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F Grosveld

Publications and source records attributed to F Grosveld.

At least 163 records · Page 9Linked to original sources
PubMed1988

The human beta-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers.

We have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta-globin gene and the murine H-2Kb gene. S1 nuclease and transcriptional run-off analysis showed that the human beta-globin gene contains at least three globin specific transcriptional control elements. One is a promoter element and is located 160 bp upstream from the transcription initiation site; it increases the efficiency of the promoter after MEL cells are induced to differentiate. The other two elements are tissue-specific transcriptional enhancers and are located downstream from the transcription initiation site, the first in the structural beta-globin gene and the second in the 3' flanking sequences.

Amino Acid Sequence↗
PubMed1988

The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein.

We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor.

Base Sequence↗
PubMed1988

A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced murine erythroleukemia cells.

We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.

Animals↗
PubMed1988

Transcriptional unit of the murine Thy-1 gene: different distribution of transcription initiation sites in brain.

Structural analysis of the mouse Thy-1.2 gene has shown that the major promoter of the gene is characterized by a tissue-specific DNase I-hypersensitive site and is located within a methylation-free island. The gene is regulated at the transcriptional level, and steady-state mRNA analysis reveals that the previously reported exon Ib contributes at most 5% of the total mRNA. The major promoter uses several transcription initiation sites within a region of 100 base pairs. The frequency of usage of these sites in brain is markedly different from that in other tissues.

Animals↗
PubMed1987

Position-independent, high-level expression of the human beta-globin gene in transgenic mice.

We have constructed a "minilocus" that contains the 5' and 3' flanking regions of the human beta-globin locus and the beta-globin gene. These regions are characterized by erythroid-specific DNAase I-superhypersensitive sites and are normally located approximately 50 kb 5' and 20 kb 3' of the beta-globin gene. This minilocus is expressed tissue-specifically in transgenic mice at a level directly related to its copy number yet independent of its position of integration in the genome. Moreover, the expression per gene copy is the same in each mouse and as high as that of the endogenous mouse beta-globin gene. These results indicate that the DNA regions flanking the human beta-globin locus contain dominant regulatory sequences that specify position-independent expression and normally activate the complete human multigene beta-globin locus.

Animals↗
PubMed1987

Ectopic expression of Thy-1 in the kidneys of transgenic mice induces functional and proliferative abnormalities.

Hybrid human--mouse Thy-1.1 genes were injected into pronuclei of Thy-1.2 mice to produce transgenic animals. A hybrid gene composed of the 5' part of the mouse Thy-1.1 gene combined with the 3' human untranslated regions was expressed abnormally in the kidney podocytes, which resulted in severe protein-uria and subsequent death in several founder mice. A hybrid Thy-1 gene composed of the human coding region with the 5' and 3' flanking regions of the mouse gene was expressed abnormally in a different part of the kidney (the tubular epithelia), which resulted in a proliferative kidney disorder. In addition, a neoplasm was found in the brain of one of these mice. These results show that the Thy-1 protein can play an important role in the activation, proliferation, and differentiation of many different cell types.

Animals↗
PubMed1987

The human beta-globin gene contains a downstream developmental specific enhancer.

The human beta-globin gene is part of a multigene family and is expressed specifically in adult human erythroid tissue (for review, 1). When the human beta-globin is introduced into fertilized mouse eggs, it is first activated in foetal liver and remains expressed in adult erythroid tissues. It therefore mimicks the pattern of expression of its murine counterpart. It has previously been shown in tissue culture and transgenic mice that sequences downstream from the beta-globin promoter are involved in this regulation. We now show that at least part of these sequences are located 0.5-1.2kb downstream from the poly A addition site and constitute a transcriptional enhancer element that is erythroid and developmental specific.

Animals↗
PubMed1987

The structure of a human neurofilament gene (NF-L): a unique exon-intron organization in the intermediate filament gene family.

We have cloned and determined the nucleotide sequence of the human gene for the neurofilament subunit NF-L. The cloned DNA contains the entire transcriptional unit and generates two mRNAs of approx. 2.6 and 4.3 kb after transfection into mouse L-cells. The NF-L gene has an unexpected intron-exon organization in that it entirely lacks introns at positions found in other members of the intermediate filament gene family. It contains only three introns that do not define protein domains. We discuss possible evolutionary schemes that could explain these results.

Amino Acid Sequence↗
PubMed1987

Progress towards construction of a total restriction fragment map of a human chromosome.

