identification of transcription stop sites at the 5'and 3'ends of the

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 21, Issue of July 25, pp. 12513-12519.1990 Printed in U.S.A.

Identification of Transcription Stop Sites at the 5’ and 3’ Ends of the Murine Adenosine Deaminase Gene*

(Received for publication, September 14, 1989)

Ming-Chei MaaS, Jeffrey M. Chinskypll, V. RamamurthySII, Brita D. Martin& and Rodney E. Kellems$#** From the PVerna and Marrs McLean Department of Biochemistry and the $Znstitute for Molecular Genetics, Baylor College of Medicine, Houston, Te.&.s 77030

We report here the identification and nucleotide sequence of two transcription termination regions associated with the murine adenosine deaminase gene. One region is situated within or very near exon one and in mouse fibroblasts accounts for more than a 50-fold drop in the abundance of nascent transcripts. This termination region is believed to be involved in the regulation of adenosine deaminase gene transcription. The other termination region is located approximately 3.5 kilobases beyond the major polyadenylation site and defines the 3’ boundary of the adenosine deaminase transcription unit.

By comparison to the amount of information available concerning transcription initiation, relatively little is known about the process of transcription termination in mammalian cells (Platt, 1986; Proudfoot and Whitelaw, 1988; Proudfoot, 1989). This is due in part to the fact that sites of transcription termination have been difficult to identify because they do not correspond to the 3’ ends of stable transcripts. The use of in uiuo pulse labeling (Ford and Hsu, 1978; Nevins and Darnell, 1978), or more typically, in vitro nuclear run-on experiments (Citron et al., 1984; Groudine et al., 1981; Hag- enbuchle et al., 1984; LeMeur et al., 1984; Rohrbaugh et al., 1985; Xu et al., 1986) has made it possible to map the distribution of nascent transcripts at genetic loci of interest, and in this way identify regions where transcription is arrested. These approaches have usually involved the analysis of genes with a high level of transcriptional activity (Citron et al., 1984; LeMeur et al., 1984; Rohrbaugh et al., 1985; Xu et al., 1986) or situations where the genetic locus of interest is present in many copies (Ford and Hsu, 1978; Frayne et al., 1984; Nevins and Darnell, 1978). With these strategies it has been possible to map the position of transcription termination to within

* This research was supported by Grants GM30204 and AI25255 from the National Institutes of Health. Robert A. Welch Foundation Grant Q-893, and a grant from Eli Lilly & Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tkement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505523.

n Support,ed by Postdoctoral Fellowship GM12059 from the Na- tional Institutes of Health.

(1 Supported by Postdoctoral Fellowship Q-893 from the Robert A. Welch Foundation.

** To whom correspondence should be addressed: Dept. of Bio- chemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

specific regions of no more than a few hundred base pairs (Citron et al., 1984; Frayne and Kellems, 1986; LeMeur et al., 1984; Rohrbaugh et al., 1985; Whitelaw and Proudfoot, 1986; Xu et al., 1986). Functional analysis of transcription termination sites based on gene transfer studies has shown that a functional polyadenylation signal is required for transcription termination (Connelly and Manley, 1988, 1989; Logan et al., 1987), even though the actual site of transcription termination may be more than several thousand base pairs downstream from the polyadenylation site (Connelly and Manley, 1989; Hagenbuchle et al,, 1981). For immunoglobulin genes the site of transcription termination is developmentally regulated and the choice of termination sites governs in part the sequences present at the 3’ end of the stable transcription product (Galli et al., 1987). These sequences in turn determine the protein product encoded by the transcript (Danner and Leder, 1985; Galli et al., 1987). Thus, the differential utilization of transcription termination sites at the 3’ ends of transcription units can play an important role in regulating gene expression.

The expression of genetic information may also be con- trolled by the utilization of transcription arrest sites which exist at the 5’ end of a gene. The use of nuclear run on experiments has revealed that some mammalian genes have an intragenic block to transcription elongation which is subject to control as a function of cell proliferation or differen- tiation (Bender et al., 1987; Bentley and Groudine, 1986; Hofer et al., 1982). Similarly, the HSP70 gene in Drosophila has a pronounced block to transcription elongation which is relieved as a result of heat shock (Rougvie and Lis, 1988). Control of transcription elongation has also been observed in several viral transcription units and has been associated with regulating the expression of specific transcription units during viral life cycles (Hay et al., 1982; Kao et al., 1987; Mok et al., 1984; Resnekov and Aloni, 1989). In all of these cases the block to elongation is situated near the 5’ end of the transcription unit. The number and variety of these examples suggest that control of transcription elongation is not an unusual feature of transcriptional regulation.

