Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

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Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽

10.1182/blood.v78.9.2433.2433 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2433-2437 ◽

Cited By ~ 15

Author(s):

SZ Huang ◽

GP Rodgers ◽

FY Zeng ◽

YT Zeng ◽

AN Schechter

Keyword(s):

Polymerase Chain Reaction ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Beta Globin ◽

Chain Reaction ◽

Globin Mrna ◽

Gamma Globin ◽

Polymerase Chain ◽

Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

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Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2698 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2698-2707 ◽

Cited By ~ 31

Author(s):

N M Yeilding ◽

W M Lee

Keyword(s):

Myogenic Differentiation ◽

Globin Gene ◽

Regulatory Elements ◽

C2c12 Cells ◽

Regulatory Function ◽

Mrna Levels ◽

Beta Globin ◽

Globin Mrna ◽

Exon 2 ◽

Coding Sequences

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

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Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽

10.1182/blood.v78.9.2433.bloodjournal7892433 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2433-2437

Author(s):

SZ Huang ◽

GP Rodgers ◽

FY Zeng ◽

YT Zeng ◽

AN Schechter

Keyword(s):

Polymerase Chain Reaction ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Beta Globin ◽

Chain Reaction ◽

Globin Mrna ◽

Gamma Globin ◽

Polymerase Chain ◽

Alpha Beta

We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

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Transcriptional regulation of a Xenopus embryonic epidermal keratin gene

Development ◽

10.1242/dev.106.2.399 ◽

1989 ◽

Vol 106 (2) ◽

pp. 399-405 ◽

Cited By ~ 7

Author(s):

E.A. Jonas ◽

A.M. Snape ◽

T.D. Sargent

Keyword(s):

Gene Expression ◽

Developmental Stages ◽

Globin Gene ◽

Regulatory Sequences ◽

Type I ◽

Beta Globin ◽

Globin Mrna ◽

Keratin Gene ◽

Sequence Elements ◽

Specific Accumulation

XK81A1 is a type I epidermal keratin gene expressed in early developmental stages of Xenopus (Jonas et al. 1985). Fusion of the keratin promoter (−5900 to +26) to a human beta globin gene led to fully epidermis-specific accumulation of human globin mRNA and protein when this DNA was injected into fertilized eggs. Further localization of regulatory sequences was accomplished by injecting marked, 5′-deleted keratin gene DNA into fertilized eggs and evaluating tissue specificity of expression. All 5′ flanking DNA upstream from −487 could be removed without interfering with keratin gene expression or regulation. These results suggest that the primary mode of regulation of epidermis-specific keratin gene expression is at the level of transcription, and that sequence elements in the 5′ flanking region of the keratin gene, between −487 and +26, are responsible for this regulation.

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Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.887-897.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 887-897

Author(s):

R D Cone ◽

A Weber-Benarous ◽

D Baorto ◽

R C Mulligan

Keyword(s):

Dimethyl Sulfoxide ◽

Globin Gene ◽

Control Point ◽

Mrna Levels ◽

Beta Globin ◽

Transcriptional Enhancer ◽

Enhancer Element ◽

Beta Globin Gene ◽

Murine Erythroleukemia ◽

Enhancer Sequences

We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.

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Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.887 ◽

1987 ◽

Vol 7 (2) ◽

pp. 887-897 ◽

Cited By ~ 78

Author(s):

R D Cone ◽

A Weber-Benarous ◽

D Baorto ◽

R C Mulligan

Keyword(s):

Dimethyl Sulfoxide ◽

Globin Gene ◽

Control Point ◽

Mrna Levels ◽

Beta Globin ◽

Transcriptional Enhancer ◽

Enhancer Element ◽

Beta Globin Gene ◽

Murine Erythroleukemia ◽

Enhancer Sequences

We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.

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Role for DNA replication in beta-globin gene activation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1301 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1301-1308 ◽

Cited By ~ 14

Author(s):

T Enver ◽

A C Brewer ◽

R K Patient

Keyword(s):

Dna Replication ◽

Transcriptional Activation ◽

Globin Gene ◽

Gene Activation ◽

Simian Virus 40 ◽

Simian Virus ◽

Covalent Modification ◽

Beta Globin ◽

Beta Globin Gene ◽

Template Copy

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

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Characterization of the major regulatory element upstream of the human alpha-globin gene cluster

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4679-4689.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4679-4689

Author(s):

A P Jarman ◽

W G Wood ◽

J A Sharpe ◽

G Gourdon ◽

H Ayyub ◽

...

Keyword(s):

Regulatory Element ◽

Globin Gene ◽

Major Elements ◽

Beta Globin ◽

Globin Mrna ◽

Expression Studies ◽

Primary Element ◽

Alpha Globin ◽

Binding Sequence

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

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Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction [published erratum appears in Blood 1994 Aug 15;84(4):1357]

Blood ◽

10.1182/blood.v83.5.1412.1412 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1412-1419 ◽

Cited By ~ 8

Author(s):

T Furukawa ◽

G Zitnik ◽

K Leppig ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Single Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Beta Globin ◽

Chain Reaction ◽

Globin Mrna ◽

Polymerase Chain ◽

In Cells

Abstract We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.

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