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Alpha‐thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3′ end processing in the human alpha 2 globin gene.

Alpha‐thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is... The EMBO Journal vol.5 no. 11 pp.2915-2922, 1986 oa-Thalassaemia caused by a poly(A) site mutation reveals that in transcriptional termination is linked to 3' end processing the human globin gene cu2 Emma Whitelaw and Nick Proudfoot region of the gene within which transcriptional termination oc- curs is not sufficient to cause termination when it is introduced Sir William Dunn School of Pathology, University of Oxford, South Parks into another gene, the adenovirus EIA gene. However, a larger Road, Oxford, OXI 3RE, UK fragment (1.6 kb) containing both the (3 globin gene poly(A) site Communicated by N.J.Proudfoot and the termination region does cause termination of transcrip- We have investigated the process of transcriptional termina- tion of the ElA gene (Falck-Pedersen et al., These ex- 1985). tion in the duplicated human a globin genes which lie 4 kb raise the that termination of periments possibility transcription apart on chromosome 16. In the human erythroleukemic cell may only occur downstream of an active site. polyadenylation line, K562, which expresses high levels of a globin, nuclear have addressed this a thalassaemic We question directly using run-off experiments suggest that termination occurs within a with a mutation at the human globin gene point AATAAA a region of 600 bp past the poly(A) site of both al and a2 which results sequence in a failure to generate correct globin genes. However, a thalassaemic a2 globin gene with et polyadenylated 3' ends (Higgs al., 1983). By comparing the a non-functional poly(A) site AAUAAG, when transfected into of this mutant a2 transcriptional termination globin gene with HeLa cells, not only fails to 3' end process but also fails to of a2 we show that that the wild-type globin gene, transcrip- terminate transcription. Studies on both steady-state RNA and does a tional termination of the a2 globin gene require functional nuclear run-off analysis of the primary transcripts show that polyadenylation site. transcription of the mutant a2 globin gene reads through in- to the intergenic sequence past the normal termination site. Results These results demonstrate that transcriptional termination termination the a2 and oc- Transcriptional of al globin genes and 3' end processing of mRNA are coupled events for the curs close to their addition sites poly(A) a2 globin gene. To of determine the position transcriptional termination for the Key words: a2 globin gene/mRNA processing/transcription ter- human a2 a and l globin genes, we carried out nuclear run-off mination/poly(A) site/cx-thalassaemia on analysis nuclei isolated from K562 cells (Lozzio and Lozzio, 1975), a human erythroleukaemia cell line that expresses high Introduction amounts of at globin following haemin induction et (Rutherford The of in development vivo and in vitro systems that accurately al., 1979; Charnay and Maniatis, 1983). The subline of K562 initiate the transcription of cloned genes has brought considerable used in these experiments expresses equivalent amounts of a2 insight into the promoter sequences of eukaryotic genes and the and acl globin mRNAs (data not shown). protein factors associated with initiation (Corden et al., 1980; Figure IA shows a diagram of the human a globin genes with Dynan and Tjian, 1985). However, far less is known about the the positions of the DNA probes used in the nuclear run-off events involved in transcriptional termination. RNA polymerase analysis indicated on the gene map. Thus a 1.6-kb PstI fragment I terminates transcription 500 bp downstream of the 3' end of (probe A) was used to detect both a2 and a1 gene transcripts the mature 28S rRNA at a set of repeated elements interspersed while two DNA fragments (probes B and C) 3' to the a2 globin by runs of pyrimidines (Grummt et al., 1985). With RNA poly- and one 3' to the a 1 were gene fragment (probe D) globin gene, merase III, the termination process appears to be controlled by used to detect gene transcripts that extend into the 3'-flanking a short run of T residues (Bogenhagen and Brown, 1981). The region. Figure lB shows the nuclear run-off data obtained using termination sites of RNA polymerase II transcription units have these a The left side is an frac- gene probes. panel agarose gel not been accurately identified. For a number of genes, such as tionation of B and while the middle and probes A, C, right panels mouse globin (Hofer et al., 1982; Citron et al., 1984), rabbit are of 32P-labelled nuclear RNA to blots of these hybridizations globin (Rohrbaugh et al., 1985), Drosophila histone fractionated Probe A is a PstI of the a 1 PstI 1.6-kb (Price and probes. digest Parker, 1984), chicken histone (Krieg and Melton, 1984), sea subcloned into Both vector band and fragment pBR322. probe urchin histone (Birchmeier et al., 1984), adenovirus major late are in this lane. As a present indicated, strong positive signal (Fraser et al., 1979; Moore and Sharp, 1985), mouse ca amylase was obtained for probe A while the vector band gave a back- (Hagenbuchle et al., 1984) and the chicken ovalbumin gene ground hybridization signal. Probes B, C and D are purified gel for (LeMeur et al., 1984), the transcribing RNA polymerases read bands and lower than gave significanfly signals probe A, only through the sites on the DNA which correspond to the sequence than Probe C is cross-contaminated slightly higher background. of that contains an at the 3' end the stable mRNA. Formation of correct 3' ends with a larger sized DNA fragment Alu repeat concomitant is in trace requires endonucleolytic cleavage of the primary transcript. this sequence. Although only present Nuclear in is was obtained to this run-off experiments, which the primary transcript a amounts, significant hybridization signal with which illustrates the of low level labelled [32P]UTP, show that transcriptional termination can fragment danger contaminating occur a long way [up to 2.0 kb in the case of the mouse a Alu in nuclear run-off amylase sequence probes. et downstream of the site. Table IA the obtained for gene (Hagenbuchle al., 1984)] poly(A) quantifies signals probes A, B, because the number Recent studies on the mouse termination and D. Each is ,B globin gene pro- signal directly comparable in all four is kb. cess indicate that the 0.8-kb DNA in the of transcribed nucleotides -1 fragment 3'-flanking potentially probes IRL Press Limited, Oxford, England E.Whitelaw and N.J.Proudfoot 1 Kb 0 1 2 3 4 5 6 a2 al Pvu EI Pst I Pst I I ALBgII I Pst Pst I Dra I It Sma I Pvu I Hinc Bgl IL II Jl Probe A C A D A B C A B A B C D Vector\ _ i, A/ Fig. 1. a Nuclear run-off analysis on human a2 and globin gene transcription in a haemin-induced K562 nuclei. (A) Map of the human globin genes. Genes are divided into exons as filled-in boxes, and non-coding or introns as a open boxes. Direction of gene transcription is indicated by arrows. Position of an Alu repeat region between a2 and ac1 is indicated. The positions of the four DNA probes used in the analysis are drawn under the map. (B) The hybridization signals obtained to blots of probes A, B, C and D versus [32P]nuclear RNA from K562 cells. On the left-hand panel is shown the ethidium bromide-stained probes separated by agarose gel Probe A electrophoresis. is a PstI digest of the a2 globin gene 1.6-kb PstI fragment in at the PstI pBR322 site. Probes B, C and D (agarose gel fractionation not are shown) purified restriction fragments of rai2W3'PS (B and C) and from palRB (D): (see Materials and methods for details of These fractionations plasmids). probe were transferred to cellulose nitrate and hybridised to RNA from [a-32P]UTP-labelled K562 nuclei. After extensive washing the autoradiographs were exposed for 3 days at -70°C (see Materials and methods) as shown on the middle and right-hand panels. Duplicate experiments with probes A and B and single experiments wtih probes C and D are shown. The larger 1.6-kb PstI probe A which contains 600 bp of Table of in nuclear run-off I. Quantitation hybridization assays 5'-flanking sequence, similarly covers 1 kb of transcribed gene sequence for both the a2 and al A. K562 globin genes. Therefore the expression (Figure 1) signal to probe A represents approximately twice the true signal DNA fragment % Hybridization for a2 or a1 globin gene transcripts separately. The hybridiza- tions in A Table IA are normalized to one half of the A PstI-PstI (a2 and al) 100 probe signal. PvuII-DraI (3Y'a2) Av 30 These data demonstrate that probes B and C hybridize at 30 and C BglII-SmaI (3'a2) 15 15% of the a2 gene signal, while probe D hybridizes at 13% D PvuII-HincII (3'al) 13 of the a 1 gene signal. This implies that termination of transcrip- tion occurs Pvul close to the sites of both a2 and al globin genes, from of the % Hybridization was calculated as densitometry autoradiographs soon after their sites. respective poly(A) a % of half the signal obtained with A since this probe probe hybridizes equally to both the a2 and ac1 Each value has the globin genes. background Ternination oftranscription is linked to polyadenylation in a tran- signal to the vector subtracted from it. (hybridization pBR322 band) sient expression system Because termination of a B. HeLa transient expression (Figure transcriptional the human globin 2) gene occurs close to the 3' of unusually end the mRNA, we decided M13 clone % Hybridization to the that 3' investigate possibility end and termina- processing a2W a2M tion were coupled events, at least for the human a2 globin gene. Sense: et al. have described a Higgs (1983) Saudi Arabian thalassaemic NcoI-BstEII 100 a2 globin gene with a base that single change mutates the BstEII-BstEII 108 AATAAA sequence to AATAAG. demon- Furthermore, they BstEII-DraI 84 strated that this gene fails to mRNA 3' generate ends when Antisense: transfected into tissue culture cells. To this mutant exploit gene NcoI-BstEII 29 57 for on studies ai gene termination, we carried out run- nuclear BstEllI-BstEII 15 off on HeLa analysis cells transfected with either the thalassaemic BstEII-DraI 18 38 or (a2M) wild-type oa2 (a2W) globin gene, together with exten- sive These 3'-flanking sequences. two constructs were cloned Background was estimated as the amount of to alone. hybridization Ml13 into the transient expression vector pSVed (Proudfoot et al., The signal minus background of sense to Ml13 clone transcripts NcoI-BstEII is 1984) and are called ca2M3'PS and ai2W3'PS, 100% hybridization. where 3'PS refers to the presence of extensive 3'-flanking sequence (see Materials and methods and Figure 4). These two associated with plasmids were then tran- transient we subcloned expression, three a gene in siently expressed HeLa cells and nuclei from these transient fragments, one from within the gene and two in the 3'-flanking expression experiments were subjected to nuclear run-off analysis. region into an M 13 vector. Clones in both orientations were ob- Because high amounts of non-specific transcription are often tained for each a gene fragment. The a gene sequence in each 2916 termination and mRNA processing in the a2 globin gene Transcriptional a2W Sense Transcrpt M13 Antisense Transcript li--i Nco I Bst E Dra I Bst E E E Sense Transcript UT M13 *.A aAntisense Transcript a2M Fig. 2. Nuclear run-off analysis on nuclei of HeLa cells transfected with a2W3'PS and a2M3'PS pSVed. M13 clones were made in both orientations for the three restriction of the a2 as indicated. 