We have recently provided evidence for the existence of a transcription arrest site near the 5’ end of the murine ADA gene and have shown that tissue specific control of transcription elongation beyond the promoter proximal region is involved in the regulation of ADA gene expression (Chinsky et al., 1989). Specifically, we found that in a tissue such as the placenta which is characterized by a high level of ADA mRNA, transcriptional activity is observed throughout the gene. How- ever, in a tissue such as the liver, which has a low level of ADA mRNA, transcriptional activity is found near the 5’ end of the gene only, with little detectable transcription occurring throughout the remainder of the gene (Chinsky et al., 1989). It has been difficult to accurately identify the location and

12513

12514 Adenosine Deaminase Gene Transcription

characterize the strength of this 5’ transcription arrest site in liver nuclei because of problems in routinely obtaining an adequate quantity of radioactively labeled nascent RNA from the single copy ADA genes present in these tissue samples. To facilitate the identification and characterization of the promoter proximal and other transcription arrest sites which may function to control transcription elongation at the murine ADA locus, we report here the use of a mouse fibroblast cell line with a 4300-fold amplification of the ADA genes (Yeung et al., 1983a, 1983b). We have provided a substantial amount of information which provides strong evidence that these cells have amplified a structurally and functionally normal ADA gene (Al-Ubaidi et al., 1990). In addition we have also shown that approximately 10 kb’ of 5’-flanking sequence and at least 15 kb of 3’-flanking sequence has been coamplified with the ADA gene (Maa, 1989). Transcription- ally active nuclei are readily prepared from these cells and the abundant ADA transcriptional activity is easily and accurately determined. Because the amplification of ADA genes is accompanied by a corresponding increase in the abundance of ADA protein, the level of expression per ADA gene in the amplified cells is indistinguishable from the low level of expression per ADA gene found in the parental cells (Yeung et al., 1983b). For this reason we believe that the analysis of ADA gene transcription in the B-1/50 cells should reveal features characteristic of ADA genes which are expressed at a low level, such as ADA gene expression in the liver. Because of the increased ADA transcriptional signal that results from ADA gene amplification the use of these cells has enabled us to identify and characterize two regions of transcription arrest associated with the murine ADA gene. One region is situated within or very near exon one and is associated with more than a 50-fold drop in the abundance of nascent transcripts. The other region is located approximately 3.5 kb beyond the major polyadenylation site and serves to define the 3’ boundary of the ADA transcription unit.

MATERIALS AND METHODS

Cell Lines and Culture Conditions-Cl-1D is a LM-TK- derived mouse cell line. Cl-1D cells selected for resistance to 1lAAU medium and 50 pM deoxycoformycin are designated B-1/50 (Yeung et al., 1983a). B-1/50 cells are characterized by a 4300-fold amplification of ADA genes (Yeung et al., 198313). Cl-1D and B-1/50 cells were grown as previously described (Yeung et al., 1983a).

Plasmid DNAs-All ADA DNA fragments used in this study were derived from cosmid clones which encompass the murine ADA gene (Ingolia et al., 1986; Al-Ubaidi et al., 1990). From these cosmids we prepared an ordered array of single-stranded Ml3 based subclones spanning the ADA locus (Fig. 1). The name of each subclone consists of one or two letters followed by a number. The number refers to the size of the subclone in kilobases. The letters refer to the restriction enzyme site at one or both ends of the subclone: E, EcoRI; H, HindIII; K, KpnI; N, NcoI; P, PstI; S, S&II; X, XbaI or XhoI (*PX1.5 and *XXI.4 only). DNA fragments used in nuclear run-on experiments were subcloned into either pTZ vectors (Pharmacia LKB Biotech- nology Inc.) or Bluescript KS vectors (Stratagene, La Jolla, CA) and transformed into BB4 cells (Stratagene, La Jolla, CA). When cells were superinfected with helper phage M13K07, single-stranded DNA was produced. All clones used were determined to be free of highly repetitive sequence elements as judged by lack of detectable hybridization when probed with radioactively labeled genomic DNA from mouse fibroblast cell line, Cl-1D.

