtained, and the two containing the largest EcoRI inserts (2.0 and 2.1 kb) were subcloned into plasmid vectors and sequenced in their entirety (data not shown; GenBank accession No. X61576). The sequences of the two clones were identical and showed extensive homol-
ogy to adult rat and mouse Cx43 cDNA, in both the coding (Beyer et al., 1987; Beyer and Steinberg, 1991; Lang et al., 1991; Henneman et al., 1992b) and noncoding regions (Lang et al., 1991). Both cDNAs encode the same polypeptide consisting of 382 aa (43 kDa; data not shown). The coding region of our Cx43 clones is identical to the mouse genomic Cx43 sequence reported by Henneman et al. (1992b). Although we observed three conservative nt changes when compared with the Cx43 cDNA sequence of Beyer and Steinberg (199 1) (positions 9, 15, and 1146 relative to the start codon at position I), these substitutions all lie within the primers used by Beyer and Steinberg (1991) to PCR amplify and clone the mouse Cx43 coding region. Our mouse Cx43 peptide sequence and also that of Beyer and Steinberg (1991) and Henneman et al. (199217)differ from adult rat Cx43 by a single aa (Lang et al., 1991); in mouse Cx43, Asn is found in place of Ser 341. It is interesting to note that another previously published mouse Cx43 sequence (Nishi et al., 1991) was reported to contain a Ser341 and, in addition, a second aa difference - a Thr in place of Met3** (data not shown). As the cDNA sequence of Nishi et al. (1991) showed numerous mismatches when compared with the mouse Cx43 sequence from this study and that of the other two groups (Beyer and Steinberg, 1991; Henneman et al., 1992b), it is not possible to evaluate the basis for the latter aa differences. The cDNA clones we have isolated do not represent full-length cDNAs, as Cx43 transcripts are over 3.0 kb in size (Beyer et al., 1987). Given that both cDNA inserts lacked a polyadenylation signal sequence and were terminated at the same position within the 3’-UTR (911 nt from the stop codon; data not shown), it is likely that the 3’ ends of these cDNA clones represent truncations at an endogenous EcoRI site (generated during cDNA library construction). This was subsequently confirmed in our genomic cloning studies (see below). (b) Northern analysis Northern blot analysis was carried out using riboprobes generated from the 2.1-kb cDNA clone (~132). RNA from adult mouse heart, a tissue known to express Cx43 abundantly (Beyer et al., 1987), exhibited a 3.2-kb band which is typical for Cx43 transcripts (lane H in Fig. 1B). This same band was observed in RNA from blastocyst embryos, embryos at 7.5-9.5 days of gestation, and decidual and placental tissue (Fig. 1B). A @actin probe (P-Act) was also included in these hybridizations to control for RNA integrity and loading, and as expected, all samples exhibited the 2.0-kb P-Act band (Giebelhaus et al., 1983). As our previous in situ hyb~dization studies suggested that Cx43 transcripts are expressed in a striking spatially
193
B. 4.5
8.5
7.5 F-z
IE
9.5 E
12.5 days PH
Fig. 1. Cx43 transcripts in mouse embryos and various adult tissues. (A) The 7.5-day embryo was manually dissected and separated into three parts for RNA isolation. This included the ectoplacental cone (C) (which gives rise to the placenta), the extraembryonic region (X) (which gives rise to the yolk sac), and the embryo proper (E). The decidua, a tissue which arises from the proliferation of maternal tissue at the implantation site, is not represented in this diagram. (B) RNA from various adult tissues and mouse embryos at different developmental stages (7.5, 8.5, and 9.5 days) were separated by electrophoresis in 2.2 M formaldehyde-1.3% agarose gels (Sambrook et al., 1989), blotted onto Nytran membranes (Stratagene, La Jolla, CA, USA), and then hybridized with a mouse Cx43 or mouse B-Act (encoding p-actin) riboprobe (made from plasmid pgAct, kindly provided by M. Goldrick, Ambion, Inc. Austin, TX, USA). For the 7.5-day mouse embryos, three fractions were obtained as described above. The 8.5-day conceptuses were separated into embryonic (E), placental (P), and decidual (D) tissues, while the 9.5-day embryo sample consisted of the embryo proper with the surrounding yolk sac membranes (E). Note that RNA from the 12.5-day placenta was also included in this analysis but exhibits no Cx43 transcripts. As expected, the 2.0-kb P-Act was observed in all samples.