5 of single strand DNA for each M13 subclone as well as M13 without an insert fragment regions globin gene jig were immobilized on dot blots and hybridized to [32P]nuclear RNA as described in Materials and methods. The sense and antisense dot blot signals obtained with either a2W or ca2M plasmids are placed above and below the (x gene map so that their positions correspond to the three different DNA fragments used to make the M13 probes. M13 clone is - 600 in so that the intensity of hybridiza- of the termination or its exact loca- bp length ciency transcriptional process tion is directly comparable. Single strand DNA prepara- tion. This may give rise to an apparent discrepancy between the signal from these six as as the were K562 a2W nuclear run-off experiments. For K562 tions clones, well parental M13, and transient immobilized on cellulose nitrate, as 'dot blots' and hybridised some signal was obtained for the immediate 3'-flanking signal to 32P-labelled nuclear RNA from a2M or a2W transient ex- (probe B) suggesting that termination occurs at least several pression experiments under conditions of DNA excess. 100 bp past the poly(A) site, while for a2W the signal detected Figure 2 shows the hybridization data obtained for these six in the immediate 3'-flanking M13 probe was not above the non- M 13 a gene probes and Table lB quantifies these hybridization in two of out of three experiments. These specific transcription signals. A striking difference was obtained between a2W and results clearly demonstrate some degree of transcriptional ter- a2M nuclear run-off signals. a2W gave a 3-fold greater signal mination for az2W but not for a2M and therefore strongly argue for the entirely genic NcoI-BstEH sense probe than for the two for a direct link between a2 globin mRNA 3' end processing region sense probes, indicating significant levels of or polyadenylation and transcriptional termination. 3'-flanking termination. In contrast with a2M, all three probes transcriptional with a a-Thalassaemic globin gene non-functional poly(A) site gave closely similar signals indicating that no transcriptional utilises a site the hwnan and a]I cryptic poly (A) between a2 globin discontinuity occurs between gene and 3'-flanking sequence. genes Similar data were also obtained from two further nuclear run- above that in the absence of a func- off experiments (data not shown). However, the amount of anti- The data presented suggest a2W a2M tional the a-thalassaemic gene fails to ter- sense throughout both the and genes and poly(A) site, globin transcripts as much as minate transcription. To confirm these data, we analysed flanking sequences was quite high, approaching 50% for a2M and to the mRNA the transient of the of the sense signal nearly equal 3'-flanking steady-state produced by expression of the a2W These anti-sense a2M transfected into Cos7 cells to determine how far the region signals experiment. gene which is a2 globin mRNA extended past the non-functional termination/ transcripts may be due to the SV40 early promoter pre- in vector and transcribes off the strand of the mutant ct2 gene. a2M3'PS sent the pSVed opposite polyadenylation region globin a from initiation of was transfected into Cos7 cells and the RNA, obtained after 48 h to the gene promoter or random transcrip- The of transient was S1 tion around the plasmid. non-specific transcription expression, analysed by mapping (Figure 3). BstEII- from both strands of the transient No mRNA 3' ends were detectable using the 600-bp presumably expression plasmid obscure the of the a2 BstEII a2 gene 3' fragment probe. Instead, complete protection may partly specific transcription globin an accurate assessment of the effi- of the was that extended all gene and therefore prevent probe seen, indicating transcripts 2917 E.Whitelaw and N.J.Proudfoot A B C D Co G G Co G Co A- A+ Co P_ _ 309 650 * 4-S 527 ,, _201 _190 ._ 527 ----_ 650 _ * -4-A 403 -_ _ 160 527 _ 4 403 4:7 .;f : S-. 122 309 v 4_ v * _w .--I _ 76 _b Bst ElI Bst E [I AATAAAB9I U Avav I lL A,B,C t -- -- --Kb. _ S *- Fig. 3. SI nuclease (A,B,D) and exonuclease VII (C) mapping of the 3' ends of globin mRNA present in cytoplasmic RNA from Cos7 cells purified transfected with ct2M3'PS pSVed. The two probes used in these experiments are shown in the were a and diagram. They BstEII-BgIIl fragment an AvaII-BglIl fragment purified from the ci2M3'PS DNA by acrylamide The were end labelled in with Klenow gel electrophoresis. probes by filling DNA polymerase and [a-32P]dNTP. A line diagram indicating the sequences the is shown at the bottom of the SI of protected by probes figure. (A) analysis RNA a2M3'PS to the Positions of and artefact bands are indicated. The cytoplasmic hybridised BstEII-BglII probe (lane G). probe (P), signal (S) (A) two are the of AT-rich in the RNA-DNA the SI reaction. 44 higher artefact bands due to 'breathing' sequences hybrid during The lower artefact band at bp is a is in B and C. RNA is in probe alone band and also apparent The minus control shown lane Co. (B) SI analysis of poly(A)-selected ca2M3'PS cytoplasmic RNA. Poly(A)+ (lane A+), poly(A)- (lane A-). The probe used was the BstEII-BglII fragment. (C) Exonuclease VII digests of cs2M3'PS cytoplasmic RNA hybridized to the BstEII-BglI probe. (D) S1 analysis of a2M3'PS cytoplasmic RNA against the smaller Avall-BgIII probe. A doublet band is obtained at 122 -20 1 bp according to restriction fragment size markers. This position is bp 3' to an AATAAA sequence midway between the ct2 and a globin genes et The two artefact bands at 100 and 72 to bands from (Hess al., 1983). bp bp correspond the artefact arising AT-rich sequences seen in panels A and B with the BstEII-BglII probe. the this first of the ct2 -a1 se- forms an SI way through portion intergenic nuclease-sensitive site in the RNA-DNA duplex quence (data not the a2-a1 because it was absent when exonuclease VII was used (Figure shown). However, using adjacent intergenic probe a band was obtain- and so it cannot RNA (BstEII-BglII), strong (S) 3C) represent with true 3' ends. ed 600 nucleotides lane The other smaller To define the 3' end of the a2 gene transcript more precisely, long (Figure 3A, G). bands (A) are SI as discussed in the This we used a smaller DNA probe labelled at an site within artefacts, figure legend. AvaII result obtained by SI analysis was confirmed using the single the BstEII-BglII DNA fragment (see Figure 3). A 122-bp 3' exonuclease VII strand-specific (Figure 3C) which gave the doublet band was obtained together with the same two artefact same 600 nucleotide in long band, absent the control lane. This bands probably caused by AT-rich sequences (Figure 3D). This 600-nucleotide band was polyadenylated (Figure 3B). Although result positions the 3' end of the mutant a2 gene transcript in a band at - 800 was seen in the this bp RNA, cor- the middle of the z2 -ac 1 intergenic sequence - 20 bases 3' to poly(A)- responds to a AT-rich DNA this an AAUAAA very sequence. Presumably, sequence, the only AAUAAA in the whole in- 2918 Transcriptional termination and mRNA processing in the a2 globin gene a2M3*S(AATAAG) a2W*S(AATAAA) a2M1H e2MS a2MD a2MB VV V Q I'K a2W AATAAA I I I I I I I I L I I l l 0 1 2 7 3 4 5 Kb Fig. 4. Line diagram depicting the various a2 globin gene constructs with respect a to the human globin gene map. The a2- and cxl-globin genes are indicated by rectangles with the filled in exons. The positions of the poly(A) addition V sequences are indicated. denotes sequence added. M denotes mutant and W denotes wild-type. tergenic sequence (Hess et al., 1983). 4 Figure shows a detailed 5A (lanes 2-5), each construct gave normal levels a of mRNA of the map human a globin genes and indicates the position of four times higher than with a2M3'PS. As a measure of transfec- the cryptic poly(A) site in the a2 -(xl intergenic sequence. In tion efficiency, the plasmid ,B pSVed (see Materials and methods) summary, these data indicate that when the normal was poly(A) site used as a co-transfection control with each a2M construct. of the a2 gene is non-functional, a2 gene transcripts extend S1 analysis using a globin 3' a band of 210 probe, gave bp 1.5 kb into the 3'-flanking region to form a new polyadenylated corresponding to the distance between the EcoRI site and the 3' 3' end at a cryptic poly(A) site. end of the : globin mRNA (Figure 5B). The signal obtained was equivalent in all tracks so that we could The direcfly compare the of the cryptic poly(A) site provides a inefficiency molecular amounts of a mRNA shown in globin Figure SA. explanation for the a-thalassaemia phenotype To confirm that the amounts of a globin mRNA were rescued Two sets of data suggest that the transcriptional termination pro- by utilizing the added al gene poly(A) site, we carried out 3' cess of the thalassaemia a2 globin gene is disrupted. First nuclear end SI analysis of RNA from cell transfected with a2MB, where run-off analysis indicates that nascent transcripts extend past the the added site is poly(A) placed 3' to the cryptic site poly(A) normal termination region and second 1 a cryptic poly(A) site kb (Figure 4). Figure 5C (lane 1) shows the 3' S1 analysis ofa2MB into the 3'-flanking region is utilized by the a2M gene at signifi- RNA a