Nuclear Run-on Transcription Analysis-Nuclei from 10’ cells were isolated and nascent RNA chains labeled with 0.5 mCi of [3ZP]UTP (3000 Ci/mmol; ICN) using the procedure described by Xu et al. (1986). RNA was extracted as described and hybridized to DNA samples that had been immobilized on nylon membranes (Bio-Rad, Zeta-probe). DNA probes (5 gg of each sample) were denatured in 0.3 N NaOH and transferred to nylon membranes using a dot blot

1 The abbreviations used are: kb, kilobase; bp, base pair.

manifold (Schleicher & Schuell). Filters were hybridized, washed, and RNase-treated as described (Xu et al., 1986).

Quantitution of Nuclear Run-on Transcription Analysis-Five fig of single-stranded DNA probes was bound to each 25mm nitrocel- lulose filter circle without prior base treatment of the DNA. Hybrid- izations of each experiment were done in triplicate as described (Frayne et al., 1984). After hybridization and washing, each filter was dried and counted directly in Scintilation counter.

DNA Sequencing-The nucleotide sequence determination was performed using the dideoxy-chain termination procedure (Sanger et al., 1977). Unambiguous sequence was obtained from both strands of DNA in all regions reported.

RESULTS

Identification and Sequence Determination of a Transcrip- tion Termination Site at the 3’ End of the ADA Gene-A large collection of single-stranded clones (Fig. 1) was prepared for use in determining the location and strand specificity of transcriptional activity at the murine ADA locus. In uitro labeled nascent transcripts were prepared from the nuclei of the ADA amplified cell line, B-l/50 (Yeung et al., 1983a), and were hybridized to single-stranded subclones which had been immobilized on nylon membranes. With regard to the ADA transcribed strand the results (Fig. 2, top panel) indicate that no transcriptional activity was detected by clones Xl.3 and XE0.6 which correspond to genomic segments situated immediately 5’ of the transcription initiation site of the ADA gene (Ingolia et al., 1986; Chinsky et al., 1989). Beginning with the clone El.4 transcriptional activity was detected from the template strand and continued throughout the ADA gene in the regions tested, extending 3’ to subclone EX2.4. No hybridization signal was associated with the next three probes, X1.0, XP2.5, and PX1.0, suggesting that the 3’ boundary of the ADA transcription unit was located within EX2.4. Incor- poration of radioactivity into nascent transcripts which hybridized to probes El.4 through EX2.4 was completely inhib- ited by 1 rg/ml a-amanitin (Fig. 2, middle panel), indicating that the signals detected resulted from RNA polymerase II transcripts.

As a control experiment we also mapped the distribution of nascent ADA transcripts in the murine placenta, a tissue which has high levels of ADA protein and mRNA. The placenta was chosen for this purpose because nuclei suitable for transcriptional analysis are readily prepared (Chinsky et al., 1989) and because a relatively high level of transcriptional activity/ADA gene permits the distribution of nascent transcripts to be determined. The results (Fig. 2, lower panel) indicate that in murine placenta, just as in B-1/50 cells, nascent transcripts are distributed throughout the ADA gene

, 5kb ,

I I I I I 1111 II I GC?tM

. * . ECDRI

1 Hindlll

Xbal

I I 1 Pstl x1.3 Y Px2.0 - “x.z4 - y IUD.6

XE0.6 Y lm.8 - *PXI.S - u x1.0 El.1 u tuz.8 - Ex7.4 - Y xp2.5

S,“)w Y EH4.1U .xI(, , - - F-m.2 sP25 - H(E),.0 Y

FIG. 1. The structure of the mouse ADA gene. The positions and relative sizes of the 12 exons of the ADA gene are indicated by the solid uertical bars (Al-Ubaidi et al., 1990). Restriction maps are provided for four enzymes. The small gap within the PstI map is a region that was not examined for Pst sites. Shown below the restriction maps are the names and locations of single-stranded probes used in this manuscript. See “Materials and Methods” for additional information concerning subclones.

