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PubMed1994

Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.

We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.

Affinity Labels↗
PubMed1994

Expression, purification, and functional characterization of the two zinc-finger domain of the human GATA-1.

The DNA-binding domain of the erythroid transcription factor GATA-1 consists of two closely related, but distinct zinc-fingers which are highly conserved among the members of the growing family of GATA-like factors. The DNA-binding domain of the human GATA-1 (F1F2) was expressed as a histidine-tagged fusion protein in Escherichia coli. The denaturated protein was purified by Ni(2+)-chelate affinity chromatography and renaturated in situ. The active recombinant protein was purified by DNA affinity chromatography. F1F2 displayed GATA-1 specific binding activity toward its DNA recognition sequences within the hypersensitive site 3 of the human locus control region and the human gamma-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F1F2 bound also the CCAAT-box region of the human gamma-globin promoter.

Base Sequence↗
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↗
PubMed1993

Hb F in sickle cell anemia.

We have reviewed the methodology for an accurate quantitation of Hb F in the blood of patients with sickle cell anemia, values observed in hundreds of patients of different (racial or ethnic) backgrounds and with differences in severity of the disease, and the various factors that affect the level of Hb F. The latter include sex, age, genetic background or chromosomal haplotypes, variations in the sequences of the locus control region(s) 5' to the epsilon-globin gene, and the presence of an alpha chain deficiency or alpha-thalassemia. Finally, a few remarks about agents effective in increasing the in vivo Hb F synthesis are also included.

Adolescent↗
PubMed1994

The in vivo expression of the globin genes of the beta cistron in gamma-, delta-, and delta beta-thalassemia heterozygotes.

There is considerable evidence suggesting that the switch from gamma to delta and beta chain production after birth is due, in part, to silencing of the gamma genes by stage-specific factors which bind to their promoters and to the competition from the adult (delta and beta) genes for a common enhancer element located in the locus control region. As a consequence one can expect that the increased Hb F production in adults with hereditary persistence of fetal hemoglobin or delta beta-thalassemia is directed mainly by gamma-globin genes in cis to the deletion(s) responsible for these conditions. Here we review data on heterozygotes with gamma-, delta- or delta beta-thalassemia, who also had an A gamma T mutation, in cis or in trans, which was used as a marker of gamma gene expression. The results show that a deletion affecting adult beta genes favors the expression of gamma genes in cis, while the deletion of a single gamma gene does not affect the expression of the beta gene in cis but leads to a faster gamma-->beta switch postnatally.

Gene Deletion↗
PubMed1994

A novel mosaic Bantu/Benin/Bantu beta s haplotype found in several African populations.

In a survey of the chromosomal backgrounds associated with the sickle cell gene in Portuguese-speaking populations from Europe and Africa, a discordance between the classical haplotype and the predicted allele at the RsaI polymorphism 5' to the beta globin gene was observed in four patients. Extensive typing of the corresponding beta s chromosomes at simple polymorphic repeat motifs revealed a novel "extended" haplotype that appeared to be a mosaic of (1) a Bantu-type DNase I hypersensitive site 2 within the beta globin gene cluster locus control region, (2) a Benin 5' subhaplotype, and (3) a Bantu 3' subhaplotype. We propose two alternative schedules for the generation of yet another chromosomal background of the sickle cell gene.

Africa↗
PubMed2004

Blue cone monochromatism: clinical findings in patients with mutations in the red/green opsin gene cluster.