alI using globin gene 3' probe. The presence of a band cant levels. However, we suspected that the amount of globin of 220 indicated that bp the added al gene poly(A) site was be- mRNA that utilises the cryptic poly(A) site is abnormally low, utilized ing while the 120-bp band corresponded to the mismatch because the patient with this thalassaemic gene expresses reduc- between the a2 sequence in present the construct and the ca 1 se- ed levels of a2 mRNA globin (Higgs et al., 1983). quence in the As RNA probe. expected, from cells transfected To measure the level of stable a2 globin mRNA produced from with a2M3'PS gave only the 120-bp mismatch band (lane 2). a2M3'PS directly, a quantitative assay of 5' ends was carried Figure SD shows the co-transfected ,B globin gene mRNA signal. out using primer extension analysis as shown in Figure 5. The We found that the a2MB transfection was twice as efficient as amount of stable mRNA was - 3-4 times lower in Cos7 cells a2M3'PS so that the amount of 3' ends from a2MB was 3- to transfected with a2M in 3'PS than those transfected with a2W 4-fold those bands in a2M3'PS, verifying the at quantitation the 3'PS lanes 1 (Figure 5A, and 6). The amount could be lower 5' end (Figure SA). The 3' end of the RNA from cells transfected for two reasons: most (i) transcripts are before the terminating with a2MH, a2MS, a2MD were also analysed with probe C site and are cryptic poly(A) unstable, or are not (ii) transcripts and were all found to be utilizing the added poly(A) site (results terminating before the cryptic poly(A) site but the cryptic poly(A) not shown). These data reveal two important features of the a12M site is not as effective as normal so that only 20-30% of the gene. Firstly the of the site at inefficiency cryptic poly(A) least transcripts this reaching point are stabilized by cleavage and in accounts part for the reduced levels of a2 globin gene ex- To polyadenylation. test these alternatives, we constructed in pression observed this type of thalassaemia. no Secondly, a2MD a2MH, a2MS, and a2MB, in which a 300-bp fragment significant level of termination can occur at transcriptional least the a 1 containing wild-type poly(A) site was at the placed HpaI, up to the BglII site in the a2 -a 1 This intergenic sequence. lat- DraI or Sacd, BglII sites, 3' to the a2M globin gene (see Figure ter result confirms our nuclear run-off data and indicates analysis 4). If all these constructs could raise the amounts of mRNA from a between 3' end linkage processing and ter- transcriptional the a2M gene to equal those of the wild-type gene, this would mination. suggest that termination was not occurring prior to the site of the added but that the poly(A) site, cryptic poly(A) site was four times less efficient in mRNA than stabilizing the normal poly(A) Discussion site. their transfection into Cos7 Following cells, each of these four a2M3'PS constructs with an added mRNA 3' end in normal poly(A) site was formation eukaryotes involves both endo- tested for levels of a2 mRNA As in and shown nucleolytic cleavage polyadenylation. The conserved synthesis. Figure highly 2919 E.Whitelaw and N.J.Proudfoot 1 2 3 4 5 6 7 M M 1 2 3 4-Cl Cap-.-_ a I _ ot am 122 t. a .-a 2 118 * Primer-_ _~n 1 M 2 3 or 3 4 5 6 7 M 20O p signal -_ -~l _ 180 Psignal-_. %qp _ 160 ' 144 BstEl PvulI HaeM BstEfl Hinfl I I -I 60 220 ^,120 ae 3 primer probe " RI LEco { __d... I.- -VI ' 3' "', \ P'.9e 5. Primer extension and SI nuclease C and of 5' and 3' ends of a mRNA in RNA from Cos7 cells Fig. (A) (B, D) mapping globin present cytoplasmic of DNA with to these are indicated in the transfected with a2MB and a2M3'PS. The the a2MH, a2MS, positions probes respect plasmids diagram. RNA from cells transfected with a2W a2MH a2MS a2MD a2MB and minus RNA Primer extension of Cos7 c2M3'PS (A) (1), (2), (3), (4), (5), (6) from the and exon of control with A. The a is an antisense strand 3'-end-labelled Hinfl-HaeIII 1st (7) probe primer single fragment 5'-non-coding region to site a band of 74 SI of RNA from Cos7 cells transfected with a2W at2. The is extended 20 the 54-bp primer by bp Cap giving bp. (B) analysis (1), and minus RNA control with the 3' 3' Probe B is a 3' end double- a2MH a2MS a2MD a2MB a2M3'PS (2), (3), (4), (5), (6) (7) , probe. , labelled, the at the EcoRI site and in with Klenow DNA and SI stranded obtained probe by linearizing plasmid pSVod unique filling polymerase [a-32P]dATP. (C) with ae2MPS and a minus RNA control with 3'a Probe was obtained analysis of RNA from cells transfected c2MB ail (1), (2) (3) probe. by linearizing BstEII site and in with Klenow DNA and of RNA from cells transfected with a2MB at the SI pSVed unique filling polymerase [a-32P]dGTP. (D) analysis a minus RNA control with 3'3. (1), a2M3'PS (2) and (3) probe 2920 termination and mRNA in the a2 Transcriptional processing globin gene AAUAAA found 10-30 bases of most in this case is than would be greater for a with expected patient sequence upstream poly- sites forms of the for two normal com- apparently cal globin genes (D.Higgs, personal adenylation part recognition signal cleavage of the and and two active Fitz- munication) a2 Indeed an (Proudfoot partially globin genes. primary transcript Brownlee, 1976; and et et a of Montell three functional a would Shenk, 1981; al., globin output nearly globin gerald Higgs 1983; al., genes 1983). Recent evidence indicates that be located expected for this rather than the observed patient single sequences immediately downstream of the site are also An and globin gene output. for these intriguing explanation apparent poly(A) required (Simonsen and McDevitt et Levinsen, Gil in a levels in the Saudi Arabian 1983; Proudfoot, 1984; discrepancies globin a- al., 1984). When the DNA to the AAUAAA thalassaemia be that the of the a2 to may inability sequence corresponding globin gene signal is altered to AAGAAA in adenovirus et terminate or results in a2 transcription (Montell al., 1983) gene transcripts reading AACAAA in a human f3-thalassaemic et the into and through intergenic sequence a1 globin gene (Orkin al., thereby inhibiting RNA are observed. a 1 globin gene expression. A recent Proudfoot 1985) paper by respectively, elongated transcripts (1986) it has been shown that when the AATAAA that such a is mutated suggests transcriptional interference effect can occur Similarly, to as in the case of the human a-thalassaemic between two AATAAG, and that this effect is alleviated adjacent genes globin by correct 3' end is abolished if the mutant termination between the two placing transcriptional gene, processing signals gene genes. is transfected into tissue culture cells et We al., (Higgs 1983). Materials and methods have extended this observation to show that of the transcription mutant a continues at least 1.0 kb a Globin and constructs into the (3 globin gene globin gene 3'-flanking to + reduced levels of a2W3'PS. The a2W3'PS contains the entire which plasmid a2 globin gene from the end region give poly(A) transcripts PvuII site 1.5 kb 5' from the site to the site 2.0 kb 3' from the downstream of cap SnaI the AATAAA found in the poly(A) just only intergenic site. This 4.5-kb was inserted into the vector fragment pSVed between the EcoRI between the a two sequence globin genes. Furthermore, by plac- and PvuI sites. The vector contains the pSVed pBR322 replication and origin the normal site from a the 1 ing polyadenylation and the SV40 and enhancer globin gene tetracycline gene replication origin sequence (Proud- downstream of this site we find that the foot et al., 1984). cryptic poly(A) transcripts read the site and as far as the inserted The a2M3'PS is beyond cryptic poly(A) a2M3'PS. plasmid identical to a2W3'PS that the except 0.6-kb site. BstEII the fragment containing wild-type poly(A) site, was AATAAA, poly(A) replaced the identical The fact that the normal by fragment from the mutant et al., in which at gene (Higgs 1983) 1 site functions globin gene poly(A) the site is AATAAG. poly(A) when 3' to the site that efficiently placed cryptic poly(A) argues a2MH, a2MS, a2MD, a2MB. These plasmids from a2M3'PS. The the site is originated a2 mRNA cryptic inefficient, only stabilizing globin 300-bp fragment from BstEll to Pvull containing the human a I globin poly(A) to 20% of the level. the absence of func- wild-type Presumably, site was inserted into a2M3'PS at the HpaI site the SacI site (c2MH), (a2MS), tional downstream of the site the DraI site signals (a2MD) and the site as shown in 4. cryptic poly(A) (McDevitt BglII (a2MB) Figure et Gil and al., its low 1984; effi- Proudfoot, 1984) The may explain paJRB. plasmid, in which a 4.0-kb from the human EcoRI-BglJI fragment The amount of a2 mRNA in al globin gene has been inserted between the EcoRI ciency. and BamHI sites of globin present reticulocytes pBR322, of the thalassaemic - has been is % described by Lauer et al. 10-20 that found in (1980). a normal patient individual et The of the a Globin gene M13 clones. Restriction from the al., enzyme fragments human a2 (Higgs 1983). inefficiency cryptic and 3' site in gene sequences indicated in were flush-ended and account for the (as Figure 2) purified, a-thalassaemic poly(A) may part phenotype ligated into the SmaI site of M13 mp8 (Maniatis et al., 1982). Clones of both of the mutant gene. orientations were isolated and the single-stranded DNA was phage grown up by Nuclear run-off a 1 to assess and a2 experiments standard globin gene procedures. in the cell show that transcription line, erythroleukemic K562, (3pSVed. The human , globin gene from in the HpaI 5'-flanking sequence to termination of both the transcriptional PstI in the 3'-flanking sequence was inserted into wild-type globin genes pSVed between the EcoRI site occurs within 100-300 of their In- and the PstI site (Proudfoot et sites. al., 1984). bp respective poly(A) the a mouse also pf35'SV. This was a from terestingly, gift Dr F.Grosveld. The rabbit (3 globin gene is in- globin gene transcription appears to terminate in a serted into a plasmid containing the SV40 and 50-250 3' of origin enhancer sequences and the transcription region bp poly- large T antigen (Grosveld et al., 1982). The presence of these sequences site allows et In both these cases the adenylation al., (Sheffery 1984). for replication in HeLa cells of any plasmids containing the SV40 origin. termination sites are closer to the site than transcriptional poly(A) Transient expression in most other