Adenosine Deaminase Gene Transcription

co lnoooy~qq L”c‘! m~~oioirNPv-N”cv7- ‘W--0xxxxwx’Px xxwfJtlPxxwxwxxP

+ a

a*aa * l Ribo

B-l I 50

. B-l / 50 + l .

(a-Amanitin) .

G2

+ dOta Placenta

On Xba Xba

I I I II 1111 II I X 1.3~ HX 1.8~ EX 2.4 u XE 0.6 LI HX 2.5 u Xl.OU

E 1.4U EH 4.1 I XP 2.5 u

SP 2.5 U HE 1.0~ PX 1.2u PX 2.0 u

FIG. 2. Strand-specific analysis of transcriptional activity at the ADA locus in cell lines and mouse placenta. Depicted at the bottom of the figure is the map of the murine ADA gene, including the locations and sizes of the single-stranded genomic clones used as hybridization probes to measure transcriptional activity. Transcrip- tion from the ADA template strand is indicated as +. Transcription from the complementary strand is indicated as -. Autoradiographic analysis of hybridization of in vitro labeled transcripts obtained from B-1/50 cell nuclei (Top); B-1/50 cell nuclei preincubated with 1.0 pg/ ml a-amanitin (Middle); mouse placenta nuclei (Bottom). The 5’ portion of the placental panel has appeared in a previous publication (Chinsky et al., 1989). In each case from 10 to 30 X lo6 cpm were added to the hybridization reactions. Included in the autoradiographs are probes corresponding to human 18 S rDNA (Ribo) and pGem2 (G2). In an effort to detect strong hybridization signals with the 3’ clones, the signal intensity associated with some of the 5’ clones (e.g. El.4 and SP2.5) exceeded the linear range of the autoradiographic film. The relative abundance of nascent transcripts associated with the 5’ probes E1.4, S(H)0.2 and SP2.5. is more accurately shown in Figs. 4A and 5.

beginning with El.4 and extending to EX2.4. As with B-1/50 cells no hybridization was associated with the last three 3’ probes, X1.0, XP2.5, or PX1.2. These results indicate that the 3’ boundary of the ADA transcription unit is the same in B-1/50 cells and placenta and is most likely situated within EX2.4.

To localize more precisely the region of transcription termination within the 3’ clone EX2.4, two smaller subclones, XK1.4 and KX0.6, were prepared (Fig. 3). Nuclear run-on transcription measurements were conducted using nascent transcripts from B-1/50 cells and the results (Fig. 3) indicate that while a strong hybridization hybridization signal was associated with XK1.4, a much weaker signal was associated with KX0.6. In multiple independent experiments probe KX0.6 always showed significant signal above background (see also Fig. 5). No hybridization was ever detected with the adjacent downstream subclone X1.0. Similar results have been observed when using in vitro labeled nascent transcripts from placental nuclei (data not shown). These findings, as well as the quantitative analysis shown in Fig. 5 (see below), suggest that transcription terminates within, or very near, the 550 nucleotide fragment, KX0.6. The putative termination region

B,: 121 181 241 301 361 421 481 541 601 661 721 781 841

ATAGCTGGCC TCTGTCCTTR GGAGGACCCA AGGGGACTAG CAGAGC*C*G CTGCTCATTC TGTTAGTCAC GAGATGGGG~ ACTAAAAGCA GCTGCAAAGA CATTGTGCAG CTCAGCCTGG TAGAGRCTGT CCTTGACCTC TGTTCCGTGT