restricted manner in the early postimplantation mouse embryo (Ruangvoravat and Lo, 1992), we further examined the distribution of Cx43 transcripts by dissecting the 7.5day mouse embryo into three fractions (Fig. 1A) and analyzing the RNA isolated from each fraction by Northern blotting (Fig. 1B). This analysis showed no Cx43 transcripts in the ectoplacental cone region (lane
C in Fig. lB), while low transcript levels were observed in the extraembryonic fraction (lane X in Fig. lB), and higher levels were found in the embryo proper (7.5 days/ lane E in Fig. 1B). This pattern of Cx43 transcript localization is consistent with the results of the in situ hybridization studies, which showed abundant hybridization signal both in the embryo proper and in cells of the extraembryonic endoderm (hatched region in Fig. 1A). We also examined the expression of Cx43 transcripts in the decidua, a tissue derived from uterine stromal cells. In agreement with the in situ hybridization (Ruangvoravat and Lo, 1992) and immunolocalization studies (Yancey et al., 1992), a very high abundance of Cx43 transcripts was observed (see lane D in Fig. 1B). Note that in the decidual RNA, the intensity of the Cx43 band was actually greater than that of actin. An examination of RNA from 8.5 and 12.5day placenta revealed no transcripts in the 12.5-day placenta and only a very low level of transcripts in the 8.5-day placenta. Given the difficulty in removing all decidual tissue from the 8.5-day placenta, it is likely that Cx43 transcripts in the 8.5-day placental samples are derived from contaminating decidual cells. In contrast, with the 12.5-day placenta, as most of the decidua is resorbed, the placenta can be readily retrieved without adherent decidual cells. Thus, it is likely that the placenta, which is mostly derived from cells in the ectoplacental cone region, remains devoid of Cx43 transcripts. This result is in agreement with our in situ hybridization analysis which showed no Cx43 hybridization signal in the placenta proper (C.R. and C.W.L., unpublished observations). (c) Cloning and characterization of Cx43 We screened a mouse genomic DNA library with the ~132 cDNA probe and identified a number of positive clones, two of which were isolated and further characterized. Restriction mapping and DNA sequencing revealed that these two clones together contained the entire Cx43 gene (Fig. 2). Genomic Southern analysis was carried out using hybridization probes derived from the cDNA or the cloned genomic DNA, and in most restriction enzyme digests, only a single hybridizing band was released (data not shown). These results suggest that there may be only a single Cx43 gene in the mouse genome. The restriction patterns observed in the genomic Southern analyses are in complete agreement with the restriction map of the cloned genomic DNA, thereby further confirming that the cloned mouse genomic DNA contained the authentic Cx43 gene gene. We sequenced 7.5 kb of the cloned genomic DNA insert, and all coding sequences obtained were in complete agreement with those of the cDNAs. The sequencing carried out included the entire coding region, the 5’-UTR
194 13.2 kb 14 kb i I
f
I
Clone 1
1 Clone 2 pXbaiXmn4WTR
Fig. 2. Restriction map and structure of the mouse Cx43 gene, A mouse NIH 3T3 genomic library (Stratagene, La Jolla, CA) was screened with the mouse Cx43 cDNA probe (p132), and two overlapping h phage clones were isolated which in total contained the entire mouse CM3 gene. Cx43 is composed of two exons (open boxes denoted I and II) separated by a 10.Skb intron (thick line) in the 5’-UTR. The entire coding region (cross hatched) is found within exon II. Regions of the cloned inserts that were sequenced are indicated by the arrows. Dots next to arrows denote where sequencing was carried out using primers. In other regions, sequence info~at~on was obtained by sequencing deletion clones generated by exam&ease III digestion. The X&I-XmnI fragment indicated below the clone-2 insert as p~a/XrnnI*~-~T~ was subcloned and used for mapping tlte transcription initiation site in the RNase protection assay. E, EcoRI sites; X, Xhol sites.
