J. Mol. Bio2. (1991) 217, 595-598
COMMUNICATIONS
Transcriptional Enhancer in the Vicinity of a Replication Origin within the 5’ Region of the Chicken a-Globin Gene Domain S. V. Razin’, Y. S. Vassetzky Jr’, A. I. Kvartskhaval N. F. Grinenko’ and G. P. Georgievl ‘Institute of Molecular Biology Academy of Xciences of the USSR 32 Vavilov St, Moscow 117984 USSR 21nstitute of General Genetics USSR Academy of Sciences, Moscow (Received 25 August
1990; accepted 9 October 1990)
A transcriptional enhancer is located near a replication origin within an upstream area of chicken domain of a-globin genes. Minimal region possessing enhancer properties is located about 4 kb upstream from the a-~ gene. Addition of the neighbouring 500 base-pair DNA fragment, including the constitutive DNase I hypersensitive site, increases the enhancer activity approximately twofold.
A class of DNA sequences that allow autonomous replication of plasmids in yeast cells (ARS) has been identified. The role of these sequences in the replication of yeast chromosomes is being studied now (for a review, see Umek et aE., 1988). Much less is known about the initiation of replication in higher eukaryotes, although replication of nuclear viruses, e.g. adenovirus or simian virus (SV40) has been studied in detail (DePamphilis & Wasserman, 1982). However, replication of most of these viruses requires virus-specific proteins that are absent in normal cells. Multiple attempts to find ARS in cells of higher eukaryotes led to highly controversial results (Holst et al., 1988; Grummt, 1989;$ McWhinney & Leffak, 1990). In this situation, study of the structure of functioning replication origins in higher eukaryotes may shed light on the mechanism of initiation of replication. Previously, we mapped a replication origin in the upstream region of the domain of chicken a-globin genes (Razin et al., 1986) and determined its primary structure (Kalandadze et al., 1990). Our data have been confirmed by others (Umek et al., 1988).
A number of publications indicate that transcriptional enhancers may also enhance replication (DeVilliers et al., 1988; O’Connor & Subramani, 1988). This made it interesting to inquire whether our replication origin contained a transcriptional enhancer. 595 002%2836/91/040595-04
$03.00/O
Plasmids carrying different subfragments of the domain of chicken a-globin genes were constructed on the basis of the pUSVLlCAT plasmid, containing the SV40 late promoter, the bacterial chloramphenicol acetyltransferase gene and the SV40 late polyadenylation region (XphI-BamHI subfragment of pSV2CAT; Gorman et aZ.: 1982) inserted into the polylinker of pUC18 (Fig. 1). Transfection of cells was carried out using the calcium phosphate method (Graham & van der Eb, 1973). The CAT assay was carried out as described (Gorman et al., 1982) at 48 hours after transfection. A plasmid, containing the CAT gene, the late SV40 promoter, the polyadenylation region and the 1720 base fragment of the chicken a-globin gene domain in different orientations (we designated it a5HR) was transfected into RAT-2 cells. At 48 hours after infection the activity of the enzyme was estimated by its ability to acetylate chloramphenicol . An RSV LTR-containing plasmid (pRSVCAT) was selected as a positive control. a5HR was found to contain a transcriptional enhancer that has approximately 2% efficiency of the RSV LTR promoter (Fig. 1). Its activity did not depend on the orientation of the insert. This enhancer is not tissue-specific, since transfection of the plasmid into NIH 3T3 cells also revealed the presence of the enhancer, although the enhancer activity was higher when the construct was transfected into embryonic chicken fibroblasts (data not 0 1991 Academic Press Limited
S. V. Razin
596
SV4Q late promoter
CAT gene
SV40 paiyadenyiation signal
pUSVLlCAT
CAT gene
------
SV40 polyadenylotion signal
pSVOCAT
pUSVLOCAT
et ai.