BACKGROUND: X-linked blue cone monochromatism (BCM) has to be differentiated from x-linked cone dystrophy and autosomal recessive rod monochromatism. METHODS: In nine male patients with congenital cone dysfunction (one family, six single cases; age range: 9-55 years), mutations in the red/green opsin gene cluster were confirmed. Clinical findings were analyzed retrospectively. RESULTS: In one family and three single cases, a single red-green hybrid gene was found carrying a Cys203Arg mutation. Two patients had multiple opsin genes, a red/green hybrid gene and at least one green pigment gene, all carrying the Cys203Arg mutation. In one patient, a large deletion of the locus control region and parts of the red pigment gene were detected. Two patients (ages: 45 and 55 years) complained about progression. Two patients presented with nystagmus. Refractive errors (+8.0 and -11.0 D) and visual acuity were variable (0.05-0.3). Only four patients had a visual acuity > or = 0.1. In two patients, visual acuity could be improved using blue filter glasses. Four of five patients > or = 25 years had dystrophic alterations in the macula. Severe color vision defects and relative central scotoma were present in all patients. In the electroretinogram, residual cone responses were detected in 2/8 patients. CONCLUSIONS: Hybrid red/green opsin genes carrying the Cys203Arg mutation are a frequent cause of BCM in German patients. Molecular genetic evaluation is mandatory for adequate diagnosis of patients since from the clinical data only two patients were diagnosed as having BCM. In the other patients, either rod monochromatism or cone-rod dystrophy could not be excluded with certainty. The patients should be cautioned that macular dystrophy may develop in adults older than 30 years.

Adolescent↗
PubMed1996

A new mechanism in blue cone monochromatism.

Blue cone monochromatism (BCM) is a rare X-linked colour vision disorder characterized by the absence of both red and green cone sensitivity. Most mutations leading to BCM fall into two classes of alterations in the red and green pigment gene array at Xq28. In one class the red and green pigment genes are inactivated by deletion in the locus control region. In the second class genetic rearrangements have created an isolated pigment gene that carries an inactivating point mutation. Here we describe a clinical case of BCM caused by a new mutation where exon 4 of an isolated red pigment gene has been deleted. The finding represents the first intragenic deletion yet described among red and green pigment genes.

Base Sequence↗
PubMed1999

Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.

Animals↗
PubMed1996

The use of the rat LAP LCR and promoter for the high-level constitutive expression of K+ channel cDNAs in a rat liver cell line.

Locus control regions (LCRs) are cis-acting elements that confer position-independent and copy-number-dependent expression upon associated genes in transgenic mice. Here we show the second example of the use of an LCR (the rat LAP LCR) in a stable expression vector system, used here in conjunction with the rat liver (NRLM) cell line. Non-transfected NRLM cells are electrically silent and highly suitable for patch clamp electrophysiology. We report reliable constitutive expression from two different K+ channel cDNAs; the voltage-gated rat clone Kv3.4 and the inward rectifier mouse clone Kir2.1. We further show that constitutive expression levels are stable for at least 8 weeks from initial recording.

Animals↗
PubMed1995

Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice.

A human CD2 minigene cassette has been used in the past to express several reporter genes in the T cell lineage of transgenic mice. However, in order to achieve appreciable levels of expression it has been necessary to integrate many copies of the transgene. In this report we describe an improved version of this cassette. The new cassette directs the expression of a reporter mouse CD8 alpha cDNA on all T cells of transgenic mice with an efficiency ten times higher than the previous cassette. The new cassette includes 5 kb 5' and 5.5 kb 3' flanking sequences containing, respectively, the promoter and locus control region (LCR) of the human CD2 gene. The LCR confers position independent, transgene copy number dependent expression of the genes linked to it in transgenic mice. The cassette also provides a transcription initiation site, the first intron of human CD2 gene and the two polyadenylation signals found in the 3' untranslated region of hCD2 gene.

Animals↗
PubMed1993

A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila.

We have characterized an element near the 5' boundary of the chicken beta-globin domain that insulates a reporter gene from the activating effects of a nearby beta-globin locus control region (5'HS2) when assayed in the human erythroid cell line K562. We show that the insulation mechanism is directional, that it operates at the level of transcription, and that it involves the alteration of chromatin structure over the promoter of the gene. The insulator has no significant stimulatory or inhibitory effects of its own. In transgenic Drosophila, the insulator protects the white minigene from position effects. The action of the insulator thus is not restricted to erythroid or mammalian cells, suggesting that such elements may serve an important and widely distributed function in the organization of chromatin structure.

Animals↗
PubMed1993

Chromatin structure and the expression of globin-encoding genes.