and upstream sequences, 3.2 kb of in&on sequences, and 2.5 kb of sequences encompassing the entire 3’-UTR (Fig. 2). Analysis of the sequence data revealed the presence of two exons (exon I an& II in Fig. 2). Exon I contains most of the 5’-UTR, while the larger 3,5-kb exon II includes the remaining 13 nt of the S-UTR and all of the coding region and 3’-UTR. The coding region in exon II encodes a 43-kDa polyp~ptide which is identical to that of the mouse embryo-derived Cx43 cDNAs characterized above (data not shown). This result, in conjunction with the fact that our aa sequence is identical to the adult mouse Cx43 sequence reported by Beyer and Sternberg (1991), would suggest that Cx43 transcripts in the 7.5 day mouse embryo are likely the same as those expressed in adult tissues. As expected, the single intron which is 10.5 kb in size is bordered by typical splice junctions (see Fig. 4 for splice donor site; splice acceptor site and remaining sequences are in CenBank database, accession No. L10388). Overall, this gene structure appears to be conserved in the connexin gene family, as a single intron in the SUTR has also been reported for human and mouse Cx43 (Fishman et al., 1990; 1991; Hennemann et al., 1992b) and for various other connexin genes (Miller et al., 1988; Willecke et al., 1991; Hennemann et al., 1992a). The conservation of this gene structure suggests that sequences in the intron may be functionally important to connexin gene regulation. In some genes, enhancer like elements in the intron specify the developmental regulation of gene
expression (Kassis et al., 1986; Konieczny and Emerson, 1987; Renucci et al,, 1992), while in a gene such as the ribosomal gene rpL32, DNA in the first intron actually contributes to promoter function (Atchison et al., 1989; Chung and Perry, 1989; Nakano et al., 1991). In future studies, it will be interesting to determine if the intron of the Cx gene family harbors such regulatory elements. (d) Mapping the tsp To determine the tsp for Cx43, we carried out a RNase protection assay using a riboprobe generated from the plasmid pXba~Xmn~5’-~~R (see Fig. 2). A single major protected band was obtained (Fig. 3A), and this same band was observed with RNA isolated from the decidua, ovary, heart, and 7.5”day mouse embryo (Fig. 3A). Yeast tRNA was used as a negative control and, when incubated with the riboprobe, did not produce protected bands as obtained with the mouse RNA samples (data not shown). Compa~son with the posi~on of a 186-nt RNA marker (position denoted by arrowhead in Fig. 34, data not shown) indicates that the protected RNA fragment is 193 nt in length. This would suggest that the tsp is 192 nt upstream from the splice donor site in exon 1. However, as RNA is known to migrate anomalously in sequencing gels, we further used primer extension analysis to map the tsp more precisely. For this analysis, deciduaI RNA was subjected to primer extension analysis using a 40-mer primer derived from exon 1 (nt 350-389 in Fig. 4). The primer extension products obtained consisted of a
195
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B. ACGT
GenOmlC
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Sewencmg
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I
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193
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-
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-
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prtmer (40 trier>
Extension
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nt,
(18 mer)
Pig. 3. Determination of the tsp for Cx43. (A) Identification of tsp by RNase protection. RNase protection assays were carried out using total RNA isolated from the 75day embryo (E), decidua (Dl,D2,D3), adult ovary (0), or adult heart (H). These assays were performed with the RNase protection kit from Ambion, Inc. (Austin, TX), using a riboprobe generated from the pXba/Xmn-S-UTR plasmid. Note that the same protected band was obtained in all samples. Negative control samples did not display this band. The slight difference in mobility of sample D2 is due to a distortion of gel migration during electrophoresis. The migration