shown). The insert has an A+T-rich block at its 3: end with several TATA-like sequences; and it was interesting to t,est whether this box could serve as a promoter. To do this, we cloned the fragment, into the Hind111 site of the plasmid pUSVLOCAT, which was similar to pUSVLlCAT, but did not contain the SV40 late promot,er (Fig. 1). A relatively strong CAT activity was observed in an extract of transfected cells, indicating that the A f T-rich block can serve as a promoter element (Fig. 1). a5HR contains many putative binding sites for different regulatory proteins, areas of homology with replication origins of papovaviruses, a constitutive DNase I hypersensitivity site, etc. (Kalandadze et al., 1990). Hence, it was interesting to determine the exact position of t’he enhancer. We have subcloned a5HR into four CAT-containing plasmids (Fig. 2). One of the plasmids carrying an XmaI-PvuIT subfragment exhibited enhancer activity. Further subcloning allowed the activity to be mapped to a XmaI-KpnI subfragment (Fig. Z), although it was half as strong as the enhancer activity of the whole a5HR and the Xmal-PvuII subfragment. One can conclude that there is a minimal enhancer and a sequence that increases the strength of the enhancer, though it does not have the properties of the enhancer itself (data not shown). The sequence of the minimal enhancer is given in Figure 2. Computer analysis of the XmaI-PvuII fragment revealed that it contained several imperfect repeats (Kalandadze et al., 1990). It contains several recognition sequences for sequence-specific DNA-binding proteins (Fig. 2). The role of these recognition sequences is under investigation. It is known that the initiation of replication involves the synthesis of an RNA primer, i.e. transcription. Therefore, the presence of a transcription enhancer near the replication origin can be readily understood.
References DePamphihs,
M.
& Wasserman, P. M. (1988). In of viral DNA (Kaplan, A., ed.), pp. 37-114, CRC Press, Boca Raton. DeVilliers, J.; Schaffner, W., Tyndall, C., Lupton, S. $ Kamen, R. (1988). Nature (London), 312, 2422246. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982). Mol. Cell. Biol. 2, 1044-1051. Graham, F. & van der Eb, A. (1973). ViroZogy, 52> 456-463. Grummt, F. (1989). GeZZ, 56, 7-8. Holst, A., Muller, F., Zastrow, G., Zentgraf, H.; Schwender, S., Dinkl, E. & Grummt, F. (1988). Cell, 52, 355-365. Kaiandadze, A. G., Bushara, S. A., Vassetzky, Y. S. $ Razin, S. V. (1990). Biochem. Biophys. Res. Common. 168, 9-15. McWhinney, C. & Leffak, M. (1990). Nucl. Acids Res. 18.
Organization
E
2
3
4
5
Figure 1. Top, scheme of the plasmid used in the CAT assay. The tested DNA fragments were cloned into the HindTII site. Bottom, CAT assay of cells transfected with pUSVLlCAT and pUSVLOCAT plasmids containing a5HR in different orientations. Cells were transfected with pUSVLlCAT containing n5HR in different orientations (lanes 4 and 5) and with pUSVLOCAT containing a5HR (lane 3); lane 2, negative control (pUSVLOCAT); lane 1, positive control (pRSVCAT).
L.
and replication
1233-1241. O’Connor, D. T. & Subramani, 16, 11207-11222.
S. (1988). Nuel.
Acids Res.