II so far studied et polymerase genes (Birnstiel al., Transfection into Cos7 cells or HeLa cells were carried out as described previously 1985). (Mellon et Whitelaw and al., 1981; Proudfoot, 1983). Cos7 cells contain a defective The fact that termination of of the c2 transcription SV40-transformed CVI wild-type monkey cell line that expresses sufficient levels of SV40 occurs close to the site in K562 and T antigen to allow globin replication of plasmid-containing SV40 origin sequences, such gene poly(A) yet of the as with HeLa mutant in pSVod. Experiments cells were carried out in the of the HeLa cells presence ca2 transcription globin gene goes plasmid p,B5'SV (see above), which allows for replication of other plasmids con- at least 1.5 kb this that termination does beyond region, suggested taining the SV40 origin. Plasmid DNA was precipitated with calcium phosphate not occur without active 3' end Nuclear run-off ex- processing. and added to subconfluent dishes of cells. After 10-16 h, the medium was changed on tissue culture cells transfected with either the mu- periments and the cells allowed to grow for another 30 h. The cells were harvested, lysed tant or the confirm this in NP-40 and the since detergent buffer, cytoplasmic and nuclear fractions separated wild-type gene suggestion termination does occur with by centrifugation through a sucrose cushion. Following incubation with the but not with the mu- proteinase wild-type K, cytoplasmic RNA was purified by phenol/chloroform extraction and ethanol tant Because these two differ one nucleotide gene. genes by only precipitation (Whitelaw and Proudfoot, 1983). Poly(A) selection was carried out A-G in the AATAAA we must assume that sequence, by standard transcrip- procedures using an oligo(dT) cellulose column (Maniatis et al., 1982). tional termination does a functional site. Such require poly(A) RNA mapping would make sense as it would requirement good SI nuclease. physiological Probe DNAs (either double or were annealed single stranded) to termination of within How such a prevent cytoplasmic RNAs (10-20 in 30 of 80% transcription genes. 11 formamide, 0.04 M Pipes pH ILg) 6.4, could work at the mechanistic level is harder to 0.4 M NaCl, 0.1 mM EDTA by denaturation at 80°C for 10 min, then system 53°C, envisage but further double strand probe or 30°C single strand to delineate the probe overnight. 300 of ice-cold exact JlI experiments sequences required SI buffer (0.25 M NaCl, 0.03 M NaAcetate 2 mM 50 pH 4.6, ZnSO4, Agg/ml for termination of RNA polymerase II genes should clarify this denatured sonicated carrier DNA) plus S (3000 units) was added to each quickly issue. hybridisation and incubated for 1 h at 30°C. SI reactions were ethanol precipitated It is that the of a-thalassaemia and fractionated on interesting severity denaturing, 7 M urea polyacrylamide homozygous gels. 2921 E.Whitelaw and N.J.Proudfoot Exonuclease VII. Hybridisations were carried out as for the SI experiments. 500 Falck-Pedersen,E., and Logan,J., Shenk,T. Darnell,J.E.,Jr (1985) Cell, 40, Al of exonuclease VII buffer (30 mM KCI, 10 mM Tris pH 7.8 and 10 mM EDTA) 897-905. with 4 U of exonuclease VII (Bethesda Research Laboratories) was added to and 251-260. Fitzgerald,M. Shenk,T. (1981) Cell, 24, hybridisations and incubated at 37°C for 2 h. The reactions were then ethanol and Darnell,J.E. J. Mol. Biol., Fraser,N.W., 129, Nevins,J.R., Ziff,E. (1979) precipitated and fractionated as for SI experiments. 643. and 473-474. Gil,A. Proudfoot,N.J. Primer (1984) Nature, 312, extension. DNA primer and RNA were annealed in 10 of 10 mM Pipes ptl de and Shewmaker,C.K. Flavell,R.A. Nature, Grosveld,G.C., Boer,E., (1982) pH 6.4, 0.4 M NaCl at 80°C for 10 min and 63°C overnight. 50 of reverse 120-126. 295, transcriptase buffer (50 mM Tris, pH 8.2, 10 mM DTT, 6 mM MgCI2, 0.5 mM and Mol. Cell 281 -288. Weintraub,H. Biol., 1, Groudine,M., Peretz,M. (1981) dATP, dCTP, dTTP, dGTP) plus reverse transcriptase (5 units) were added to and Hassouna,N. Bachellerie,J.-P. Cell, Grummt,I., Maier,U., Ohrlein,A., (1985) hybridisations and incubated at 42°C for I h. The reactions were ethanol 801-810. 43, precipitated twice and fractionated by electrophoresis on 7 M urea polyacrylamide and Cribbs,D.L. Schibler,U. Cell, 38, Hagenbuchle,O., Wellauer,P.K., (1984) gels. 737-744. Nuclear isolation, transcription and RNA purification and Proc. Acad. Hess,J.F., Fox,M., Schmid,C.W. Shen,C.-K.J. Natl. (1983) A variant K562 line which forms an adherent monolayer (M.V.Chao, unpublished) Sci. 5970-5974. USA, 80, was grown in DMEM supplemented with 10% fetal calf serum in the presence and Weatherall,D.J. Higgs,D.R., Goodbourn,S.E.Y., Lamb,J., Clegg,J.B., was of 50 uM haemin for 3-4 days. Nuclear isolation and transcription carried 398-400. Proudfoot,N.J. (1983) Nature, 306, out according to Groudine et al. (1981) but with some modifications. Cells 585-593. and Hofer,E. Darnell,J.E. (1981) Cell, 23, (- 1 x 107) were spun 1 x M M down, washed in SSC (0.15 NaCI 0.015 and 887-893. Cell, 29, Hofer,E., Hofer-Warbinek,R. Darnell,J.E.,Jr (1982) sodium citrate, pH 7.0). Nuclei were isolated by lysis of cells in RSB (0.01 M and Nucleic Acids Res., 7, Kafatos,F.C., Jones,C.W. Efstratiadis,A. (1979) Tris pH 7.4, 0.01 M NaCI, 3 mM MgCl2) containing 0.5% NP-40 and centrifuga- 1541-1551. for 10 2000 in without NP-40 tion min, r.p.m. The nuclei were washed RSB and 203-206. Krieg,P.A. Melton,D. (1984) Nature, 308, and resuspended in 2 x transcription buffer (5 mM DTT, 180 mM KCl, 10 mM and 119-130. Cell, 20, Lauer,J., Shen,C.-K. Maniatis,T. (1980) MgCl2, 20 mM Tris pH 7.8, 50% glycerol). Elongation was carried out at 30°C and EMBO 2779-2786. J., 3, LeMeur,M.A., Galliot,B. Gerlinger,P. (1984) for 10 min in the presence of 0.2 mM ATP, GTP, CTP, 200 [a-32P]UTP 321-334. and /Ci Lozzio,C.B. Lozzio,B.B. (1975) Blood, 45, (3000 Ci/mmol, Amersham). Reactions were terminated by the addition of RNase- and Molecular A Maniatis,T., Fritsch,E.F. Sambrook,J. (1982) Cloning. free DNase I (Miles) to 100 ,ug/ml and incubation at 30°C for a further 5 min. Manual. Cold Harbor NY. Press, Laboratory Spring Laboratory Under these conditions, pre-initiated RNA chains are elongated 100-300 and by Cell, 37, McDevitt,M.A., Imperiale,M.J., Ali,H. Nevins,J.R. (1984) nucleotides (Weber et al., 1977). The RNA was purified according to the method 993-999. to Treisman and Maniatis (1985). Basically, the reactions were in and 279-288. deproteinized Cell, Mellon,P., Parker,V., Gluzman,Y. Maniatis,T. (1981) 27, a solution of 0.1 M Tris pH 7.5, 0.15 M NaCl, 1% SDS 10 mM EDTA with and Nature, 305, Montell,C., Fisher,E.F., Caruthers,M.H. Berk,A.J. (1983) 100 of proteinase K per ml and subsequent -chloroform extraction phenol 600-605. ytg and ethanol precipitation. The was taken in 300 1l 10 mM 845-855. pellet up Tris, pH 7.5, and Cell, Moore,C.L. Sharp,P.A. (1985) 41, 10 mM MgCl2 and incubated at 37°C for 30 min in the presence of 50 and EMBO Antonarakis,S.E. Kazazian,H.H.,Jr (1985) gltg/ml Orkin,S.H., Cheng,T.-C., RNase-free DNase I (Miles). The reaction was extracted with 453-456. phenol/chloroform 4, J., and ethanol precipitated. 423-429. and Price,D.H. Parker,C.S. (1984) Cell, 38, Protocols for nuclear and RNA for 730-731. isolation transcription purification experiments Proudfoot,N.J. (1986) Nature, 321, using HeLa cells in which plasmids were were iden- 211-214. being transiently expressed and Proudfoot,N.J. Brownlee,G.G. (1976) Nature, 263, tical to those used for the nuclear isolation, and RNA and EMBO transcription purification J., 3, Rutherford,T.R. (1984) Proudfoot,N.J,. Partington,G.A. of K562. The transfection of HeLa cells with plasmids was carried out as described 1533-1540. above. h later. 2 x 106 cells were and Mol. Cell. Nuclei were purified 40-48 Approximately Biol., Johnson,J., James,M. Hardison,R. (1985) Rohrbaugh,M.L., used for each experiment. 147-160. 5, 164-165. and Weatherall,D.J. Nature, 280, Rutherford,T.R., Clegg,J.B. (1979) Hybridization to nitrocellulose filters Mol. 417-436. and 172, Riflind,R.A. J. Biol., Sheffery,M., Marks,P.A. (1984) bound to nitrocellulose filters to M13 single strand DNA (5 was according Rg) Mol. Cell. 2250-2258. and 3, Biol., Simonsen,C.C. Levinson,A.D. (1983) the method of Kafatos et al. (1979) with a miniblot filtration unit. 72-75. and Treisman,R. Maniatis,M. Nature, 315, (1985) first the Experiments on DNA fragments were done by purifying fragments 611-616. and Damell,J. Weber,J., Jelinek,W. (1977) Cell, 10, from a large them on a small agarose gel and then running (0.2 ,ig) agarose gel. Nucleic Acids 7717-7733. and Res., Whitelaw,E. Proudfoot,N.J. 11, (1983) The DNA nitrocellulose to Maniatis et al. was then transferred to according (1982). to the of Hofer and Alkali breakage of RNA was performed according technique Received on 28 1986 July Damell was 1 ml. was car- (1981). The volume of hybridization Hybridization ried out at of formamide and 42°C in the presence 50% (Treisman Maniatis, 1985) for the filters were washed: 30 48 h. After hybridization, min, 25°C, 2 x 0.1I% SDS. Then the filters 2 x SSC, 0.1% SDS; 30 min; 65°C, SSC, were x with RNAse at a concentra- incubated in 2 SSC at 37°C (DNase-free) The filters were washed in 0.2 x 0.1% SDS at tion of 2 finally SSC, 14g/ml. 68°C for 30 min. Acknowledgements We gratefully acknowledge Dr for the cs2 thalassaemic Doug Higgs providing globin gene We thank both and used throughout these experiments. Doug Higgs Dean Jackson for discussion. we are indebted to Zena Werb for helpful Finally, her critical reading of the manuscript. EW and NJP were a Medical supported by Research Council project grant no. G8108316CB. References Birchmeier,C., Schumperli,D., Sconzo,G. and Proc. Natl. Birnstiel,M.L. (1984) Acad. Sci. USA, 81, 1057-1061. Birnstiel,M.L., Busslinger,M. and Strub,K. (1985) Cell, 349-359. 41, Bogenhagen,D.F. and Brown,D.D. (1981) 261-270. Cell, 24, Charnay,P. and Maniatis,T. 1281 - 1283. (1983) Science, 220, and Citron,B., Falck-Pedersen,E., Salditt-Georgieff,M. Darnell,J.E.Jr (1984) Nucleic Acids Res., 12, 8723-873 1. Corden,J., Wasylyk,B., Buchwalder,A., and Sassone-Corsi,P., Kedinger,C. Chambon,P. (1980) Science, 290, 1406-1414. 774-778. Dynan,W.S. and Tjian,R. (1985) Nature, 316, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Alpha‐thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3′ end processing in the human alpha 2 globin gene.

Whitelaw, E.; Proudfoot, N.