position of a 186-m RNA marker (not shown; position denoted by arrowhead) in conjunction with a DNA sequencing ladder, both of which were run in parallel with the RNA samples, suggest that the protected fragment is 193 nt in length. The sequencing ladder was generated using a primer in the S-UTR (nt 372-389 in Fig. 4). Samples Dl, D2, and D3 contain I, 3, and 5 pg of decidua RNA, respectively. Methods: Total RNA was hyb~d~~d with the riboprobe at 45°C for 14 h prior to RNase treatment. Following RNase treatment, the protected products were loaded on 8 M urea/6% acrylamide sequencing gels. DNA sequencing ladders and RNA markers were electrophoresed in parallel to allow the sizing of the protected fragments. The amount of RNA and the RNase concentration used were titrated to optimize for the detection of the protected fragment. Yeast tRNA was used as a negative control and, when incubated with the riboprobe, did not generate the protected band obtained with the mouse RNA samples. (B) Identification of the Cx43 tsp by primer extension. Primer extension performed with decidual (D) RNA using a 40-mer primer (complementary to nt 350-389; Fig. 4) resulted in a doublet which, when compared with the sequencing ladder, was determined to be 172/171 nt in length. The sequencing ladder spans DNA in exon I and was obtained using primer to nt 372-389 in Fig. 4, Methods: ‘*P-radiolabeled primer was hybridized with total RNA from the decidua for 12 h at 40°C in hybridization buffer containing 80% formamide (Sambrook et al., 1989). Following hybridization, primer extension was carried out for 2 h at 40°C using AMV reverse transcriptase (Promega, Madison, WI, USA) under standard conditions (except 50 mM TrisI-ICl pH 8.5 was used to optimize enzyme activity). The samples were then ethanol-precipitated and run on 8 M urea/6% polyacrylamide sequencing gels. Yeast tRNA was used as a negative control.
closely spaced doublet of 172/173 nt (Fig. 3B). Note that the finding of a doublet probably results from the fact that the primer extension assay entailed first generating cDNAs using AMV reverse transcriptase, an enzyme previously shown to transcribe the cap structure of mRNA (Gupta and Kingsbury, 1984). Consequently, both the authentic cDNA product and one that is longer by one nt are expected (Gupta and Kingsbury, 1984). Thus, the primer extension data place the tsp at 189/188 nt 5’ of the exon 1 splice donor site, i.e., 4 or 5 nt downstream from the tsp suggested by the RNase protection assay (see asterisks in Fig. 4). The near identity of the tsp indicated by these RNase protection and primer extension assays would suggest that there are probably no addi-
tional exons upstream from exon I. Hence, we assign the tsp for Cx43 to nt 218 (Fig. 4), i.e., 188 nt 5’ of the exon 1 splice donor site. However, it should be noted that a mouse ovary Cx43 cDNA sequence characterized by Nishi et al. (GenBank accession No. M63801) contained 12 additional nt 5’ of this tsp. As these additional nt are identical to immediately adjacent sequences found in the corresponding position of the Cx43 genomic DNA, it is possible that Cx43 may have more than one tsp. Consistent with this possibility is our finding of a slightly larger protected fragment upon greatly overexposing gels containing products of the RNase protection assays (data not shown). This minor band likely accounts for less than 5% of the total products. Thus, the tsp site we have
CCGcclTllmccrcCcrcCCcrrrclcclAticcccTccl-rc
GGClTGAM~MMGCTCTGTGf
,tGCTmAtC*CGF*iC*CUtT 240
498
GWATCCGT
Fig. 4. The nt sequence from exon 1 and flanking regions of the mouse Cx43 gene. Exon 1 (underlined) contains only the first 188 nt from UTR of the Cx43 transcript. The fsp corresponds to the C residue denoted by the asterisk. The region of the 5’-UTR included in the Cx43 clone is indicated by a horizontal bracket. Note the presence of a palindromic sequence (divergent arrows) just upstream from the end of the sequence. The small vertical arrow denotes the position where premature te~ination was observed in primer extensions carried out with a complementary to nt 363-402 (see section g). (Sequence submitted to GenBank, accession No. L10387).