l3GGGCTCCCA
GTACGACCAG
CAGGTCACGT
TGGAGTCTCT
~GTCCI’CAAG
ACTGCGCAGT
i=2 575
GTCTCAL:CTl FZA
1GAGCCTTGT
GCCCCCCATT
CAGCCCASCA
Protein
635
AGCCTCACCA C/ESP
CATCACACIG TAGCCCl TAC binding site ot human AHS ACGTCAAGAT GGAGCCCTGT
CAGCACAGCA
CCTCACGTlC
AGGCCCCAGC
AGCCAGCATG
GAACCATCAA
ATCCTTAGAG
TTGGAAGATG
TCTGAATCCT
GTTCAGCCCG
GCACCTCTCA
CACCCCACTC
AACACIKITC
AGCCAAGAGC
CCCGCCATCA
GCCCAGTGCC
695
GCCCCCAGAC AP-2 755
TGTGCCCCCA
CACCC-binding
protein
815 CTACAGCTCA
ACCCAGCACC
TCACGCCACC
CAGCAGCACT
892
875
CCCAGTCCGG AP-2
ATCGGTAC
Bose-pairs 0
5
xIOe3 IO
15
HindXl Xmal
XmoI
XmoI
PVOlI
KpnI
PsfI
”
I.0 Bose-pole
I
x lO-3
2
3
4
pRSVCAT
t
Figure 2. CAT assay of cells transfected with the pUSVLlCAT plasmid containing different subfragments of a5HR. Control (mock transfection, non-transfected RAT-Z cells, and cells transfected with pUSVL1CAT); cells were transfected with the first XmaIIXmaI subfragment (lane l), the second XmaI-XmaI subfragment (lane 2) XmaIIPwuII subfragment (lane 3), and PvuII-Hind111 subfragment (lane 4) (see the scheme above); lane pRSVCAT, cells were transfected with the plasmid containing a strong RSV LTR enhancer. The scheme indicates the position of cl5HR and its subfragments in the domain of the chicken a-globin genes. The sequence of the XmaI-KpnI subfragment of a5HR is given above the autoradiogram. The underlined nucleotides contain recognition sites for different DNA-binding proteins (Turpaev & Vassetzky, 1990).
598
S. Y. Razin
Razin, S. V., Kekelidze, M. G., Lukanidin, E. M., Scherrer, K. & Georgiev, G. P. (1986). Nucl. Acids Res. 14, 8189-8207. Turpaev, K. T. & Vassetzky, Y. S., Jr (1990). Genetika (USSR), 26, 804-817. Edited
et al. Umek, R. M., Linskens; M. H. K., Kowalski, D. & Huberman, J. A. (1988). Biochim. Biophys. Acta, 1007, 1-14.
by J. Karn
COMMUNICATIONS
Transcriptional Enhancer in the Vicinity of a Replication Origin within the 5’ Region of the Chicken a-Globin Gene Domain S. V. Razin’, Y. S. Vassetzky Jr’, A. I. Kvartskhaval N. F. Grinenko’ and G. P. Georgievl ‘Institute of Molecular Biology Academy of Xciences of the USSR 32 Vavilov St, Moscow 117984 USSR 21nstitute of General Genetics USSR Academy of Sciences, Moscow (Received 25 August
1990; accepted 9 October 1990)
A transcriptional enhancer is located near a replication origin within an upstream area of chicken domain of a-globin genes. Minimal region possessing enhancer properties is located about 4 kb upstream from the a-~ gene. Addition of the neighbouring 500 base-pair DNA fragment, including the constitutive DNase I hypersensitive site, increases the enhancer activity approximately twofold.
A class of DNA sequences that allow autonomous replication of plasmids in yeast cells (ARS) has been identified. The role of these sequences in the replication of yeast chromosomes is being studied now (for a review, see Umek et aE., 1988). Much less is known about the initiation of replication in higher eukaryotes, although replication of nuclear viruses, e.g. adenovirus or simian virus (SV40) has been studied in detail (DePamphilis & Wasserman, 1982). However, replication of most of these viruses requires virus-specific proteins that are absent in normal cells. Multiple attempts to find ARS in cells of higher eukaryotes led to highly controversial results (Holst et al., 1988; Grummt, 1989;$ McWhinney & Leffak, 1990). In this situation, study of the structure of functioning replication origins in higher eukaryotes may shed light on the mechanism of initiation of replication. Previously, we mapped a replication origin in the upstream region of the domain of chicken a-globin genes (Razin et al., 1986) and determined its primary structure (Kalandadze et al., 1990). Our data have been confirmed by others (Umek et al., 1988).