The EMBO Journal , Volume 5 (11) – Nov 1, 1986
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Springer Journals
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Copyright © European Molecular Biology Organization 1986
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0261-4189
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1460-2075
DOI
10.1002/j.1460-2075.1986.tb04587.x
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Abstract

The EMBO Journal vol.5 no. 11 pp.2915-2922, 1986 oa-Thalassaemia caused by a poly(A) site mutation reveals that in transcriptional termination is linked to 3' end processing the human globin gene cu2 Emma Whitelaw and Nick Proudfoot region of the gene within which transcriptional termination oc- curs is not sufficient to cause termination when it is introduced Sir William Dunn School of Pathology, University of Oxford, South Parks into another gene, the adenovirus EIA gene. However, a larger Road, Oxford, OXI 3RE, UK fragment (1.6 kb) containing both the (3 globin gene poly(A) site Communicated by N.J.Proudfoot and the termination region does cause termination of transcrip- We have investigated the process of transcriptional termina- tion of the ElA gene (Falck-Pedersen et al., These ex- 1985). tion in the duplicated human a globin genes which lie 4 kb raise the that termination of periments possibility transcription apart on chromosome 16. In the human erythroleukemic cell may only occur downstream of an active site. polyadenylation line, K562, which expresses high levels of a globin, nuclear have addressed this a thalassaemic We question directly using run-off experiments suggest that termination occurs within a with a mutation at the human globin gene point AATAAA a region of 600 bp past the poly(A) site of both al and a2 which results sequence in a failure to generate correct globin genes. However, a thalassaemic a2 globin gene with et polyadenylated 3' ends (Higgs al., 1983). By comparing the a non-functional poly(A) site AAUAAG, when transfected into of this mutant a2 transcriptional termination globin gene with HeLa cells, not only fails to 3' end process but also fails to of a2 we show that that the wild-type globin gene, transcrip- terminate transcription. Studies on both steady-state RNA and does a tional termination of the a2 globin gene require functional nuclear run-off analysis of the primary transcripts show that polyadenylation site. transcription of the mutant a2 globin gene reads through in- to the intergenic sequence past the normal termination site. Results These results demonstrate that transcriptional termination termination the a2 and oc- Transcriptional of al globin genes and 3' end processing of mRNA are coupled events for the curs close to their addition sites poly(A) a2 globin gene. To of determine the position transcriptional termination for the Key words: a2 globin gene/mRNA processing/transcription ter- human a2 a and l globin genes, we carried out nuclear run-off mination/poly(A) site/cx-thalassaemia on analysis nuclei isolated from K562 cells (Lozzio and Lozzio, 1975), a human erythroleukaemia cell line that expresses high Introduction amounts of at globin following haemin induction et (Rutherford The of in development vivo and in vitro systems that accurately al., 1979; Charnay and Maniatis, 1983). The subline of K562 initiate the transcription of cloned genes has brought considerable used in these experiments expresses equivalent amounts of a2 insight into the promoter sequences of eukaryotic genes and the and acl globin mRNAs (data not shown). protein factors associated with initiation (Corden et al., 1980; Figure IA shows a diagram of the human a globin genes with Dynan and Tjian, 1985). However, far less is known about the the positions of the DNA probes used in the nuclear run-off events involved in transcriptional termination. RNA polymerase analysis indicated on the gene map. Thus a 1.6-kb PstI fragment I terminates transcription 500 bp downstream of the 3' end of (probe A) was used to detect both a2 and a1 gene transcripts the mature 28S rRNA at a set of repeated elements interspersed while two DNA fragments (probes B and C) 3' to the a2 globin by runs of pyrimidines (Grummt et al., 1985). With RNA poly- and one 3' to the a 1 were gene fragment (probe D) globin gene, merase III, the termination process appears to be controlled by used to detect gene transcripts that extend into the 3'-flanking a short run of T residues (Bogenhagen and Brown, 1981). The region. Figure lB shows the nuclear run-off data obtained using termination sites of RNA polymerase II transcription units have these a The left side is an frac- gene probes. panel agarose gel not been accurately identified. For a number of genes, such as tionation of B and while the middle and probes A, C, right panels mouse globin (Hofer et al., 1982; Citron et al., 1984), rabbit are of 32P-labelled nuclear RNA to blots of these hybridizations globin (Rohrbaugh et al., 1985), Drosophila histone fractionated Probe A is a PstI of the a 1 PstI 1.6-kb (Price and probes. digest Parker, 1984), chicken histone (Krieg and Melton, 1984), sea subcloned into Both vector band and fragment pBR322. probe urchin histone (Birchmeier et al., 1984), adenovirus major late are in this lane. As a present indicated, strong positive signal (Fraser et al., 1979; Moore and Sharp, 1985), mouse ca amylase was obtained for probe A while the vector band gave a back- (Hagenbuchle et al., 1984) and the chicken ovalbumin gene ground hybridization signal. Probes B, C and D are purified gel for (LeMeur et al., 1984), the transcribing RNA polymerases read bands and lower than gave significanfly signals probe A, only through the sites on the DNA which correspond to the sequence than Probe C is cross-contaminated slightly higher background. of that contains an at the 3' end the stable mRNA. Formation of correct 3' ends with a larger sized DNA fragment Alu repeat concomitant is in trace requires endonucleolytic cleavage of the primary transcript. this sequence. Although only present Nuclear in is was obtained to this run-off experiments, which the primary transcript a amounts, significant hybridization signal with which illustrates the of low level labelled [32P]UTP, show that transcriptional termination can fragment danger contaminating occur a long way [up to 2.0 kb in the case of the mouse a Alu in nuclear run-off amylase sequence probes. et downstream of the site. Table IA the obtained for gene (Hagenbuchle al., 1984)] poly(A) quantifies signals probes A, B, because the number Recent studies on the mouse termination and D. Each is ,B globin gene pro- signal directly comparable in all four is kb. cess indicate that the 0.8-kb DNA in the of transcribed nucleotides -1 fragment 3'-flanking potentially probes IRL Press Limited, Oxford, England E.Whitelaw and N.J.Proudfoot 1 Kb 0 1 2 3 4 5 6 a2 al Pvu EI Pst I Pst I I ALBgII I Pst Pst I Dra I It Sma I Pvu I Hinc Bgl IL II Jl Probe A C A D A B C A B A B C D Vector\ _ i, A/ Fig. 1. a Nuclear run-off analysis on human a2 and globin gene transcription in a haemin-induced K562 nuclei. (A) Map of the human globin genes. Genes are divided into exons as filled-in boxes, and non-coding or introns as a open boxes. Direction of gene transcription is indicated by arrows. Position of an Alu repeat region between a2 and ac1 is indicated. The positions of the four DNA probes used in the analysis are drawn under the map. (B) The hybridization signals obtained to blots of probes A, B, C and D versus [32P]nuclear RNA from K562 cells. On the left-hand panel is shown the ethidium bromide-stained probes separated by agarose gel Probe A electrophoresis. is a PstI digest of the a2 globin gene 1.6-kb PstI fragment in at the PstI pBR322 site. Probes B, C and D (agarose gel fractionation not are shown) purified restriction fragments of rai2W3'PS (B and C) and from palRB (D): (see Materials and methods for details of These fractionations plasmids). probe were transferred to cellulose nitrate and hybridised to RNA from [a-32P]UTP-labelled K562 nuclei. After extensive washing the autoradiographs were exposed for 3 days at -70°C (see Materials and methods) as shown on the middle and right-hand panels. Duplicate experiments with probes A and B and single experiments wtih probes C and D are shown. The larger 1.6-kb PstI probe A which contains 600 bp of Table of in nuclear run-off I. Quantitation hybridization assays 5'-flanking sequence, similarly covers 1 kb of transcribed gene sequence for both the a2 and al A. K562 globin genes. Therefore the expression (Figure 1) signal to probe A represents approximately twice the true signal DNA fragment % Hybridization for a2 or a1 globin gene transcripts separately. The hybridiza- tions in A Table IA are normalized to one half of the A PstI-PstI (a2 and al) 100 probe signal. PvuII-DraI (3Y'a2) Av 30 These data demonstrate that probes B and C hybridize at 30 and C BglII-SmaI (3'a2) 15 15% of the a2 gene signal, while probe D hybridizes at 13% D PvuII-HincII (3'al) 13 of the a 1 gene signal. This implies that termination of transcrip- tion occurs Pvul close to the sites of both a2 and al globin genes, from of the % Hybridization was calculated as densitometry autoradiographs soon after their sites. respective poly(A) a % of half the signal obtained with A since this probe probe hybridizes equally to both the a2 and ac1 Each value has the globin genes. background Ternination oftranscription is linked to polyadenylation in a tran- signal to the vector subtracted from it. (hybridization pBR322 band) sient expression system Because termination of a B. HeLa transient expression (Figure transcriptional the human globin 2) gene occurs close to the 3' of unusually end the mRNA, we decided M13 clone % Hybridization to the that 3' investigate possibility end and termina- processing a2W a2M tion were coupled events, at least for the human a2 globin gene. Sense: et al. have described a Higgs (1983) Saudi Arabian thalassaemic NcoI-BstEII 100 a2 globin gene with a base that single change mutates the BstEII-BstEII 108 AATAAA sequence to AATAAG. demon- Furthermore, they BstEII-DraI 84 strated that this gene fails to mRNA 3' generate ends when Antisense: transfected into tissue culture cells. To this mutant exploit gene NcoI-BstEII 29 57 for on studies ai gene termination, we carried out run- nuclear BstEllI-BstEII 15 off on HeLa analysis cells transfected with either the thalassaemic BstEII-DraI 18 38 or (a2M) wild-type oa2 (a2W) globin gene, together with exten- sive These 3'-flanking sequences. two constructs were cloned Background was estimated as the amount of to alone. hybridization Ml13 into the transient expression vector pSVed (Proudfoot et al., The signal minus background of sense to Ml13 clone transcripts NcoI-BstEII is 1984) and are called ca2M3'PS and ai2W3'PS, 100% hybridization. where 3'PS refers to the presence of extensive 3'-flanking sequence (see Materials and methods and Figure 4). These two associated with plasmids were then tran- transient we subcloned expression, three a gene in siently expressed HeLa cells and nuclei from these transient fragments, one from within the gene and two in the 3'-flanking expression experiments were subjected to nuclear run-off analysis. region into an M 13 vector. Clones in both orientations were ob- Because high amounts of non-specific transcription are often tained for each a gene fragment. The a gene sequence in each 2916 termination and mRNA processing in the a2 globin gene Transcriptional a2W Sense Transcrpt M13 Antisense Transcript li--i Nco I Bst E Dra I Bst E E E Sense Transcript UT M13 *.A aAntisense Transcript a2M Fig. 2. Nuclear run-off analysis on nuclei of HeLa cells transfected with a2W3'PS and a2M3'PS pSVed. M13 clones were made in both orientations for the three restriction of the a2 as indicated. 