mapped likely represents the major tsp for Cx43, while a minor population of Cx43 transcripts may be derived from another tsp slightly further upstream. (e) Analysis of sequences at the 5’ end An examination of sequences upstream from the tsp (Fig. 4) for promoter/enhancer elements revealed a degenerate TATA box positioned 22 bp upstream from the tsp (Fig. 4) (Bucher, 1990). In addition, a consensus APlbinding site was found (41 bp upstream from the tsp; Fig. 4) (Angel et al., 1987; Lee et al., 1987). The presence of an API -binding site in Cx43 is particularly interesting, as these elements exhibit enhancer activity that can specify inducibility by the tumor promoter TPA (Angel et al., 1987; Lee et al., 1987), and gap junctional communication and the expression of Cx43 transcripts are observed to be modulated by TPA (see for example Brissette et al., 1991). Other members of the connexin gene family, specifically genes encoding Cx26 and Cx32, exhibit TATA-less promoter regions (Hennemann et al., 1992a). For Cx26, the promoter region is observed to be G + C-rich and to contain several GC boxes that are potential SPl binding sites (Hennemann et al., 1992a). In contrast, Cx32 exhibits only 49% G + C and has no GC boxes in its 5’ upstream sequences (Hennemann et al., 1992a). Both Cx26 and Cx32 have numerous putative regulatory motifs in the regions 5’ to tsp. Such motifs include consensus recognition sequences for NFKB and CAMP-response elements. Although such consensus sequences were not found in our 2 16 bp of 5’ flanking sequences, it will be interesting to determine if such sites exist in more distal 5’ sequences, especially in light of the complex physiological regulation of Cx43 expression reported by others (see, for example regulation by cyclic nucleotides; Mehta et al., 1992). However, functional assays, either in vitro or in uiuo, will be needed to demonstrate which of these various sequence elements in the different Cx genes are truly of physiological importance. Analysis of the nt sequence within the Cx43 5’ UTR revealed that it is highly A+T-rich, being composed of
the ScDNA cDNA primer
only 30% G + C residues (Fig. 4). This would suggest that this region is likely relatively unstructured and therefore could provide for the efficient translation of the Cx43 message (Kozak, 1991a,b). Moreover, nt surrounding the AUG closely match the Kozak (1991a) consensus sequence for translation initiation. This includes the presence of a purine at position -3 (relative to the start codon) and a G at position +4. This further suggests that the Cx43 transcript is likely to be translated efficiently. Interestingly, we noted that embedded within this A+T-rich S-UTR is a 30-nt palindrome (Fig. 4). Although the theoretical AG for this hairpin structure is only -9 kcal, we observed a primer extension block that maps precisely to the beginning of this palindrome (nt 336, see arrow in Fig. 4; data not shown) in the course of our attempts to map the tsp using a 40-mer primer (nt 363-402 in Fig. 4). This extension block was not observed with primers derived from sequences situated further upstream from this 40-mer (such as that used for the experiment shown in Fig. 3). It is also interesting to note that the cDNA insert in the Cx43 cDNA clone, ~132, terminates downstream from this palindrome (see the horizontally bracketed region in Fig. 4), thereby further suggesting that a secondary structure may be formed in this region at least under some conditions. (f) Analysis of the 3’-UTR At the 3’ end