Biomedical subjects

F Grosveld

Publications and source records attributed to F Grosveld.

At least 127 records · Page 7Linked to original sources

PubMed1992

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.

Animals↗

PubMed1992

Selective inhibition of neurite outgrowth on mature astrocytes by Thy-1 glycoprotein.

THY-1, the smallest member of the immunoglobulin superfamily, is a major cell-surface component expressed by several tissues. The protein, carbohydrate and gene structures of this molecule are known, yet its function is not. It is highly expressed in nervous tissue, where it appears on virtually all neurons after the cessation of axonal growth. Here we show that expression of Thy-1 by a neural cell line inhibits neurite outgrowth on mature astrocytes, but not on other cellular substrata which include Schwann cells and embryonic glia. This inhibition of neurite extension on astrocytes can be reversed by low concentrations (nanomolar) of soluble Thy-1. If a similar interaction between neuronal Thy-1 and astrocytes occurs in vivo, it could stabilize neuronal connections and suppress axonal regrowth after injury in the astrocyte-rich areas of adult central nervous system.

Animals↗

PubMed1992

Developmental regulation of a complete 70-kb human beta-globin locus in transgenic mice.

We have used a linker-based ligation strategy to combine two 35-kb cosmid inserts from the human beta-globin locus into one linear fragment containing the entire locus. This 70-kb fragment was introduced into transgenic mice by microinjection of fertilized eggs. Southern blot analysis showed that a single complete transgene locus can be introduced into the germ line with high efficiency. Analysis of the expression patterns of the locus during development shows that the epsilon-globin gene behaves as a purely embryonic gene, the gamma-globin gene as an embryonic and early fetal gene, and the beta-globin gene as a fetal adult gene. Quantitation of expression showed that the levels of transcription of the epsilon- and gamma-globin genes are reversed relative to their mouse homologs but that the total output of the human and mouse loci is constant during development. These results suggest that multiple changes in DNA sequences and transcription factor balance must have occurred for the human gamma-globin gene to have evolved into a fetal gene.

Animals↗

PubMed1991

Expression of adult and tadpole specific globin genes from Xenopus laevis in transgenic mice.

Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching.

Aging↗

PubMed1991

Hypersensitive site 4 of the human beta globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.

Animals↗

PubMed1991

Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.

Animals↗

PubMed1991

The 5'HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites.

The locus control region (LCR) of the human beta-globin locus consists of four hypersensitive regions (5'HS 1-4). One of these sites, 5'HS2, is active in both transient and stable transfection assays and transgenic mice. It has previously been shown that the jun/fos consensus binding sites in 5'HS2 are required for high levels of transcription. In this paper we show that it is the 5' of the two consensus sites that is required for this function with a contribution of the 3' site to the overall activity. The functional complex at both sites includes NF-E2. Its role in HS2 is to provide 'enhancer' activity but is not required for position independent activation. High levels of enhancement are achieved by interaction of the NF-E2 sites with three downstream elements. One of these sites binds the known factor GATA-1, whereas the other two interact with two novel DNA binding factors (H-BP and J-BP).

Base Sequence↗

PubMed1991

Importance of globin gene order for correct developmental expression.

We have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the genes. When the human beta-globin gene is linked as a single gene to the LCR it is activated prematurely in the embryonic yolk sac. We show that the correct timing of beta gene activation is restored when it is placed farther from the LCR than a competing human gamma- or alpha-globin gene. Correct timing is not restored when beta is the globin gene closest to the LCR. Similarly, the human gamma-globin gene is silenced earlier when present farthest from the LCR. On the basis of this result, we propose a model of developmental gene control based on stage-specific elements immediately flanking the genes and on polarity in the locus. We suggest that the difference in relative distance to the LCR, which is a consequence of the ordered arrangement of the genes, results in nonreciprocal competition between the genes for activation by the LCR.

Aging↗

PubMed1991

The functional domains of the murine Thy-1 gene promoter.

The Thy-1 gene promoter resembles a "housekeeping" promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.

Animals↗

PubMed1991

The effect of class II gene transfection on the tumourigenicity of the H-2K-negative mouse leukaemia cell line K36.16.