5 of single strand DNA for each M13 subclone as well as M13 without an insert fragment regions globin gene jig were immobilized on dot blots and hybridized to [32P]nuclear RNA as described in Materials and methods. The sense and antisense dot blot signals obtained with either a2W or ca2M plasmids are placed above and below the (x gene map so that their positions correspond to the three different DNA fragments used to make the M13 probes. M13 clone is - 600 in so that the intensity of hybridiza- of the termination or its exact loca- bp length ciency transcriptional process tion is directly comparable. Single strand DNA prepara- tion. This may give rise to an apparent discrepancy between the signal from these six as as the were K562 a2W nuclear run-off experiments. For K562 tions clones, well parental M13, and transient immobilized on cellulose nitrate, as 'dot blots' and hybridised some signal was obtained for the immediate 3'-flanking signal to 32P-labelled nuclear RNA from a2M or a2W transient ex- (probe B) suggesting that termination occurs at least several pression experiments under conditions of DNA excess. 100 bp past the poly(A) site, while for a2W the signal detected Figure 2 shows the hybridization data obtained for these six in the immediate 3'-flanking M13 probe was not above the non- M 13 a gene probes and Table lB quantifies these hybridization in two of out of three experiments. These specific transcription signals. A striking difference was obtained between a2W and results clearly demonstrate some degree of transcriptional ter- a2M nuclear run-off signals. a2W gave a 3-fold greater signal mination for az2W but not for a2M and therefore strongly argue for the entirely genic NcoI-BstEH sense probe than for the two for a direct link between a2 globin mRNA 3' end processing region sense probes, indicating significant levels of or polyadenylation and transcriptional termination. 3'-flanking termination. In contrast with a2M, all three probes transcriptional with a a-Thalassaemic globin gene non-functional poly(A) site gave closely similar signals indicating that no transcriptional utilises a site the hwnan and a]I cryptic poly (A) between a2 globin discontinuity occurs between gene and 3'-flanking sequence. genes Similar data were also obtained from two further nuclear run- above that in the absence of a func- off experiments (data not shown). However, the amount of anti- The data presented suggest a2W a2M tional the a-thalassaemic gene fails to ter- sense throughout both the and genes and poly(A) site, globin transcripts as much as minate transcription. To confirm these data, we analysed flanking sequences was quite high, approaching 50% for a2M and to the mRNA the transient of the of the sense signal nearly equal 3'-flanking steady-state produced by expression of the a2W These anti-sense a2M transfected into Cos7 cells to determine how far the region signals experiment. gene which is a2 globin mRNA extended past the non-functional termination/ transcripts may be due to the SV40 early promoter pre- in vector and transcribes off the strand of the mutant ct2 gene. a2M3'PS sent the pSVed opposite polyadenylation region globin a from initiation of was transfected into Cos7 cells and the RNA, obtained after 48 h to the gene promoter or random transcrip- The of transient was S1 tion around the plasmid. non-specific transcription expression, analysed by mapping (Figure 3). BstEII- from both strands of the transient No mRNA 3' ends were detectable using the 600-bp presumably expression plasmid obscure the of the a2 BstEII a2 gene 3' fragment probe. Instead, complete protection may partly specific transcription globin an accurate assessment of the effi- of the was that extended all gene and therefore prevent probe seen, indicating transcripts 2917 E.Whitelaw and N.J.Proudfoot A B C D Co G G Co G Co A- A+ Co P_ _ 309 650 * 4-S 527 ,, _201 _190 ._ 527 ----_ 650 _ * -4-A 403 -_ _ 160 527 _ 4 403 4:7 .;f : S-. 122 309 v 4_ v * _w .--I _ 76 _b Bst ElI Bst E [I AATAAAB9I U Avav I lL A,B,C t -- -- --Kb. _ S *- Fig. 3. SI nuclease (A,B,D) and exonuclease VII (C) mapping of the 3' ends of globin mRNA present in cytoplasmic RNA from Cos7 cells purified transfected with ct2M3'PS pSVed. The two probes used in these experiments are shown in the were a and diagram. They BstEII-BgIIl fragment an AvaII-BglIl fragment purified from the ci2M3'PS DNA by acrylamide The were end labelled in with Klenow gel electrophoresis. probes by filling DNA polymerase and [a-32P]dNTP. A line diagram indicating the sequences the is shown at the bottom of the SI of protected by probes figure. (A) analysis RNA a2M3'PS to the Positions of and artefact bands are indicated. The cytoplasmic hybridised BstEII-BglII probe (lane G). probe (P), signal (S) (A) two are the of AT-rich in the RNA-DNA the SI reaction. 44 higher artefact bands due to 'breathing' sequences hybrid during The lower artefact band at bp is a is in B and C. RNA is in probe alone band and also apparent The minus control shown lane Co. (B) SI analysis of poly(A)-selected ca2M3'PS cytoplasmic RNA. Poly(A)+ (lane A+), poly(A)- (lane A-). The probe used was the BstEII-BglII fragment. (C) Exonuclease VII digests of cs2M3'PS cytoplasmic RNA hybridized to the BstEII-BglI probe. (D) S1 analysis of a2M3'PS cytoplasmic RNA against the smaller Avall-BgIII probe. A doublet band is obtained at 122 -20 1 bp according to restriction fragment size markers. This position is bp 3' to an AATAAA sequence midway between the ct2 and a globin genes et The two artefact bands at 100 and 72 to bands from (Hess al., 1983). bp bp correspond the artefact arising AT-rich sequences seen in panels A and B with the BstEII-BglII probe. the this first of the ct2 -a1 se- forms an SI way through portion intergenic nuclease-sensitive site in the RNA-DNA duplex quence (data not the a2-a1 because it was absent when exonuclease VII was used (Figure shown). However, using adjacent intergenic probe a band was obtain- and so it cannot RNA (BstEII-BglII), strong (S) 3C) represent with true 3' ends. ed 600 nucleotides lane The other smaller To define the 3' end of the a2 gene transcript more precisely, long (Figure 3A, G). bands (A) are SI as discussed in the This we used a smaller DNA probe labelled at an site within artefacts, figure legend. AvaII result obtained by SI analysis was confirmed using the single the BstEII-BglII DNA fragment (see Figure 3). A 122-bp 3' exonuclease VII strand-specific (Figure 3C) which gave the doublet band was obtained together with the same two artefact same 600 nucleotide in long band, absent the control lane. This bands probably caused by AT-rich sequences (Figure 3D). This 600-nucleotide band was polyadenylated (Figure 3B). Although result positions the 3' end of the mutant a2 gene transcript in a band at - 800 was seen in the this bp RNA, cor- the middle of the z2 -ac 1 intergenic sequence - 20 bases 3' to poly(A)- responds to a AT-rich DNA this an AAUAAA very sequence. Presumably, sequence, the only AAUAAA in the whole in- 2918 Transcriptional termination and mRNA processing in the a2 globin gene a2M3*S(AATAAG) a2W*S(AATAAA) a2M1H e2MS a2MD a2MB VV V Q I'K a2W AATAAA I I I I I I I I L I I l l 0 1 2 7 3 4 5 Kb Fig. 4. Line diagram depicting the various a2 globin gene constructs with respect a to the human globin gene map. The a2- and cxl-globin genes are indicated by rectangles with the filled in exons. The positions of the poly(A) addition V sequences are indicated. denotes sequence added. M denotes mutant and W denotes wild-type. tergenic sequence (Hess et al., 1983). 