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Results for “Locus Control Region”

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At least 55 records · Page 3Linked to original sources

PubMed1992

Human alpha-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3' of the zeta-globin gene.

The high level expression of the human alpha-globin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human alpha-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with alpha-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact alpha-globin genes. This genetic study supports the critical role of the LCR in the transcriptional activation of the human alpha-globin gene cluster and substantiates the importance of LCR deletions in the etiology of alpha-thalassemia.

Blotting, Southern↗

PubMed1992

Haplotypes in SS patients from Nigeria; characterization of one atypical beta S haplotype no. 19 (Benin) associated with elevated HB F and high G gamma levels.

We have determined the haplotypes of 669 beta S and 109 beta A chromosomes from numerous members of 297 Nigerian families of various ethnic backgrounds. Among the beta S chromosomes, haplotype 19 was detected in 93.2%, haplotype 17 in 3.4%, and haplotype 20 in 0.1%, while 2.4% represented atypical haplotypes. As many as 60.6% of the beta A chromosomes exhibited haplotype 19 mutations, 8.2% had haplotype 3, and 1.8% had haplotype 20. Two siblings with elevated Hb F and G gamma levels were heterozygous for a beta S chromosome with haplotype 19 and a second chromosome with a hybrid haplotype (termed 19 B). In this hybrid chromosome, haplotype 3-like locus control region (LCR) [hypersensitive site-2 (HS-2)] sequences are in juxtaposition to those of the 5' flanking region of the G gamma promoter of a beta S chromosome with haplotype 19. The presence of this hybrid chromosome is associated with high G gamma values in individuals with both sickle cell anemia (SS) and sickle cell trait (AS); it closely resembles another hybrid beta S chromosome, termed 19 A, observed in a previously reported Turkish SS patient who was homozygous for this chromosome and had high Hb F and high G gamma values. In both instances, it is hypothesized that the haplotype 3-like sequences of the LCR HS-2 contain genetic determinants that can combine with factors produced during hematopoietic stress, resulting in increased gamma-globin gene expression.

Adolescent↗

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

In vivo protein-DNA interactions at the beta-globin gene locus.

We have investigated in vivo protein-DNA interactions in the beta-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express epsilon- and gamma-globin but not beta-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The beta promoter was also not footprinted, while extensive footprints were observed in the promoter of the active gamma-globin gene. No footprints were seen in the A gamma and beta 3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells.

Alkylating Agents↗

PubMed1992

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.

Anemia, Sickle Cell↗

PubMed1992

High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia.

A line of transgenic mice with two cointegrated transgenes, the human beta S- and alpha 2-globin genes, linked to the beta-globin locus control region was produced and bred with mice carrying a deletion of the mouse beta major-globin gene. In transgenic mice homozygous for the beta major deletion (alpha H beta S[beta MDD]; where alpha H is human alpha-globin and MD is mouse deletion), 72.5 +/- 2.4% (mean +/- SD) of the beta-chains are beta S and the ratio of alpha H- to beta S-globin was 0.73. Introduction of a heterozygous mouse alpha-globin deletion into mice homozygous for the beta major deletion (alpha H beta S[alpha MD beta MDD]) resulted in 65.1 +/- 8.5% beta S and a human alpha/beta ratio of 0.89 +/- 0.2. Sickling occurs in 95% of erythrocytes from alpha H beta S[beta MDD] mice after slow deoxygenation. Transmission electron microscopy revealed polymer fiber formation but not fascicles of fiber. Increased organ weight was noted in lung, spleen, and kidney of transgenic mice vs. controls that may be due to hypertrophy or increased blood volume in the lungs and/or increased tissue water content. The hemoglobin content of lung, spleen, and kidney was also elevated in transgenic animals due to trapped hemoglobin and/or increased blood volume. When transgenic and control mice were examined by magnetic resonance imaging at 9.4 tesla, some transgenic animals had enlarged kidneys with prolonged relaxation time, consistent with increased organ weight and water content. The glomerular filtration rate was elevated in transgenic animals, which is characteristic of young sickle cell patients. Furthermore, exposure to hypoxia resulted in significantly decreased hematocrit, increased erythrocyte density, and induced a urine-concentrating defect. We conclude that the transgenic mouse line reported here has chronic organ damage and further hematological and organ dysfunction can be induced by hypoxia.