of Cx43, we observed two polyadenylation signal sequences 1696 and I71 8 bp 3’ of the stop codon (data not shown). This positioning of the poly(A)addition sites is consistent with the 3.2-kb size of the Cx43 transcript. Note the presence of two closely spaced EcoRI sites in the 3’-UTR (Fig. 2). The more 5’ of these two EcoRI sites is positioned precisely where our cDNAs terminated, thereby confirming that our cDNA clones were truncated at an endogenous EcoRI site as discussed above. A comparison of the 3’4JTR of the mouse Cx43 with that of rat (Lang et al., 1991), human (Fishman et al., 1990), and cow (Lash et al., 1990) revealed a surprisingly
197
A. FBS Cl-IX
-++++++++++++++++ -
-+-
Time(hrs)
0
.25.25
.5
+
-
+
-
+
.5
1
1
2
2
-
+
3.53.5
-
+-
6
6
10
+
-
+
10
24
24 kb
Probes
- 3.3
CX43
-
“I 0
0.15
0.5 Time
1
2
3.5
6
10
24
(hrs) after 20% serum addition
Fig. 5. Analysis of c-fos, c-myc, and Cx43 transcripts in tissue culture cells after serum stimulation. (A) Total cellular RNA isolated from quiescent (-FBS) or serum stimulated BALB/c 3T3 cells (+FBS) in the presence or absence of CHX were resolved by gel electrophoresis, transferred to a nylon membrane, and hybridized with c-fos, c-myc, C&3, or ribosomal protein L32-encoding cDNA (rpL32) probes. To monitor for possible induction typical of the immediate early-type genes, this analysis included RNA isolated at various time points after serum addition. Methods: BALB/c 3T3 cells (Mohn et al., 1991; a gift of R. Taub, University of Pennsylv~ia, Philadelphia, PA) were grown in Dulbecco’s modified Eagle’s medium supplements with 10% fetal bovine serum/2 mM ~ut~ine/lO units ~~cillin~i0 pg str~tomycjn per ml. To obtain quiescent cells, the cultures were grown to su~nfluen~ in the above medium, then changed to a medium containing 0.5% serum (Lau and Nathans, 1987). After culturing for another 48-72 h, the quiescent cells were stimulated with a medium containing 20% serum in the presence or absence of 10 pg CHX/ml, then harvested at various time points for RNA isolation (Chomczynski and Sacchi, 1987) and Northern blotting. Hybridization was carried out sequentially with c-myc (encoding exons 2 and 3; see Stone et al., 1987; gift of W. Lee, University of Pennsylvania, Philadelphia, PA), c-fos (Mohn et al., 1991; gift of R. Taub, University of Pennsylvania, Philadelphia, PA), rpL32 (Meyuhas and Perry, 1980; gift of R. Perry, Fox Chase Cancer Institute, Philadelphia, PA), and the mouse Cx43 cDNA probe. Prior to each rehybridization, the blot was stripped by boiling in 10 mM TrisHCl pH 7.5/l mM EDTA/l% SDS for 20 min. Hybridization and wash conditions used for detecting the c-myc, c-fos, and ribosomal protein L32 transcripts have been described previously (Wang et al., 1992). Hybridization for Cx43 detection was carried out using riboprobes derived from ~132 at 1 x lo6 cpm/ml at 65°C overnight, and then the blot was washed in four changes of 0.12 x SSC/O.l% SDS at 65°C (30 min each). After washing, blots were autoradiographed at -70°C for l-3 days on Kodak X-Omat film with a DuPont Cronex Lightning Plus intensifying screen. SSC is 0.15 M NaCl/O.OlS M Nascitrate pH 7.6. (B). The relative transcript abundance detected by the Northern analysis was quantitated densitometrically using Image QuantTM software ~3.0 (Molecular Dynamics, Sunnyville, CA) and plotted graphically. The amount of RNA loaded in each lane was normalized with the rpL32 transcript. The Northern blot and the quantitation data for c-fos and c-myc were adopted from Wang et al. (1992).