A number of publications indicate that transcriptional enhancers may also enhance replication (DeVilliers et al., 1988; O’Connor & Subramani, 1988). This made it interesting to inquire whether our replication origin contained a transcriptional enhancer. 595 002%2836/91/040595-04
$03.00/O
Plasmids carrying different subfragments of the domain of chicken a-globin genes were constructed on the basis of the pUSVLlCAT plasmid, containing the SV40 late promoter, the bacterial chloramphenicol acetyltransferase gene and the SV40 late polyadenylation region (XphI-BamHI subfragment of pSV2CAT; Gorman et aZ.: 1982) inserted into the polylinker of pUC18 (Fig. 1). Transfection of cells was carried out using the calcium phosphate method (Graham & van der Eb, 1973). The CAT assay was carried out as described (Gorman et al., 1982) at 48 hours after transfection. A plasmid, containing the CAT gene, the late SV40 promoter, the polyadenylation region and the 1720 base fragment of the chicken a-globin gene domain in different orientations (we designated it a5HR) was transfected into RAT-2 cells. At 48 hours after infection the activity of the enzyme was estimated by its ability to acetylate chloramphenicol . An RSV LTR-containing plasmid (pRSVCAT) was selected as a positive control. a5HR was found to contain a transcriptional enhancer that has approximately 2% efficiency of the RSV LTR promoter (Fig. 1). Its activity did not depend on the orientation of the insert. This enhancer is not tissue-specific, since transfection of the plasmid into NIH 3T3 cells also revealed the presence of the enhancer, although the enhancer activity was higher when the construct was transfected into embryonic chicken fibroblasts (data not 0 1991 Academic Press Limited
S. V. Razin
596
SV4Q late promoter
CAT gene
SV40 paiyadenyiation signal
pUSVLlCAT
CAT gene
------
SV40 polyadenylotion signal
pSVOCAT
pUSVLOCAT
et ai.
shown). The insert has an A+T-rich block at its 3: end with several TATA-like sequences; and it was interesting to t,est whether this box could serve as a promoter. To do this, we cloned the fragment, into the Hind111 site of the plasmid pUSVLOCAT, which was similar to pUSVLlCAT, but did not contain the SV40 late promot,er (Fig. 1). A relatively strong CAT activity was observed in an extract of transfected cells, indicating that the A f T-rich block can serve as a promoter element (Fig. 1). a5HR contains many putative binding sites for different regulatory proteins, areas of homology with replication origins of papovaviruses, a constitutive DNase I hypersensitivity site, etc. (Kalandadze et al., 1990). Hence, it was interesting to determine the exact position of t’he enhancer. We have subcloned a5HR into four CAT-containing plasmids (Fig. 2). One of the plasmids carrying an XmaI-PvuIT subfragment exhibited enhancer activity. Further subcloning allowed the activity to be mapped to a XmaI-KpnI subfragment (Fig. Z), although it was half as strong as the enhancer activity of the whole a5HR and the Xmal-PvuII subfragment. One can conclude that there is a minimal enhancer and a sequence that increases the strength of the enhancer, though it does not have the properties of the enhancer itself (data not shown). The sequence of the minimal enhancer is given in Figure 2. Computer analysis of the XmaI-PvuII fragment revealed that it contained several imperfect repeats (Kalandadze et al., 1990). It contains several recognition sequences for sequence-specific DNA-binding proteins (Fig. 2). The role of these recognition sequences is under investigation. It is known that the initiation of replication involves the synthesis of an RNA primer, i.e. transcription. Therefore, the presence of a transcription enhancer near the replication origin can be readily understood.
References DePamphihs,
M.