The developmental regulation of globin gene expression in the chicken has been studied. All of the genes are regulated by a small number of general erythroid factors. In addition, expression of individual members of the family must be controlled in a lineage (stage)-specific manner. In some cases, the relevant factors may be stage specific, but in others they are not confined to one stage, but exert their control through developmentally regulated changes in their abundance within the nucleus. Chromatin structural elements, such as locus control regions and insulators, are also involved in control of eukaryotic gene expression. Because so much is understood about regulation of individual genes, the globin family has proven valuable in investigating control of transcription at the level of chromatin structure.

Animals↗
PubMed1995

The position of integration affects expression of the A gamma-globin-encoding gene linked to HS3 in transgenic mice.

Proper expression of the human beta-globin (beta Glb) locus is dependent on the presence of a major regulatory element located upstream from the beta Glb gene cluster, the locus control region (LCR). The LCR, as well as the individual DNase-I-hypersensitive sites from which it is composed, have been shown to provide position-of-integration-independent expression in transgenic mice. Here, we report that a transgenic founder carrying multiple integrations of a hypersensitive site 3::A gamma globin gene (HS3::A gamma) construct produced three types of progeny, one with zero A gamma expression in the adult stage, one with minimal A gamma expression (1% of A gamma-expressing cells) and one with abundant A gamma expression (100% A gamma-expressing cells). The possibility that these phenotypes were due to parental imprinting or to DNA rearrangements of the transgene or to point mutations of the HS3 core or the A gamma promoter were excluded. The pattern of inheritance of the three HS3::A gamma transgene phenotypes indicate that the transgene has integrated into three different chromosomes. These results provide direct evidence that the HS3 of the LCR is not sufficient to protect the A gamma gene from position effects excerted by the surrounding chromatin.

Animals↗
PubMed1996

High-level regulated expression of the human G6PD gene in transgenic mice.

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.

Animals↗
PubMed1993

New ultra-micro high-performance liquid chromatographic method for determining the gamma chain composition of hemoglobin F in normal adults.

The difficulty in isolating the minute quantity of Hb F (< 1%) present in the red blood cells of normal adults greatly complicates the determination of its gamma chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the gamma chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb from as little as 50 microliters of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known beta-globin gene cluster haplotypes, and confirmed the association of high, low, and very low G gamma levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the G gamma and beta promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the beta-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the G gamma levels that have been observed.

Adult↗
PubMedDate not supplied

Nuclear topography of beta-like globin gene cluster in IL-3-stimulated human leukemic K-562 cells.

The beta-like globin genes, Ggamma, Agamma, delta and beta, forming specific clusters on chromosome 11, are transcriptionally regulated by the locus control region (LCR). The members of beta-like globin gene cluster (11p15.4) are variously switched during ontogenetic dependent erythropoiesis; however, changes of globin gene expression can be also observed during erythroid differentiation of bone marrow cells. In our experiments, interleukin-3 (IL-3)-stimulated human leukemic K-562 cells were used as a model system in which nuclear organization and expression of the beta-like globin gene cluster was investigated. In addition, the influence of IL-3 on the arrangement of chromosome 11 territory was analyzed. We observed that the beta-globin gene is not expressed in progenitor (nondifferentiated) K-562 cells, but is, however, activated after IL-3 stimulation of the K-562 population. A similar nuclear location of beta-like globin gene clusters was found in both control and IL-3-treated cells, which indicates that changes in cluster gene expression are accompanied by conserved nuclear topography of the gene cluster studied. On the other hand, the studied type of cell differentiation was characterized by relocation of chromosome 11 and its centromeric regions closer to the nuclear periphery, which seems to be a general feature of many pathways of cellular maturation. The beta-like globin gene cluster was observed on chromatin loops extended away from compact chromosome 11 territories that were more condensed in regions closer to the nuclear membrane. The relocation of chromosome 11 territories towards the nuclear periphery and simultaneous appearance of chromatin loops may explain the conserved nuclear positioning of the gene cluster studied.

Cell Differentiation↗