B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

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Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5386-5393.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5386-5393 ◽

Cited By ~ 1

Author(s):

N Schek ◽

C Cooke ◽

J C Alwine

Keyword(s):

Consensus Sequence ◽

Simian Virus 40 ◽

Simian Virus ◽

Polyadenylation Signal ◽

Upstream Region ◽

Core Element ◽

The Core ◽

Core Elements ◽

Upstream Element

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

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Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5386 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5386-5393 ◽

Cited By ~ 48

Author(s):

N Schek ◽

C Cooke ◽

J C Alwine

Keyword(s):

Consensus Sequence ◽

Simian Virus 40 ◽

Simian Virus ◽

Polyadenylation Signal ◽

Upstream Region ◽

Core Element ◽

The Core ◽

Core Elements ◽

Upstream Element

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

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PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

AJP Cell Physiology ◽

10.1152/ajpcell.00356.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. C528-C534 ◽

Cited By ~ 7

Author(s):

Shiuhyang Kuo ◽

Ann L. Chokas ◽

Richard J. Rogers ◽

Harry S. Nick

Keyword(s):

Binding Sites ◽

Manganese Superoxide Dismutase ◽

Consensus Sequence ◽

Dimethyl Sulfate ◽

Site Directed Mutagenesis ◽

Cellular Respiration ◽

Point Analysis ◽

Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

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Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4484 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4484-4491 ◽

Cited By ~ 17

Author(s):

A Mayeda ◽

Y Ohshima

Keyword(s):

Consensus Sequence ◽

Donor Site ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

T Antigen ◽

Beta Globin ◽

Vitro Assay ◽

Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

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Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2911 ◽

1984 ◽

Vol 4 (12) ◽

pp. 2911-2920 ◽

Cited By ~ 8

Author(s):

H Mishoe ◽

J N Brady ◽

M Radonovich ◽

N P Salzman

Keyword(s):

Rna Polymerase Ii ◽

Dna Sequences ◽

Base Pair ◽

Transcriptional Control ◽

Simian Virus 40 ◽

Simian Virus ◽

Control Element ◽

Late Promoter ◽

Transcription Control

We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

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Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.541 ◽

1989 ◽

Vol 9 (2) ◽

pp. 541-550 ◽

Cited By ~ 46

Author(s):

S E Porter ◽

J J Champoux

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Unknown Origin ◽

Simian Virus ◽

Asymmetric Distribution ◽

Potential Break

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.

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Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3667 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3667-3676 ◽

Cited By ~ 14

Author(s):

T A Firak ◽

K N Subramanian

Keyword(s):

Base Pair ◽

Simian Virus 40 ◽

Simian Virus ◽

Proximal Region ◽

Slight Reduction ◽

Transcriptional Enhancer ◽

Core Element ◽

Enhancer Activity ◽

Simplex Virus

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.

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Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3667-3676.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3667-3676

Author(s):

T A Firak ◽

K N Subramanian

Keyword(s):

Base Pair ◽

Simian Virus 40 ◽

Simian Virus ◽

Proximal Region ◽

Slight Reduction ◽

Transcriptional Enhancer ◽

Core Element ◽

Enhancer Activity ◽

Simplex Virus

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.

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Class II box consensus sequences in the HLA-DR alpha gene: transcriptional function and interaction with nuclear proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.50 ◽

1989 ◽

Vol 9 (1) ◽

pp. 50-56 ◽

Cited By ~ 25

Author(s):

P A Sherman ◽

P V Basta ◽

T L Moore ◽

A M Brown ◽

J P Ting

Keyword(s):

Protein Binding ◽

Nuclear Protein ◽

Consensus Sequence ◽

Regulatory Elements ◽

Class Ii ◽

Promoter Function ◽

Hla Dr ◽

Alpha Gene

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.

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