-

EX 2.4 I

XK 1.41

KX 0.6~ x 1.01

ACCTTGCCTT GRTTCTATCR RRGGCATTCC AGTCCCAGCA TGCCTCCCTT TCRGACI\TCA GTCATGGTGG CTAGC**GC* A*T*c*CAA* GRCTCTCAGT AAGGCAAACG GTGTTGTARA TGATGGCTAA GGATGAAGGC AGGCRGCAAR CAAAGCTGAT GAGCCTTGAA GAGCCCCTTG GCACTGTGCC CGGTGTGCCA GGTTGGACAG TGAGCAGCCG TTG*GT*m ACCCGTCACG AGAGGGCATG AGACAGCCTG TGCTGGAACA GCTCAGTTCA GAAAGGGGCG CCTCTGTCAG AGACAAGCTG TGGTCTGGGA CCACAATGAG AAGCAACCGC CAGAAGCAGG GCCCGCAGGC GAARGGAAAA AGTGGGGAAT AI\GRGTTGGG GCTGCGGAGG GTTCCGT*CT TGAACAGAAA GCAGGGACCA ACACAAGGAA GAAACTTTGT GAGGCTATGG AGGTAAGGGG CAGGGTATTC CAT- ATGGTCATTG GCTGGGCGAG CCTAAGAAGA RGTTAAATCT ATTACRTGRG AAACTGGGTC CCTGTCCCCA TCCTAAGTCA TTTCTGGGCA ATAAGGACTG AGAACTCTAG

TCRAGCACAT TCRTGTACTG GGCCTACRTG GAGAGCTGAG GCTGGGTCCA TGCCAAACAC TAGGGC- AGGCCTTAGC CTTTGTTCCA GGGTGGGGAG GGCCAAAGGG TCCCGAGGCA CTCCRGTTTC ACATTGTCCC

n

FIG. 3. Identification and sequence determination of a transcription termination site at the 3’ end of the ADA gene. A, autoradiographic analysis of hybridization of in vitro labeled nascent transcripts isolated from B-1/50 cell nuclei (5 x lo6 cpm). Shown below the dot blot is a schematic illustration depicting the locations and sizes of the single-stranded hybridization probes used. B, nucleotide sequence of the murine ADA transcription termination region. The region sequenced is indicated by the double-headed arrow and includes the entire KX0.6 clone (550 bp) and 343 bp from the 3’ end of XK1.4. Both the KpnI site (GGTACC) and XbaI site (TCTAGA) are underlined. The CCAAT sequence is boxed.

(shown as the double arrowhead in Fig. 3A) has been sequenced and the approximately 900 nucleotide sequence is presented in Fig. 3B. When this sequence was compared with that of nine other termination regions identified by nuclear run-on analysis, the most conserved region is a 15-nucleotide sequence shown in Table I. This sequence corresponds to the first 15 positions of a 20 nucleotide consensus sequence previously associated with RNA polymerase II termination regions (Frayne and Kellems, 1986). The corresponding sequence in the ADA termination region is boxed in Fig. 3B. Another sequence of potential importance is the CCAAT box element (Fig. 3B, boxed), which is believed to play a role in transcription termination in the adenovirus genome (Con- nelly and Manley, 1989).

The observations presented above provide strong evidence that the amplified ADA genes in the B-1/50 cells and the single copy ADA genes present in murine placenta utilize the same 3’ termination region. However, B-1/50 cells and placental tissue differ markedly in the relative abundance of ADA nascent transcripts at the 5’ end of the ADA gene. This difference is believed to result from tissue or cell type specific differences in the utilization of an exon one region transcription arrest site (Chinsky et al., 1989). Specifically, the very intense hybridization signal associated with the 5’ probes El.4 and SP2.5, when using nascent RNA from B-1/50 cell


TABLE I A partially conserved 15 nucleotide sequence associated with RNA

polymerase II termination regions

Distance

Gene from the nearest poly(A)

Conserved sequence

Matches/ Ref consensus

Rabbit &-globin Chicken ovalbumin Mouse dihydrofolate

reductase Mouse K Mouse fl,.j-globin Human a-globin (a,) Human n-globin (a,) Mouse n-globin Mouse ADA

signal

bp 1108 cTCA.UATAGGAAtA 13/15 36

904 AcCAgATTAGGAAGA 13/15 23 518 AaCgAATTAGaAAGA 12/15 11

232 -1300

333 983

ATCgAATTAGaAAtA 12/15 41 ATaAAgATAGGAtGA 12/15 5 ATCAAAAcAaaAcGA 11115 40 ATttcATcAGGAAGA 11/15 40 AgCAtAATtGGAtGc 10/15 39 ATggAgATgGGgAGA 10/15

119 -3750

A Consensus” AT&WA TAGGAAGA

T

’ Computer analysis revealed that this is the most conserved sequence shared among the nine termination regions considered.

nuclei (Fig. 2, top panel) was not observed when using RNA from placental nuclei (Fig. 2, lower panel). The pattern dis- played by the B-1/50 nuclei was more like that found in a tissue such as the liver, which has a low level of ADA (Chinsky et al., 1989).