& Wasserman, P. M. (1988). In of viral DNA (Kaplan, A., ed.), pp. 37-114, CRC Press, Boca Raton. DeVilliers, J.; Schaffner, W., Tyndall, C., Lupton, S. $ Kamen, R. (1988). Nature (London), 312, 2422246. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982). Mol. Cell. Biol. 2, 1044-1051. Graham, F. & van der Eb, A. (1973). ViroZogy, 52> 456-463. Grummt, F. (1989). GeZZ, 56, 7-8. Holst, A., Muller, F., Zastrow, G., Zentgraf, H.; Schwender, S., Dinkl, E. & Grummt, F. (1988). Cell, 52, 355-365. Kaiandadze, A. G., Bushara, S. A., Vassetzky, Y. S. $ Razin, S. V. (1990). Biochem. Biophys. Res. Common. 168, 9-15. McWhinney, C. & Leffak, M. (1990). Nucl. Acids Res. 18.
Organization
E
2
3
4
5
Figure 1. Top, scheme of the plasmid used in the CAT assay. The tested DNA fragments were cloned into the HindTII site. Bottom, CAT assay of cells transfected with pUSVLlCAT and pUSVLOCAT plasmids containing a5HR in different orientations. Cells were transfected with pUSVLlCAT containing n5HR in different orientations (lanes 4 and 5) and with pUSVLOCAT containing a5HR (lane 3); lane 2, negative control (pUSVLOCAT); lane 1, positive control (pRSVCAT).
L.
and replication
1233-1241. O’Connor, D. T. & Subramani, 16, 11207-11222.
S. (1988). Nuel.
Acids Res.
l3GGGCTCCCA
GTACGACCAG
CAGGTCACGT
TGGAGTCTCT
~GTCCI’CAAG
ACTGCGCAGT
i=2 575
GTCTCAL:CTl FZA
1GAGCCTTGT
GCCCCCCATT
CAGCCCASCA
Protein
635
AGCCTCACCA C/ESP
CATCACACIG TAGCCCl TAC binding site ot human AHS ACGTCAAGAT GGAGCCCTGT
CAGCACAGCA
CCTCACGTlC
AGGCCCCAGC
AGCCAGCATG
GAACCATCAA
ATCCTTAGAG
TTGGAAGATG
TCTGAATCCT
GTTCAGCCCG
GCACCTCTCA
CACCCCACTC
AACACIKITC
AGCCAAGAGC
CCCGCCATCA
GCCCAGTGCC
695
GCCCCCAGAC AP-2 755
TGTGCCCCCA
CACCC-binding
protein
815 CTACAGCTCA
ACCCAGCACC
TCACGCCACC
CAGCAGCACT
892
875
CCCAGTCCGG AP-2
ATCGGTAC
Bose-pairs 0
5
xIOe3 IO
15
HindXl Xmal
XmoI
XmoI
PVOlI
KpnI
PsfI
”
I.0 Bose-pole
I
x lO-3
2
3
4
pRSVCAT
t
Figure 2. CAT assay of cells transfected with the pUSVLlCAT plasmid containing different subfragments of a5HR. Control (mock transfection, non-transfected RAT-Z cells, and cells transfected with pUSVL1CAT); cells were transfected with the first XmaIIXmaI subfragment (lane l), the second XmaI-XmaI subfragment (lane 2) XmaIIPwuII subfragment (lane 3), and PvuII-Hind111 subfragment (lane 4) (see the scheme above); lane pRSVCAT, cells were transfected with the plasmid containing a strong RSV LTR enhancer. The scheme indicates the position of cl5HR and its subfragments in the domain of the chicken a-globin genes. The sequence of the XmaI-KpnI subfragment of a5HR is given above the autoradiogram. The underlined nucleotides contain recognition sites for different DNA-binding proteins (Turpaev & Vassetzky, 1990).
598
S. Y. Razin
Razin, S. V., Kekelidze, M. G., Lukanidin, E. M., Scherrer, K. & Georgiev, G. P. (1986). Nucl. Acids Res. 14, 8189-8207. Turpaev, K. T. & Vassetzky, Y. S., Jr (1990). Genetika (USSR), 26, 804-817. Edited
et al. Umek, R. M., Linskens; M. H. K., Kowalski, D. & Huberman, J. A. (1988). Biochim. Biophys. Acta, 1007, 1-14.
by J. Karn