There is much evidence to suggest that potentially immunogenic tumour cells can escape cytolytic immune destruction by loss of class I antigen expression. Many tumours are allele-specific class I negative and, in murine systems, reconstitution of class I expression by gene transfection leads to an increase in tumour immunogenicity. In many systems where mice have rejected class I transfected tumour cells they are also immune to a subsequent challenge with the untransfected parent tumour. In this study we have examined the effect of stable class II antigen expression (induced by gene transfection) on a class I loss mutant (H-2Kk negative) murine cell line, K36.16. We show that H-2Ek expression is more effective at increasing tumour immunogenicity than the reconstitution of H-2Kk expression in these cells. This suggests that the induction of class II antigen expression on tumour cells may provide an effective way of enhancing tumour-specific immune responses in vivo.

Animals↗

PubMed1990

DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression.

The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus.

Animals↗

PubMed1990

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors.

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.

Antigens, Viral↗

PubMed1990

A transgenic mouse model of sickle cell disorder.

A single base-pair mutation (beta s) in codon 6 of the human beta-globin gene, causing a single amino-acid substitution, is the cause of sickle cell anaemia. The mutant haemoglobin molecule, HbS, polymerizes when deoxygenated and causes deformation of the erythrocytes to a characteristic 'sickled' shape. Sickling of cells in small vessels causes painful crises and other life-threatening complications. Although the molecular basis for sickle cell anaemia has been known for 30 years, no definitive treatment is available. An animal model of sickle cell anaemia would not only allow a detailed analysis of the factors that initiate erythrocyte sickling in vivo and of the pathophysiology of the disease, but would also permit the development of novel approaches to the treatment of the disease. By using the dominant control region sequences from the human beta-globin locus, together with human alpha- and beta s-globin genes, we have obtained three transgenic mice with HbS levels ranging from 10 to 80% of total haemoglobin in their red cells. As observed in homozygous and heterozygous Hbs patients, the erythrocytes of this mouse sickle readily on deoxygenation. Irreversibly sickled cells, which are characteristic of sickle-cell patients homozygous for beta s, are also observed in the peripheral blood of the mouse with high levels of HbS.

Anemia, Sickle Cell↗

PubMed1990

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta-globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon-globin gene. Addition of these sites confers copy number dependent expression on the human beta-globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40-50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta-globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive 'in vivo'. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF-E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.

Animals↗

PubMed1990

Detailed analysis of the site 3 region of the human beta-globin dominant control region.

Four DNase I hypersensitive sites characterize the human beta-globin Dominant Control Region (DCR) providing position independent, high levels of erythroid specific expression to linked homologous and heterologous genes when introduced into cultured cells or in transgenic mice. We have delineated the hypersensitive site located 10.5 kbp upstream of the epsilon-globin gene by short range DNase I sensitivity mapping to a 600 bp region. Using transgenic mice and MEL cells the functional part of this region was further mapped to a 300 bp central core, which provides position independent, high level expression. It contains a number of ubiquitous and erythroid specific protein binding sites, including the previously described factors NF-E1 (GF1) and NF-E2. The latter binds to a dimer of the consensus binding sequence for jun/fos. The presence of this sequence is required for the function of the element, but single or multimerized copies of this site failed to give position independent, high levels of expression in transgenic mice or MEL cells. We therefore conclude that a combination of factor binding sites is necessary to allow site 3 to function as a strong transcriptional activator, resulting in position independent expression of the beta-globin gene.

Animals↗

PubMed1990

Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice.

A 21.5 kb DNA fragment carrying the entire chicken lysozyme gene locus was introduced into the germ line of mice. The fragment contains the transcribed region plus 11.5 kb 5'-flanking and 5.5 kb 3'-flanking sequences including all known cis-regulatory elements and the 5' and 3' attachment elements (A-elements) which define the borders of the DNase I sensitive chromatin domain. All sequences which adopt a DNase I hypersensitive chromatin conformation in vivo are present on the construct. Seven founder mice were analysed. All of these expressed chicken lysozyme RNA at high levels specifically in macrophages, as is the case in the donor species. Expression levels are dependent on the copy number of integrated genes indicating that a complete gene locus, as defined by its chromatin structure, functions as an independent regulatory unit when introduced into a heterologous genome.

Animals↗

PubMed1990

Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.

Animals↗

PubMed1990

Tissue-specific control elements of the Thy-1 gene.

We have exploited the structural homology, but different patterns of expression of the murine and human Thy-1 genes to map a number of tissue-specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally-regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent.

Animals↗