A Block to Transcription Elongation Occurs within or Very Near Exon I in B-1150 Cells-As shown in Fig. 2 (top panel), when using nascent transcripts from the B-1/50 cells a very high level of transcriptional activity was detected by El.4 (a clone which encompasses the first exon of the ADA gene) and an overlapping clone SP2.5. Much lower levels of transcriptional activity were detected by clones 3’ of SP2.5. We show below that the increased signal associated with SP2.5 is due entirely to sequences within El.4. The nascent transcripts detected by El.4 are RNA polymerase II products as indicated by the ability of a-amanitin (1 rg/ml) to completely block the incorporation of radioactivity into these transcripts during the in vitro run-on assay (Fig. 2, middlepanel). The transcripts detected by El.4 did not result from initiation in vitro as judged by the inability of a 0.5% Sarkosyl (Hawley and Roeder, 1985), 1 mg/ml heparin (Hawley and Roeder, 1985), or 800 mM KC1 (Rougvie and Lis, 1988) to prevent the incorporation of radioactivity into these nascent transcripts during the in vitro run-on assay (data not shown; Chinsky et al., 1989). The increased signal associated with El.4 is not due to the presence of a repetitive sequence element because Southern blotting experiments have demonstrated that this fragment is a single copy sequence in the mouse genome (data not shown), and nucleotide sequence analysis (see below) revealed no regions corresponding to known repetitive elements. Taken together these findings indicate that a very high abundance of preinitiated nascent transcripts is associated with the exon 1 region of the ADA gene in the B-1/50 cells.

The distribution of nascent transcripts within the El.4 probe was localized further by the use of strand-specific subclones. The results shown in Fig. 4A indicate that the high abundance of nascent transcripts which reside in this region can be localized to the first 200 nucleotides of the ADA gene (see probe S(H)0.2, Fig. 4A). These findings place a block to transcription elongation within or very near exon one. The sequence of El.4 was determined and is presented in Fig. 4B. Notable features present at the 5’ end of intron one include an 18-base pair sequence which is completely conserved be-

ATG

S(H) 0.2 I

E 1.4 -,-I

SP 2.5 I-;-

FIG. 4. Identification of a block to transcription elongation within or near exon one. A, autoradiographic analysis of in vitro labeled transcripts from B-1/50 cell nuclei (5 X lo6 cpm) hybridized to single-stranded genomic clones from the exon one region. Shown below the dot blot is a diagram of the 5’ region of the murine ADA gene including exon one (hatched box) and flanking regions. B, nucleotide sequence of El.4. The translational start site is numbered as +l. The arrows pointing downward indicate the positions of multiple transcription initiation sites. Sequences in boxes indicate the region with 85% sequence similarity when compared with the corresponding region of the human ADA gene and mismatches (*) are marked. The arrows (+) indicate 31-base pair direct repeats with a single mismatch (*).

tween mouse and human ADA (Wiginton et al,, 1986) and a 31-base pair repeat with only a single mismatch.

The Distribution of Nascent Transcripts at the ADA Locus: a Quantitative Analysis-Because of the 4300-fold amplification of ADA genes in the B-1/50 cells it was possible to accurately quantitate the distribution of nascent transcripts across the ADA genes in these cells. For this purpose in vitro labeled transcripts were prepared and hybridized to filter immobilized single-stranded clones from the ADA locus. The amount of radioactivity hybridized to individual filter-immobilized DNA clones was determined by liquid scintillation counting and normalized to account for differences in clone size. The findings (Fig. 5) indicate that there is more than a 50-fold greater abundance of nascent transcripts associated with exon one (e.g. probe S(mO.2) than with other regions of

Adenosine Deaminase Gene Transcription 12517

ADA GENE (12 eronr)

FIG. 5. Quantitative analysis of transcriptional activity at the ADA locus. In uitro labeled nascent transcripts from B-1/50 cell nuclei were hybridized to the indicated single-stranded probes encompassing the ADA locus. The amount of radioactivity hybridized to each probe was determined and normalized to the size of the probe and expressed as cpm/kb. Values represent the average of three experiments and are normalized to EH4.1.

the ADA gene. The level of transcriptional activity then remains relatively constant throughout the gene in the regions examined until reaching the termination region (Fig. 5). Here the abundance of transcripts increased substantially just prior to complete loss of nascent transcripts. No transcriptional activity extended into the region represented by the probe X1.0. Thus, as indicated above, transcription appears to terminate near the boundary of XK1.4 and KX0.6 in a region encompassing, but not extending past, that included in KX0.6.