4 Figure shows a detailed 5A (lanes 2-5), each construct gave normal levels a of mRNA of the map human a globin genes and indicates the position of four times higher than with a2M3'PS. As a measure of transfec- the cryptic poly(A) site in the a2 -(xl intergenic sequence. In tion efficiency, the plasmid ,B pSVed (see Materials and methods) summary, these data indicate that when the normal was poly(A) site used as a co-transfection control with each a2M construct. of the a2 gene is non-functional, a2 gene transcripts extend S1 analysis using a globin 3' a band of 210 probe, gave bp 1.5 kb into the 3'-flanking region to form a new polyadenylated corresponding to the distance between the EcoRI site and the 3' 3' end at a cryptic poly(A) site. end of the : globin mRNA (Figure 5B). The signal obtained was equivalent in all tracks so that we could The direcfly compare the of the cryptic poly(A) site provides a inefficiency molecular amounts of a mRNA shown in globin Figure SA. explanation for the a-thalassaemia phenotype To confirm that the amounts of a globin mRNA were rescued Two sets of data suggest that the transcriptional termination pro- by utilizing the added al gene poly(A) site, we carried out 3' cess of the thalassaemia a2 globin gene is disrupted. First nuclear end SI analysis of RNA from cell transfected with a2MB, where run-off analysis indicates that nascent transcripts extend past the the added site is poly(A) placed 3' to the cryptic site poly(A) normal termination region and second 1 a cryptic poly(A) site kb (Figure 4). Figure 5C (lane 1) shows the 3' S1 analysis ofa2MB into the 3'-flanking region is utilized by the a2M gene at signifi- RNA a alI using globin gene 3' probe. The presence of a band cant levels. However, we suspected that the amount of globin of 220 indicated that bp the added al gene poly(A) site was be- mRNA that utilises the cryptic poly(A) site is abnormally low, utilized ing while the 120-bp band corresponded to the mismatch because the patient with this thalassaemic gene expresses reduc- between the a2 sequence in present the construct and the ca 1 se- ed levels of a2 mRNA globin (Higgs et al., 1983). quence in the As RNA probe. expected, from cells transfected To measure the level of stable a2 globin mRNA produced from with a2M3'PS gave only the 120-bp mismatch band (lane 2). a2M3'PS directly, a quantitative assay of 5' ends was carried Figure SD shows the co-transfected ,B globin gene mRNA signal. out using primer extension analysis as shown in Figure 5. The We found that the a2MB transfection was twice as efficient as amount of stable mRNA was - 3-4 times lower in Cos7 cells a2M3'PS so that the amount of 3' ends from a2MB was 3- to transfected with a2M in 3'PS than those transfected with a2W 4-fold those bands in a2M3'PS, verifying the at quantitation the 3'PS lanes 1 (Figure 5A, and 6). The amount could be lower 5' end (Figure SA). The 3' end of the RNA from cells transfected for two reasons: most (i) transcripts are before the terminating with a2MH, a2MS, a2MD were also analysed with probe C site and are cryptic poly(A) unstable, or are not (ii) transcripts and were all found to be utilizing the added poly(A) site (results terminating before the cryptic poly(A) site but the cryptic poly(A) not shown). These data reveal two important features of the a12M site is not as effective as normal so that only 20-30% of the gene. Firstly the of the site at inefficiency cryptic poly(A) least transcripts this reaching point are stabilized by cleavage and in accounts part for the reduced levels of a2 globin gene ex- To polyadenylation. test these alternatives, we constructed in pression observed this type of thalassaemia. no Secondly, a2MD a2MH, a2MS, and a2MB, in which a 300-bp fragment significant level of termination can occur at transcriptional least the a 1 containing wild-type poly(A) site was at the placed HpaI, up to the BglII site in the a2 -a 1 This intergenic sequence. lat- DraI or Sacd, BglII sites, 3' to the a2M globin gene (see Figure ter result confirms our nuclear run-off data and indicates analysis 4). If all these constructs could raise the amounts of mRNA from a between 3' end linkage processing and ter- transcriptional the a2M gene to equal those of the wild-type gene, this would mination. suggest that termination was not occurring prior to the site of the added but that the poly(A) site, cryptic poly(A) site was four times less efficient in mRNA than stabilizing the normal poly(A) Discussion site. their transfection into Cos7 Following cells, each of these four a2M3'PS constructs with an added mRNA 3' end in normal poly(A) site was formation eukaryotes involves both endo- tested for levels of a2 mRNA As in and shown nucleolytic cleavage polyadenylation. The conserved synthesis. Figure highly 2919 E.Whitelaw and N.J.Proudfoot 1 2 3 4 5 6 7 M M 1 2 3 4-Cl Cap-.-_ a I _ ot am 122 t. a .-a 2 118 * Primer-_ _~n 1 M 2 3 or 3 4 5 6 7 M 20O p signal -_ -~l _ 180 Psignal-_. %qp _ 160 ' 144 BstEl PvulI HaeM BstEfl Hinfl I I -I 60 220 ^,120 ae 3 primer probe " RI LEco { __d... I.- -VI ' 3' "', \ P'.9e 5. Primer extension and SI nuclease C and of 5' and 3' ends of a mRNA in RNA from Cos7 cells Fig. (A) (B, D) mapping globin present cytoplasmic of DNA with to these are indicated in the transfected with a2MB and a2M3'PS. The the a2MH, a2MS, positions probes respect plasmids diagram. RNA from cells transfected with a2W a2MH a2MS a2MD a2MB and minus RNA Primer extension of Cos7 c2M3'PS (A) (1), (2), (3), (4), (5), (6) from the and exon of control with A. The a is an antisense strand 3'-end-labelled Hinfl-HaeIII 1st (7) probe primer single fragment 5'-non-coding region to site a band of 74 SI of RNA from Cos7 cells transfected with a2W at2. The is extended 20 the 54-bp primer by bp Cap giving bp. (B) analysis (1), and minus RNA control with the 3' 3' Probe B is a 3' end double- a2MH a2MS a2MD a2MB a2M3'PS (2), (3), (4), (5), (6) (7) , probe. , labelled, the at the EcoRI site and in with Klenow DNA and SI stranded obtained probe by linearizing plasmid pSVod unique filling polymerase [a-32P]dATP. (C) with ae2MPS and a minus RNA control with 3'a Probe was obtained analysis of RNA from cells transfected c2MB ail (1), (2) (3) probe. by linearizing BstEII site and in with Klenow DNA and of RNA from cells transfected with a2MB at the SI pSVed unique filling polymerase [a-32P]dGTP. (D) analysis a minus RNA control with 3'3. (1), a2M3'PS (2) and (3) probe 2920 termination and mRNA in the a2 Transcriptional processing globin gene AAUAAA found 10-30 bases of most in this case is than would be greater for a with expected patient sequence upstream poly- sites forms of the for two normal com- apparently cal globin genes (D.Higgs, personal adenylation part recognition signal cleavage of the and and two active Fitz- munication) a2 Indeed an (Proudfoot partially globin genes. primary transcript Brownlee, 1976; and et et a of Montell three functional a would Shenk, 1981; al., globin output nearly globin gerald Higgs 1983; al., genes 1983). Recent evidence indicates that be located expected for this rather than the observed patient single sequences immediately downstream of the site are also An and globin gene output. for these intriguing explanation apparent poly(A) required (Simonsen and McDevitt et Levinsen, Gil in a levels in the Saudi Arabian 1983; Proudfoot, 1984; discrepancies globin a- al., 1984). When the DNA to the AAUAAA thalassaemia be that the of the a2 to may inability sequence corresponding globin gene signal is altered to AAGAAA in adenovirus et terminate or results in a2 transcription (Montell al., 1983) gene transcripts reading AACAAA in a human f3-thalassaemic et the into and through intergenic sequence a1 globin gene (Orkin al., thereby inhibiting RNA are observed. a 1 globin gene expression. A recent Proudfoot 1985) paper by respectively, elongated transcripts (1986) it has been shown that when the AATAAA that such a is mutated suggests transcriptional interference effect can occur Similarly, to as in the case of the human a-thalassaemic between two AATAAG, and that this effect is alleviated adjacent genes globin by correct 3' end is abolished if the mutant termination between the two placing transcriptional gene, processing signals gene genes. is transfected into tissue culture cells et We al., (Higgs 1983). Materials and methods have extended this observation to show that of the transcription mutant a continues at least 1.0 kb a Globin and constructs into the (3 globin gene globin gene 3'-flanking to + reduced levels of a2W3'PS. The a2W3'PS contains the entire which plasmid a2 globin gene from the end region give poly(A) transcripts PvuII site 1.5 kb 5' from the site to the site 2.0 kb 3' from the downstream of cap SnaI the AATAAA found in the poly(A) just only intergenic site. This 4.5-kb was inserted into the vector fragment pSVed between the EcoRI between the a two sequence globin genes. Furthermore, by plac- and PvuI sites. The vector contains the pSVed pBR322 replication and origin the normal site from a the 1 ing polyadenylation and the SV40 and enhancer globin gene tetracycline gene replication origin sequence (Proud- downstream of this site we find that the foot et al., 1984). cryptic poly(A) transcripts read the site and as far as the inserted The a2M3'PS is beyond cryptic poly(A) a2M3'PS. plasmid identical to a2W3'PS that the except 0.6-kb site. BstEII the fragment containing wild-type poly(A) site, was AATAAA, poly(A) replaced the identical The fact that the normal by fragment from the mutant et al., in which at gene (Higgs 1983) 1 site functions globin gene poly(A) the site is AATAAG. poly(A) when 3' to the site that efficiently placed cryptic poly(A) argues a2MH, a2MS, a2MD, a2MB. These plasmids from a2M3'PS. The the site is originated a2 mRNA cryptic inefficient, only stabilizing globin 300-bp fragment from BstEll to Pvull containing the human a I globin poly(A) to 20% of the level. the absence of func- wild-type Presumably, site was inserted into a2M3'PS at the HpaI site the SacI site (c2MH), (a2MS), tional downstream of the site the DraI site signals (a2MD) and the site as shown in 4. cryptic poly(A) (McDevitt BglII (a2MB) Figure et Gil and al., its low 1984; effi- Proudfoot, 1984) The may explain paJRB. plasmid, in which a 4.0-kb from the human EcoRI-BglJI fragment The amount of a2 mRNA in al globin gene has been inserted between the EcoRI ciency. and BamHI sites of globin present reticulocytes pBR322, of the thalassaemic - has been is % described by Lauer et al. 10-20 that found in (1980). a normal patient individual et The of the a Globin gene M13 clones. Restriction from the al., enzyme fragments human a2 (Higgs 1983). inefficiency cryptic and 3' site in gene sequences indicated in were flush-ended and account for the (as Figure 2) purified, a-thalassaemic poly(A) may part phenotype ligated into the SmaI site of M13 mp8 (Maniatis et al., 1982). Clones of both of the mutant gene. orientations were isolated and the single-stranded DNA was phage grown up by Nuclear run-off a 1 to assess and a2 experiments standard globin gene procedures. in the cell show that transcription line, erythroleukemic K562, (3pSVed. The human , globin gene from in the HpaI 5'-flanking sequence to termination of both the transcriptional PstI in the 3'-flanking sequence was inserted into wild-type globin genes pSVed between the EcoRI site occurs within 100-300 of their In- and the PstI site (Proudfoot et sites. al., 1984). bp respective poly(A) the a mouse also pf35'SV. This was a from terestingly, gift Dr F.Grosveld. The rabbit (3 globin gene is in- globin gene transcription appears to terminate in a serted into a plasmid containing the SV40 and 50-250 3' of origin enhancer sequences and the transcription region bp poly- large T antigen (Grosveld et al., 1982). The presence of these sequences site allows et In both these cases the adenylation al., (Sheffery 1984). for replication in HeLa cells of any plasmids containing the SV40 