We present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human inserts were isolated by hybridisation to a human Alu sequence. DNAs from ninety-six randomly chosen cosmids were digested with either EcoRI or HindIII, end-labelled with 35S-dATP and analysed using agarose gel electrophoresis. Comparison of the restriction fragment patterns revealed two pairs of overlapping clones, that were confirmed by cross-hybridization of the overlapping fragments. The two pairs of cosmids both mapped to human chromosome 17, as shown by hybridization to a panel of somatic cell hybrids. These data demonstrate that the generation of an overlapping cosmid map along a human chromosome is feasible, representing an intermediate step towards the complete sequencing of a human chromosome.

Animals↗
PubMed1987

Site specific demethylation in the promoter of human gamma-globin gene does not alleviate methylation mediated suppression.

Human gamma A-globin genes with different patterns of methylation were synthesized in vitro. These methylated constructs were transfected into mouse L cells and their expression analyzed by S1-nuclease protection assays. We show that methylation downstream of +92 does not affect transcription while methylation downstream of -210 abolishes transcription. To test which of the six CpG dinucleotides between -210 and +92 plays a role in DNA methylation mediated suppression of transcription, we generated mutant gamma-globin genes which lack the three CpGs 5' to the cap site, the three CpGs 3' to the cap site, or all six CpGs. After in vitro methylation, we show that removal of methylated residues 5' or 3' to the cap site did not alleviate suppression, whereas removal of both 5' and 3' methylated residues restored partial expression. These results indicate that CpG methylation at different positions can cause suppression of expression and they are inconsistent with the hypothesis that site-specific demethylations in the promoter region can alleviate suppression. We propose that a minimum length of methylation-free DNA in the promoter area is the only requirement for expression of this promoter by ubiquitous transcription factors, and discuss these results with reference to methylation-free islands.

Animals↗
PubMed1987

Differential regulation of a Thy-1 gene in transgenic mice.

We have generated Thy-1.1-transgenic Thy-1.2 mice to study the developmental expression of the Thy-1 gene in detail by transcriptional and immunological methods. In brain, the expression of the injected gene was identical to that of the endogenous gene in a tissue- and development-specific manner. In lymphoid tissue, the transferred gene was also expressed correctly in the early phases of T-cell lineage development; however, as the T cells matured, the transcription of the transferred gene, but not the endogenous gene, was suppressed. This result shows that different regulatory elements are used to express the Thy-1 gene in early and late lymphoid development.

Age Factors↗
PubMed1987

The regulation of expression of human beta-globin genes.

We have analyzed the regulation of expression of the human beta-globin gene by DNA mediated gene transfer in MEL cells and microinjection in fertilized mouse eggs. The results show that the beta-globin gene contains two regulatory elements: one element in the promoter, that is required for full transcriptional activity and, secondly, a tissue and developmental specific enhancer 3' to the gene.

Animals↗
PubMed1986

The maintenance of methylation-free islands in transgenic mice.

The Thy-1 gene is expressed in a tissue- and stage-specific pattern and has a typical 1.6kb methylation-free island (MFI) covering about 600bp upstream and downstream of the two alternative first exons. By microinjection of a mouse Thy-1.1/human Thy-1 gene into fertilized eggs, we were able to show that the MFI is restored in the transgenic mice. The flanking sequence became methylated, but the MFI remains unmethylated in all tissues of transgenic mice at different developmental stages tested, irrespective of the site of expression of the gene. There is one exception, in extra-embryonal tissues of 14.5 day embryos a small percentage of the islands were methylated. We conclude that maintenance of the MFI is regulated by cis-acting sequences present within the gene, and indicates that the unmethylated state of the islands is consistent with a necessary but not sufficient condition for expression of the gene.

Animals↗
PubMed1986

T cell independent Thy-1 allo-antibody response with the use of transgenic mice.

We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.

Animals↗
PubMed1986

Beta-globin gene promoter generates 5' truncated transcripts in the embryonic/fetal erythroid environment.

We report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly. Using hybrid constructs of these two genes we demonstrate that aberrant activity is conferred by sequences 5' of the beta-globin gene. Thus beta-globin promoter attached to the H2 coding sequence produces H2 transcripts with truncated 5' ends. By introducing a series of deletions in the beta-globin promoter we restrict these sequences to the -77/+28 base pair region spanning the CAAT element to the translation initiation site. These results are consistent with the lack of recognition of the beta-globin gene major cap site in Putko and K562 cells. We suggest that inactivity of the adult globin gene in the embryonic/fetal environment is at least in part conferred by sequences within the beta-globin gene promoter.

Animals↗
PubMed1986

Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns.

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues.

Animals↗