Opposite Strand Transcription Occurs at Several Locations at the ADA Locus-When using the B-1/50 cells, opposite strand transcriptional activity was detected at three locations within or immediately flanking the ADA locus (Fig. 2, top panel). Opposite strand transcriptional activity was associated with probes X1.0 and XP2.5, which are located in the 3’- flanking region immediately downstream of the ADA transcription termination site and with probe SP2.5, which is located immediately downstream of the exon one region transcription block. Opposite strand transcriptional activity was also associated with XE0.6, which is located immediately 5’ of the ADA promoter region. The S’flanking probe X1.0 and the intragenic probe EH4.1 detected opposite strand transcriptional activity when using nuclei from placenta. All regions of opposite strand transcription were sensitive to inhi- bition by a-amanitin (1 pg/ml), suggesting that the activity was the result of RNA polymerase II transcription.

DISCUSSION

We have used a mouse fibroblast cell line having a 4300- fold amplification of ADA genes cells as a convenient model system to determine the distribution of nascent transcripts across the ADA locus. As explained in the Introduction, the analysis of ADA transcriptional activity in these cells is expected to reveal features characteristic of ADA genes which are expressed at a low level, such as that found in the liver. Using ADA gene-amplified cells, we have shown here that the abundance of nascent transcripts drops by more than 50-fold

within the first 200 nucleotides of the ADA gene. These findings provide a minimum quantitative estimate to the level of control that can be achieved through the regulation of the transcription elongation beyond the promoter proximal region. Sequence analysis of the transcription arrest region revealed the presence of a 31-base pair direct repeat having only a single mismatch and an 18-bp sequence showing complete identity to the corresponding position of the human ADA gene (Wiginton et al., 1986). In this regard it is interesting to note that a promoter proximal transcription arrest site is associated with the human ADA gene and that cell type-specific control of transcription elongation beyond the 5’ region plays a major role in regulating human ADA gene expression (Lattier et al., 1989; Chen et al., 1990). The 1%bp conserved sequence may be a component of &-regulatory elements associated with a transcription arrest signal.

Two possible models may account for the relatively high nuclear run-on transcriptional signal associated with the exon one region of the mouse ADA gene. According to one model the existence of a promoter proximal transcription pause site may cause a pileup of RNA polymerase II complexes 5’ of the pause site. If cellular factors responsible for the putative transcription pause site are lost during the isolation of nuclei, the previously blocked nascent transcripts may elongate during in vitro nuclear run-on transcription and incorporate radioactive precursors. According to another model most of the ADA transcription complexes terminate prematurely within the promoter proximal region, and the released transcripts are rapidly degraded. As a result of this process there is a constant and high flux of nascent transcripts through the exon one region of the ADA gene, with only a small percentage of transcription complexes proceeding into more 3’ portions of the gene. At this stage of our investigations we cannot distinguish between these two models.

An exon one region transcription arrest site has been characterized for the human and mouse c-myc genes (Bentley and Groudine, 1986, 1988; Nepveu and Marcu, 1986). A variety of studies have shown that control of transcription elongation through this region plays a major role in regulating c-myc gene expression. In view of the potential similarities between the control of c-myc and ADA gene expression, we compared the sequence of El.4 with that of the comparable regions of the human and mouse c-myc genes. No significant sequence similarities were found. However, recent evidence from our laboratory (Ramamurthy et al., 1990) indicates that when the DNA template El.4 is injected into Xenopus oocytes transcription initiates at the major ADA start site and terminates approximately 96 nucleotides downstream within exon one. Similar findings have been reported for the human c-myc gene, where the exon one associated transcription arrest signal is also recognized in Xenopus oocytes (Bentley and Groudine, 1986, 1988).