Anemia, Sickle Cell↗

PubMed1992

Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.

The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-like globin genes in erythroid tissues. One of these sites, designated 5'HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5'HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5'HS-2 were linked to the human beta-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5'HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in beta-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5'HS-2 fragment. In contrast, a 358-bp downstream portion of 5'HS-2, which lacked the NFE-2/AP-1 sequences, resulted in beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-globin gene alone but lower than the entire 5'HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5'HS-2 but that other sequences are required for full activity in vivo.

Animals↗

PubMed1992

The LCR-like alpha-globin positive regulatory element functions as an enhancer in transiently transfected cells during erythroid differentiation.

A positive regulatory element (PRE) similar to the locus control region (LCR) of the human beta-globin gene cluster has recently been identified 40 kb upstream of the human zeta-globin mRNA cap site (Higgs D.R. W.G. Wood, A.P. Jarman, J. Sharpe, J. Lida, I.M. Pretorius, and H. Ayyub. 1990). We investigated the influence of the alpha PRE on human alpha-globin promoter activity in transiently transfected cells. The introduction of the alpha PRE into alpha-globin promoter/CAT expression constructs increased alpha-globin promoter activity by 15-30 fold in a human erythroid cell line (Putko) as well as in mouse erythroleukemia cells (MELCs) induced with hexamethylene bisacetamide (HMBA). When these constructs were introduced into uninduced MELCs or HeLa cells, only a 2-3 fold increase in alpha-globin promoter activity was observed. Deletion of 600 bp of alpha-globin 5' flanking sequences containing six putative SP1-binding sites had no significant effect on levels of alpha-globin promoter enhancement by the alpha PRE. We further demonstrated that the alpha PRE and HS2 of the beta-LCR could similarly enhance transcriptional activity of the SV40 early promoter in HMBA induced MELCs. Finally, we showed that alpha-globin promoter activity in the presence of the alpha PRE increased with continued HMBA exposure and was coincident with transcriptional activation of endogenous globin genes.

Acetamides↗

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↗

PubMed1990

Autonomous developmental control of human embryonic globin gene switching in transgenic mice.

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.

Animals↗

PubMed1992

In vivo footprinting of the human alpha-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction.

A major regulatory element required for expression of the human alpha-globin genes is located 40 kb upstream of the embryonic zeta-globin gene. To understand how this and other locus control region (LCR) elements contribute to high-level expression in erythroid cells, we have performed high-resolution, in vivo dimethyl sulfate footprinting. In addition, we have modified the dimethyl sulfate-based ligation-mediated polymerase chain reaction in vivo footprinting procedure to permit the assessment of interactions at guanine and adenine residues, rather than guanines alone. In vivo footprinting of the human alpha-LCR element carried on chromosome 16 in a mouse erythroleukemia cell environment revealed protein occupancy at GATA-1, AP-1/NF-E2, and CACC/GGTGG motifs, specific differences compared with in vitro protein binding, and distinct changes in one region upon dimethyl sulfoxide-induced cellular maturation. No protein contacts were detected in nonexpressing hepatoma cells. In addition, we have demonstrated that two AP-1 motifs in the alpha-LCR element which are occupied in vivo bind purified mouse NF-E2 protein in vitro. Our data suggest that three proteins, GATA-1, NF-E2, and unknown CACC/GGTGG factors, are minimally required as DNA-binding proteins for the function of LCR-like elements. The juxtaposition and interaction of these factors with each other, and with accessory proteins not directly in contact with DNA, are likely to account for the relative position independence of the upstream globin regulatory elements.