high degree of sequence conservation (data not shown). This would suggest that this region is likely to be of functional importance. Included in this region are five AUUUA motifs, four of which are conserved across all four species (data not shown). The one AUUUA motif in mouse not conserved in the other species is actually located downstream from the two polyadenylation consensus sites. This A + U-rich sequence motif is suggested to play a role in message instability in immediate early genes such as c-fos (Wilson and Treisman, 1988; Shyu et al., 1989; 1991). This class of genes is rapidly induced upon growth factor or serum stimulation and is likely to
play an important role in the alteration of gene expression required for cell cycle progression (Bravo, 1990; Herschman, 1991). With regard to the possible role of gap junctions in growth control and cell cycle regulation, it is interesting to note that a cDNA encoding the gap junction gene Cx26 was isolated in a subtraction library screen designed to identify tumor suppressor genes (Lee et al., 1991). To determine whether or not the mouse Cx43 belongs to the class of immediate early genes, we examined quiescent (serum-starved) NIH 3T3 and BALBfc 3T3 cells for the transient elevation of Cx43 transcripts following
198 serum stimulation. Typical immediate-early genes, such as c-myc and c-fos, exhibit a rapid but transient rise of transcript abundance upon serum or growth factor stimulation (Bravo, 1990; Herschman, 1991). This effect is greatly accentuated in the presence of CHX (Lau and Nathans, 1987). Thus, we isolated RNA from NIH 3T3 or BALB/c 3T3 cells that were serum starved, then subsequently serum stimulated either in the presence or absence of CHX. Northern analysis of RNA harvested from these cells at various time points after the initiation of serum stimulation revealed no obvious change in Cx43 transcript abundance upon serum stimulation, even in the presence of CHX (Fig. 5; also data not shown). In contrast, c-myc and c--0s showed the expected transient sharp rise in transcript abundance, and this increase was accentuated in the presence of CHX (Fig. 5). These results demonstrate that at least in NIH and BALB/c 3T3 cells, Cx43 is not regulated in a manner characte~stic of immediate-early genes. (g) Conclusions We cloned and characterized mouse Cx43 cDNA and genomic DNA. Using the cloned mouse Cx43 probes, we further characterized the expression of Cx43 transcripts in mouse embryos and in tissue culture cells subjected to serum stimulation. These studies provide the foundation for future research in which molecular approaches will be used to manipulate the expression of Cx43 for further examining its role in development and growth regulation. The data we have presented demonstrate that : (1) Cx43 transcripts are expressed in a spatially restricted manner in the 7.5-day mouse embryo. This is indicated by the complete absence of Cx43 transcripts in the 7.5day ectoplacental cone and the 12.5-day placenta. (2) The Cx43 gene isolated from the genomic library screen encodes the same transc~pt~protein as the cloned cDNA. This finding, in conjunction with other data, suggests that Cx43 transcripts expressed in the embryo are likely identical to those in adult tissues. (3) Similar to other connexin-encoding genes, Cx43 is composed of two exons with a single large intron positioned in the S-LJTR. (4) The transcription initiation site is mapped to a C residue situated 188 nt upstream from the splice donor site of exon 1. This same initiation site is observed for Cx43 transcripts expressed in the mouse embryo and in various adult tissues. (5) Cx43 contains an APl-binding site and a degenerate TATA box. (6) The 3’-UTR of Cx43 is highly conserved. This includes four AUUUA motifs, a sequence associated with message instability in immediate early genes. (7) Cx43 expression in NIH and BALB/c 3T3 cells does
not exhibit the serum inducibility which is characteristic of immediate early genes.
ACKNOWLEDGEMENTS
We would like to thank David Paul for the generous gift of the rat Cx43 probe and Janet Rossant for the mouse embryo cDNA library. This work was supported by N.I.H. grant HD21355 and NSF grant DCB-06886. C.R. was partially supported by NRSA postdoctoral fellowship GM-141 16, and R.S. was supported by predoctoral fellowships from the March of Dimes and NIH VMSTP, 5-T32GM07 170.
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