As a result of our analysis we have identified a 3’ termination region located approximately 3.5 kb downstream from the major polyadenylation site of the ADA gene. This is presumably the transcription termination site which defines the 3’ end of the ADA transcription unit. We conclude that transcription terminates very efficiently in this region because no detectable transcriptional activity was present immediately downstream of the 550 nucleotide termination fragment even though the B-1/50 cells used in these experiments con- tain many thousands of copies of ADA 3’-flanking sequences. Thus, virtually all of the amplified ADA genes terminated transcription within, or just prior to, a very small region of less than 550 nucleotides. The functional significance of the ADA termination region identified in B-1/50 cells is under-


scored by the fact that transcription terminates within the same region in murine placenta, a tissue in which the ADA gene is actively transcribed (Chinsky et al., 1989). No transcriptional activity is detected in this region of the ADA gene in the liver, a tissue where very few transcripts extend beyond the exon one region (Chinsky et al., 1989). Potentially important sequence motifs present within the ADA termination region include a 15-nucleotide sequence which is partially conserved among termination regions (Table I) and a CCAAT sequence which is suggested to play a role in transcription termination in adenovirus (Connelly and Manley, 1989). It is interesting that in the B-1/50 cells and in placenta a substantial increase in the abundance of nascent transcripts is observed in the 1.4-kb XhoI-KpnI fragment just prior to the 550 nucleotide KpnI-X&I termination fragment. A similar finding has been observed within the termination region of the mouse immunoglobulin k gene (Xu et al., 1986). The increased abundance of nascent transcripts just prior to complete termination may reflect a slowing of the movement of RNA polymerase through such regions to allow 5’ exonucleases the op- portunity to remove nascent transcripts which remain following cleavage and polyadenylation (Connelly and Man- ley, 1988, Proudfoot, 1989). According to this model, once the nascent transcripts have been completely removed by the 5’ exonuclease activity, the polymerase is free to disengage from the template. Specific mechanisms to account for this process have been proposed (Connelly and Manley, 1988; Proudfoot, 1989).

Opposite strand transcription at the ADA locus was observed at several locations. One region was associated with the ADA promoter and may represent divergent transcriptional activity as has been observed for the human ADA promoter (Berkvens, 1988) and other G:C-rich bidirectional promoters (Huckaby et al., 1987; Linton et al., 1989; Melton, 1987). Another region of opposite strand transcription was located immediately downstream of the block to transcription elongation associated with exon one, and a third region was located immediately downstream of the 3’ transcription termination region. Opposite strand transcription downstream of the mouse K-light chain gene transcription termination site has been observed and may reflect a functional role for this activity (Xu et al., 1986). For example, such opposite strand transcription may impede the progression of RNA polymerase from each strand and cause termination to occur in both directions. Alternatively, the presence of complementary transcripts may play a functional role in the termination process.

The level of ADA protein and mRNA in mice shows a striking tissue distribution which covers a range of more than IO,OOO-fold (Chinsky et al., 1990; Knudsen et al., 1988; Ra- mamurthy et al., 1989). In some murine tissues such as the heart, brain, and muscle, ADA activity is barely detectable. Many tissues, especially those of hematopoietic origin, have intermediate levels. The highest levels are present in the placenta and in tissues of the alimentary tract (Chinsky et al., 1990, Knudsen et al., 1988; Ramamurthy et al., 1989). The level of ADA protein and mRNA is subject to pronounced prenatal and postnatal developmental control in these seem- ingly unrelated tissues. The genetic signals which govern this complex pattern of gene expression have not been identified. The results of the transcriptional analysis presented here and elsewhere (Chinsky et al., 1989) indicate that a strong intra- genie block to transcription elongation occurs within or near exon one and that control of transcription elongation through this region has the potential to play a significant role in regulating ADA gene expression.

Acknowledgments-We are grateful to Jeffrey Innis, Randy Mifflin, and Zhi Chen for critical review of this manuscript. We thank Debra Parr for help in preparing the manuscript. Alanosine was kindly provided by the Drug Synthesis and Chemistry Branch, and 2’- deoxycoformycin by the Natural Products Branch, of the Develop- mental Therapeutics Program, Division of Cancer Treatment, Na- tional Cancer Institute. We are also grateful to the Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company for providing Pentostatin (2’-deoxycoformycin).

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