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S Goodbourn

Publications and source records attributed to S Goodbourn.

29 records · Page 2Linked to original sources
PubMed1992

Identification of novel factors that bind to the PRD I region of the human beta-interferon promoter.

Treatment of cells with virus or synthetic double-stranded RNA (dsRNA) leads to the transient transcriptional activation of the beta-interferon gene. Genetic analysis has revealed that the 5' regulatory sequence responsible for this induction contains multiple positive and negative elements. One of these, Positive Regulatory Domain I (PRD I), has been shown to bind the positively-acting transcription factor IRF-1. In this study we show that this element is inducible under conditions where IRF-1 cannot be detected, suggesting that additional cellular factors are involved in the induction process. To investigate the existence of such factors we have analysed the range and properties of PRD I-binding activities present in HeLa cells. In addition to the repressor protein IRF-2, several novel factors can bind to PRD I in uninduced cells: two of these have properties consistent with a role in negative regulation; levels of two others increase upon priming, and may be alternative candidates for activators. Upon induction we also observe a novel factor whose appearance does not depend upon de novo protein synthesis, and which appears to be a truncated form of IRF-2. The potential involvement of these factors in regulating the beta-interferon gene is discussed.

Base Sequence↗
PubMed1992

A method for sequence-specific deletion mutagenesis.

We describe a novel procedure for the construction of deletion mutants. Existing exonuclease-based protocols are efficient at producing randomly positioned deletions over large regions of DNA, but are of limited use in targetted mutagenesis due to their inherent sequence-specificity. We have taken advantage of the Exonuclease III-resistant nature of alpha-thio-dNTPs, incorporated into the target DNA template by a primer extension reaction, to generate base-specific alpha-thio-dNTP terminated products. Following removal of the 5' overhanging strands, the products can be cloned to generate a nested set of deletions with single base-pair increments. We demonstrate the utility of this technique by isolating multiple deletions over a 40bp region of the human beta-interferon promoter.

Base Sequence↗
PubMed1992

Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter.

The cyclic AMP (cAMP) response elements (CREs) of the somatostatin and vasoactive intestinal peptide (VIP) promoters contain binding sites for CRE-binding protein (CREB) that are essential for cAMP-regulated transcription. Using F9 embryonal carcinoma cells, we show that the somatostatin and VIP promoters exhibit a differentiation-dependent cAMP response, demonstrating that these promoters are regulated by transcription factors that become active during differentiation. Lack of cAMP responsiveness of the somatostatin promoter in undifferentiated cells is not due to the absence of known positive-acting factors (the catalytic subunit of protein kinase A [cPKA] and CREB) or a general inhibition of protein kinase A activity. Since overexpression of exogenous cPKA and CREB is sufficient to activate the somatostatin promoter in undifferentiated cells, these findings suggest that a negative factor(s) represses endogenous cPKA and CREB. In contrast to their effects on somatostatin, exogenous CREB and cPKA do not activate the VIP promoter. Thus, despite coregulation during differentiation and the ability to bind CREB, the somatostatin and VIP promoters are not coordinately activated by CREB in undifferentiated F9 cells.

Base Sequence↗
PubMed1990

The regulation of beta-interferon gene expression.

Expression of the beta-interferon gene is induced in many cell lines by viral infection or treatment with double-stranded RNA. The sequence requirements for this induction have been analysed in detail. A minimal inducible element has a modular structure which is consistent with a model in which induction requires de-repression of a dominant negative regulatory domain and the activation of at least two positive regulatory domains. A number of cellular factors that can bind to the beta-IFN promoter have been identified, and some of these are only detectable after induction, including the transcription factor NF-kappa B. cDNA clones have recently been isolated which may encode regulatory factors for beta-IFN expression.

Animals↗
PubMed1989

Double-stranded RNA activates binding of NF-kappa B to an inducible element in the human beta-interferon promoter.

The human beta-interferon promoter contains at least two positive acting domains (PRD I and PRD II). PRD I has been previously shown to stimulate basal transcription and to respond to induction by double-stranded RNA (dsRNA). Here we show that PRD II functions independently as a constitutive element that also responds to induction. A cellular factor that specifically binds to PRD II has been identified, and the levels of this factor increase markedly in extracts from cells treated with dsRNA. The inducible factor has a binding specificity that is indistinguishable from the transcription factor NF-kappa B. As has been shown for NF-kappa B, the PRD II-specific factor can be activated in uninduced extracts by treatment with detergent, suggesting that the inactive state is due to association with an inhibitory factor. Induction by dsRNA therefore provides a novel means for the post-translational activation of NF-kappa B. Potential binding sites for NF-kappa B are present in the 5' flanking regions of a number of genes involved in the immune response, several of which are inducible by dsRNA. These findings demonstrate a role for NF-kappa B in the physiological activation of genes in non-lymphoid cells.

Animals↗
PubMed1988

Overlapping positive and negative regulatory domains of the human beta-interferon gene.

Virus or poly(I).poly(C) induction of human beta-interferon gene expression requires a 40-base-pair DNA sequence designated the interferon gene regulatory element (IRE). Previous studies have shown that the IRE contains both positive and negative regulatory DNA sequences. To localize these sequences and study their interactions, we have examined the effects of a large number of single-base mutations within the IRE on beta-interferon gene regulation. We find that the IRE consists of two genetically separable positive regulatory domains and an overlapping negative control sequence. We propose that the beta-interferon gene is switched off in uninduced cells by a repressor that blocks the interaction between one of the two positive regulatory sequences and a specific transcription factor. Induction would then lead to inactivation or displacement of the repressor and binding of transcription factors to both positive regulatory domains.

DNA Mutational Analysis↗
PubMed1987

Regulation of inducible and tissue-specific gene expression.

Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.

Animals↗
PubMed1986

The human beta-interferon gene enhancer is under negative control.

The human beta-interferon gene is regulated by an inducible enhancer element. Analysis of the effect of deletions within this element on beta-interferon transcription indicates that this enhancer is under negative control. Deletion of sequences from the 3' end of the enhancer leads to a dramatic increase in the basal level of beta-interferon mRNA and a decrease in the induction ratio. The remaining 5' region of the enhancer can act as a strong constitutive transcription element, and it shares considerable homology with sequences known to be required for the activity of constitutive viral enhancers. We conclude that the beta-interferon enhancer consists of a constitutive transcription element and a negative regulatory sequence that prevents enhancer activity prior to induction. Thus, derepression of a constitutive transcription element appears to play a key role in the control of human beta-interferon gene expression.

Animals↗
PubMed1985

Human beta-interferon gene expression is regulated by an inducible enhancer element.

We have localized the regulatory sequence required for viral or poly(I)-poly(C) activation of human beta-interferon gene expression to a region located between -37 and -77 from the mRNA cap site. This sequence has the characteristics of an inducible enhancer element: it can act upstream or downstream of the beta-interferon gene regardless of its orientation, and at distances up to approximately 1 kilobase from its normal location. Moreover, this element can confer inducibility on a heterologous promoter. Further analysis has identified a minimal regulatory element of 14 base pairs within this enhancer. Sequences closely related to this element are present five times within the 5'-flanking regions of both the alpha- and beta-interferon genes. The number of these minimal regulatory elements required for maximal beta-interferon gene expression appears to differ in different cell lines.

Animals↗
PubMed1980

The 5' flanking region of human epsilon-globin gene.

The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.

Base Sequence↗