Adenine↗

PubMed1992

Normal delta-globin gene sequences in Sardinian nondeletional delta beta-thalassemia.

In order to clarify the reasons for the reduced Hb A2 levels in Sardinian delta beta-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the delta-globin gene from an individual of Sardinian descent who is a compound heterozygote for the beta zero-thalassemia codon 39 (C-->T) nonsense mutation and the Sardinian delta beta-thalassemia [codon 39(C-->T)/-196(C-->T)A gamma]. The analysis of the delta-globin gene from the delta beta-thalassemia chromosome revealed an entirely normal sequence. The defective function of the delta-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondeletional high persistence of fetal hemoglobin mutation of the A gamma gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.

Base Sequence↗

PubMed1992

Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.

The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.

Animals↗

PubMed1991

[Current trends in hemoglobin genetic diseases].

The current aspects of genetic diseases of hemoglobin are as follows. (1) The localisation and the dimension of at-risk populations for sickle cell disease and thalassemia in metropolitan France are not exactly known. However, some recent epidemiological studies and preliminary results of studies being currently underway allow to delimitate the problem. The at-risk populations are localized in the parisian area, in Provence-Côte d'Azur, in the Lyon and Grenoble areas, in Lorraine, Alsace and the Northern part of France. The number of heterozygotes for these diseases is situated between 250,000 and 350,000. The number of homozygotes born each year is between 100 and 150. More than 250 homozygous thalassemia patients and probably 2,000 to 3,000 sickle cell patients are living at this time in metropolitan France. (2) The transgenic mouse model is an experimental tool which allowed important advances in the knowledge of the hemoglobin human genome. Thus, we now have lines of mice expressing human fetal (g genes) and human adult (b genes) hemoglobins. Recently, enhancer sequences have been identified in 5' to the embryonic gene epsilon. When this region, named locus-region-control (LCR), is inserted along with g and b globin genes, their expression is observed at a very high level. The main objective of this technique is to obtain mouse model for sickle cell disease and thalassemia for studies on the pathophysiology and the therapy of these diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Sickle Cell↗

PubMed1991

Regulation of human fetal and adult globin genes in mouse erythroleukemia cells.

We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta-globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5' end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

Adenine Phosphoribosyltransferase↗

PubMed1990

Human globin locus activation region (LAR): role in temporal control.

A region of DNA located far upstream of the human beta-globin locus is critically involved in the regulation of the beta-globin gene family. Recent experiments in transgenic mice suggest that switching from fetal to adult globin gene expression during human development results from competition among individual globin gene family members for interaction with sequences in this region. The phenotypes of patients with defined hemoglobinopathies support this hypothesis.

Animals↗

PubMed1986

A unique immune response control locus mapping with the H-2D class I region.

The immune response to the polysaccharide antigen Trinitrophenyl (TNP)-ficoll is controlled by two complementing loci in the murine major histocompatibility complex. One locus of control is present in the I-A subregion and the second is located between the S and D regions. A monoclonal antibody 48-21.7 was selected for its ability to significantly block the in vitro primary response to TNP-ficoll. This antibody bound to antigen-presenting cells does not interfere with the presentation of other antigens. Experiments are presented to demonstrate that the monoclonal antibody 48-21.7 precipitates a unique protein of approximately 40,000 daltons. Sequential immunoprecipitation with monoclonal antibodies to the class I Db antigen and to relevant Ia antigens demonstrated no cross-reactivity. Amino acid sequencing has found unique amino acids to be present at three class I N-terminal invariant positions. These observations indicate the presence of a unique class I-like molecule coded for by genes mapping between S and D and involved in the response to the polysaccharide ficoll.

Animals↗

PubMed1985

A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli.

Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome. The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions. Temperature-sensitivity conferred by mutation dicA1 in a recA background [corrected] was suppressed by the presence of hybrid plasmids carrying the wild-type gene. In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval. These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.

Escherichia coli↗