origin. termination sites are closer to the site than transcriptional poly(A) Transient expression in most other II so far studied et polymerase genes (Birnstiel al., Transfection into Cos7 cells or HeLa cells were carried out as described previously 1985). (Mellon et Whitelaw and al., 1981; Proudfoot, 1983). Cos7 cells contain a defective The fact that termination of of the c2 transcription SV40-transformed CVI wild-type monkey cell line that expresses sufficient levels of SV40 occurs close to the site in K562 and T antigen to allow globin replication of plasmid-containing SV40 origin sequences, such gene poly(A) yet of the as with HeLa mutant in pSVod. Experiments cells were carried out in the of the HeLa cells presence ca2 transcription globin gene goes plasmid p,B5'SV (see above), which allows for replication of other plasmids con- at least 1.5 kb this that termination does beyond region, suggested taining the SV40 origin. Plasmid DNA was precipitated with calcium phosphate not occur without active 3' end Nuclear run-off ex- processing. and added to subconfluent dishes of cells. After 10-16 h, the medium was changed on tissue culture cells transfected with either the mu- periments and the cells allowed to grow for another 30 h. The cells were harvested, lysed tant or the confirm this in NP-40 and the since detergent buffer, cytoplasmic and nuclear fractions separated wild-type gene suggestion termination does occur with by centrifugation through a sucrose cushion. Following incubation with the but not with the mu- proteinase wild-type K, cytoplasmic RNA was purified by phenol/chloroform extraction and ethanol tant Because these two differ one nucleotide gene. genes by only precipitation (Whitelaw and Proudfoot, 1983). Poly(A) selection was carried out A-G in the AATAAA we must assume that sequence, by standard transcrip- procedures using an oligo(dT) cellulose column (Maniatis et al., 1982). tional termination does a functional site. Such require poly(A) RNA mapping would make sense as it would requirement good SI nuclease. physiological Probe DNAs (either double or were annealed single stranded) to termination of within How such a prevent cytoplasmic RNAs (10-20 in 30 of 80% transcription genes. 11 formamide, 0.04 M Pipes pH ILg) 6.4, could work at the mechanistic level is harder to 0.4 M NaCl, 0.1 mM EDTA by denaturation at 80°C for 10 min, then system 53°C, envisage but further double strand probe or 30°C single strand to delineate the probe overnight. 300 of ice-cold exact JlI experiments sequences required SI buffer (0.25 M NaCl, 0.03 M NaAcetate 2 mM 50 pH 4.6, ZnSO4, Agg/ml for termination of RNA polymerase II genes should clarify this denatured sonicated carrier DNA) plus S (3000 units) was added to each quickly issue. hybridisation and incubated for 1 h at 30°C. SI reactions were ethanol precipitated It is that the of a-thalassaemia and fractionated on interesting severity denaturing, 7 M urea polyacrylamide homozygous gels. 2921 E.Whitelaw and N.J.Proudfoot Exonuclease VII. Hybridisations were carried out as for the SI experiments. 500 Falck-Pedersen,E., and Logan,J., Shenk,T. Darnell,J.E.,Jr (1985) Cell, 40, Al of exonuclease VII buffer (30 mM KCI, 10 mM Tris pH 7.8 and 10 mM EDTA) 897-905. with 4 U of exonuclease VII (Bethesda Research Laboratories) was added to and 251-260. Fitzgerald,M. Shenk,T. (1981) Cell, 24, hybridisations and incubated at 37°C for 2 h. The reactions were then ethanol and Darnell,J.E. J. Mol. Biol., Fraser,N.W., 129, Nevins,J.R., Ziff,E. (1979) precipitated and fractionated as for SI experiments. 643. and 473-474. Gil,A. Proudfoot,N.J. Primer (1984) Nature, 312, extension. DNA primer and RNA were annealed in 10 of 10 mM Pipes ptl de and Shewmaker,C.K. Flavell,R.A. Nature, Grosveld,G.C., Boer,E., (1982) pH 6.4, 0.4 M NaCl at 80°C for 10 min and 63°C overnight. 50 of reverse 120-126. 295, transcriptase buffer (50 mM Tris, pH 8.2, 10 mM DTT, 6 mM MgCI2, 0.5 mM and Mol. Cell 281 -288. Weintraub,H. Biol., 1, Groudine,M., Peretz,M. (1981) dATP, dCTP, dTTP, dGTP) plus reverse transcriptase (5 units) were added to and Hassouna,N. Bachellerie,J.-P. Cell, Grummt,I., Maier,U., Ohrlein,A., (1985) hybridisations and incubated at 42°C for I h. The reactions were ethanol 801-810. 43, precipitated twice and fractionated by electrophoresis on 7 M urea polyacrylamide and Cribbs,D.L. Schibler,U. Cell, 38, Hagenbuchle,O., Wellauer,P.K., (1984) gels. 737-744. Nuclear isolation, transcription and RNA purification and Proc. Acad. Hess,J.F., Fox,M., Schmid,C.W. Shen,C.-K.J. Natl. (1983) A variant K562 line which forms an adherent monolayer (M.V.Chao, unpublished) Sci. 5970-5974. USA, 80, was grown in DMEM supplemented with 10% fetal calf serum in the presence and Weatherall,D.J. Higgs,D.R., Goodbourn,S.E.Y., Lamb,J., Clegg,J.B., was of 50 uM haemin for 3-4 days. Nuclear isolation and transcription carried 398-400. Proudfoot,N.J. (1983) Nature, 306, out according to Groudine et al. (1981) but with some modifications. Cells 585-593. and Hofer,E. Darnell,J.E. (1981) Cell, 23, (- 1 x 107) were spun 1 x M M down, washed in SSC (0.15 NaCI 0.015 and 887-893. Cell, 29, Hofer,E., Hofer-Warbinek,R. Darnell,J.E.,Jr (1982) sodium citrate, pH 7.0). Nuclei were isolated by lysis of cells in RSB (0.01 M and Nucleic Acids Res., 7, Kafatos,F.C., Jones,C.W. Efstratiadis,A. (1979) Tris pH 7.4, 0.01 M NaCI, 3 mM MgCl2) containing 0.5% NP-40 and centrifuga- 1541-1551. for 10 2000 in without NP-40 tion min, r.p.m. The nuclei were washed RSB and 203-206. Krieg,P.A. Melton,D. (1984) Nature, 308, and resuspended in 2 x transcription buffer (5 mM DTT, 180 mM KCl, 10 mM and 119-130. Cell, 20, Lauer,J., Shen,C.-K. Maniatis,T. (1980) MgCl2, 20 mM Tris pH 7.8, 50% glycerol). Elongation was carried out at 30°C and EMBO 2779-2786. J., 3, LeMeur,M.A., Galliot,B. Gerlinger,P. (1984) for 10 min in the presence of 0.2 mM ATP, GTP, CTP, 200 [a-32P]UTP 321-334. and /Ci Lozzio,C.B. Lozzio,B.B. (1975) Blood, 45, (3000 Ci/mmol, Amersham). Reactions were terminated by the addition of RNase- and Molecular A Maniatis,T., Fritsch,E.F. Sambrook,J. (1982) Cloning. free DNase I (Miles) to 100 ,ug/ml and incubation at 30°C for a further 5 min. Manual. Cold Harbor NY. Press, Laboratory Spring Laboratory Under these conditions, pre-initiated RNA chains are elongated 100-300 and by Cell, 37, McDevitt,M.A., Imperiale,M.J., Ali,H. Nevins,J.R. (1984) nucleotides (Weber et al., 1977). The RNA was purified according to the method 993-999. to Treisman and Maniatis (1985). Basically, the reactions were in and 279-288. deproteinized Cell, Mellon,P., Parker,V., Gluzman,Y. Maniatis,T. (1981) 27, a solution of 0.1 M Tris pH 7.5, 0.15 M NaCl, 1% SDS 10 mM EDTA with and Nature, 305, Montell,C., Fisher,E.F., Caruthers,M.H. Berk,A.J. (1983) 100 of proteinase K per ml and subsequent -chloroform extraction phenol 600-605. ytg and ethanol precipitation. The was taken in 300 1l 10 mM 845-855. pellet up Tris, pH 7.5, and Cell, Moore,C.L. Sharp,P.A. (1985) 41, 10 mM MgCl2 and incubated at 37°C for 30 min in the presence of 50 and EMBO Antonarakis,S.E. Kazazian,H.H.,Jr (1985) gltg/ml Orkin,S.H., Cheng,T.-C., RNase-free DNase I (Miles). The reaction was extracted with 453-456. phenol/chloroform 4, J., and ethanol precipitated. 423-429. and Price,D.H. Parker,C.S. (1984) Cell, 38, Protocols for nuclear and RNA for 730-731. isolation transcription purification experiments Proudfoot,N.J. (1986) Nature, 321, using HeLa cells in which plasmids were were iden- 211-214. being transiently expressed and Proudfoot,N.J. Brownlee,G.G. (1976) Nature, 263, tical to those used for the nuclear isolation, and RNA and EMBO transcription purification J., 3, Rutherford,T.R. (1984) Proudfoot,N.J,. Partington,G.A. of K562. The transfection of HeLa cells with plasmids was carried out as described 1533-1540. above. h later. 2 x 106 cells were and Mol. Cell. Nuclei were purified 40-48 Approximately Biol., Johnson,J., James,M. Hardison,R. (1985) Rohrbaugh,M.L., used for each experiment. 147-160. 5, 164-165. and Weatherall,D.J. Nature, 280, Rutherford,T.R., Clegg,J.B. (1979) Hybridization to nitrocellulose filters Mol. 417-436. and 172, Riflind,R.A. J. Biol., Sheffery,M., Marks,P.A. (1984) bound to nitrocellulose filters to M13 single strand DNA (5 was according Rg) Mol. Cell. 2250-2258. and 3, Biol., Simonsen,C.C. Levinson,A.D. (1983) the method of Kafatos et al. (1979) with a miniblot filtration unit. 72-75. and Treisman,R. Maniatis,M. Nature, 315, (1985) first the Experiments on DNA fragments were done by purifying fragments 611-616. and Damell,J. Weber,J., Jelinek,W. (1977) Cell, 10, from a large them on a small agarose gel and then running (0.2 ,ig) agarose gel. Nucleic Acids 7717-7733. and Res., Whitelaw,E. Proudfoot,N.J. 11, (1983) The DNA nitrocellulose to Maniatis et al. was then transferred to according (1982). to the of Hofer and Alkali breakage of RNA was performed according technique Received on 28 1986 July Damell was 1 ml. was car- (1981). The volume of hybridization Hybridization ried out at of formamide and 42°C in the presence 50% (Treisman Maniatis, 1985) for the filters were washed: 30 48 h. After hybridization, min, 25°C, 2 x 0.1I% SDS. Then the filters 2 x SSC, 0.1% SDS; 30 min; 65°C, SSC, were x with RNAse at a concentra- incubated in 2 SSC at 37°C (DNase-free) The filters were washed in 0.2 x 0.1% SDS at tion of 2 finally SSC, 14g/ml. 68°C for 30 min. Acknowledgements We gratefully acknowledge Dr for the cs2 thalassaemic Doug Higgs providing globin gene We thank both and used throughout these experiments. Doug Higgs Dean Jackson for discussion. we are indebted to Zena Werb for helpful Finally, her critical reading of the manuscript. EW and NJP were a Medical supported by Research Council project grant no. G8108316CB. References Birchmeier,C., Schumperli,D., Sconzo,G. and Proc. Natl. Birnstiel,M.L. (1984) Acad. Sci. USA, 81, 1057-1061. Birnstiel,M.L., Busslinger,M. and Strub,K. (1985) Cell, 349-359. 41, Bogenhagen,D.F. and Brown,D.D. (1981) 261-270. Cell, 24, Charnay,P. and Maniatis,T. 1281 - 1283. (1983) Science, 220, and Citron,B., Falck-Pedersen,E., Salditt-Georgieff,M. Darnell,J.E.Jr (1984) Nucleic Acids Res., 12, 8723-873 1. Corden,J., Wasylyk,B., Buchwalder,A., and Sassone-Corsi,P., Kedinger,C. Chambon,P. (1980) Science, 290, 1406-1414. 774-778. Dynan,W.S. and Tjian,R. (1985) Nature, 316,

Journal

The EMBO Journal – Springer Journals

Published: Nov 1, 1986

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