Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

Trans acting regulation of beta globin gene expression in erythroleukemia (K562) cells.

K562 cells are induced by hemin to produce gamma and epsilon globin but not beta globin, although the beta globin gene is intact, and when isolated is expressed in a transient expression assay (1, 2). We have previously shown that an epsilon globin gene transferred into K562 cells is expressed and inducible (3). In this paper, we report the stable transfer of a sickle or betaS globin gene into K562 cells. Thirty-six different transformed lines were tested; 24 of 36 lines contained an intact betaS globin gene. However, using S1 nuclease, Dot blot, and Northern blotting analyses, none of these lines showed beta globin mRNA expression. These results indicate that trans acting factors are responsible for the lack of expression of the beta globin gene in K562 cells.     

Butyrate induces expression of transfected human fetal and endogenous mouse embryonic globin genes in GM 979 erythroleukemia cells

We have analyzed the expression of endogenous murine genes and of transfected human fetal A gamma globin gene in GM 979, a mouse erythroleukemia line which produces adult as well as embryonic globins. Optimal induction of the endogenous murine adult globin genes was obtained with DMSO or HMBA while the epsilon y and beta h1 embryonic genes were preferentially induced by butyrate. Similarly, the transferred human A gamma-globin gene was preferentially induced by butyrate. These results as well as previous observations in vivo or in erythroid cell cultures suggest that butyrate preferentially induces the expression of fetal globin genes.     

Distal CCAAT box deletion in the A gamma globin gene of two black adolescents with elevated fetal A gamma globin.

Point mutations in G gamma and A gamma globin gene promoters are associated with increased production of G gamma and A gamma globin, respectively. To determine whether an upstream promoter mutation could account for elevated A gamma in a Black adolescent with A gamma-beta+-HPFH and sickle cell trait, we cloned the 13 kb BglII fragment containing both gamma genes into phage lambda vector EMBL3. For one clone, the A gamma upstream promoter showed no hybridization to a 19 bp oligonucleotide whose sequence centered at -117. A gamma promoter sequence data for this mutant clone revealed a 13 bp deletion which eliminated the A gamma distal CCAAT box. Amplified A gamma genomic DNA of this and a similar case showed hybridization to both deletion-mutant and normal oligonucleotide probes. We propose that this 13 bp deletion removes part of the binding site for a repressor protein which is abundant in adult erythroid cells.     

A novel nuclear protein which binds to G gamma and A gamma globin promoters and modulates hemoglobin synthesis in K562 cells.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.     

应用实时定量RT-PCR技术检测b地中海贫血 珠蛋白基因的表达

为了定量检测 b 地中海贫血(b 地贫)的 a、b 和γ珠蛋白基因表达水平, 提取正常成人对照组、正常胎儿对照组和b 地贫患者组组成的样本 DNA,采用反向点杂交法（RDB）分析b 地贫各种突变类型;提取样本RNA用于进行针对a、b 和γ珠蛋白基因的荧光实时定量RT-PCR（FQ RT-PCR）。根据FQ RT-PCR原理,设计合成分别对应于a、b 和γ珠蛋白基因的3对引物和3条荧光探针,FQ RT-PCR在ABI 7700系统进行。用SPSS 10.0对实验数据进行统计学分析,分别计算正常对照组 (bA/bA,aa/aa),脐带血组(bA/bA,aa/aa),轻型b 地贫组(bT/bA,aa/aa),重型b地贫组(bT/bT,aa/aa)的a、b 和γmRNA比值,其中a/b分别为4.62±1.20、7.81±2.89、13.51±5.12、188.24±374.04;a/(b +γ)分别为4.43±1.17、0.56±0.49、9.62±4.37、2.14±1.58;γ/(b+γ) 分别为0.04±0.03、0.92±0.06、0.28±0.18、0.95±0.04。由于组与组之间均值变异范围较大,将其进行对数转换后再进行方差分析。结果表明： a/b与a/(b+γ)在所有组与组之间均有显著性差异。γ/(b+γ)除了在脐带血组和重型b地贫组之间无显著性差异外,在其他组与组之间均有显著性差异。实验说明,人类b珠蛋白基因的表达水平从正常对照组到重型b地贫组急剧下降且以重型b地贫组为最低;相反γ珠蛋白基因表达却明显升高,以重型b地贫组为最高。与正常成人对照组相比,胎儿期b mRNA水平较低但γmRNA 水平较高。因此,正常个体不同时期和不同类型b 地贫之间a、b与γ珠蛋白基因表达不同而且互相影响。 Abstract：whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with b-thalassemia minor (bT/bA,aa/aa) and 39 with b-thalassemia major (bT/bT,aa/aa). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for b-thalassemia genotype analysis. Different types of b globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of a, b andγglobin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of a, b andγhuman globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of a, b andγglobin mRNA were calculated and the ratios of a/b, a/(b + γ) andγ/(b + γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, b-thalassemia minor and b-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and b-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of a/b + 1, a/(b + γ) + 1 andγ/(b + γ) +1 was used for statistical analyses and intergroup comparison. The a/b globin gene mRNA ratios were determined to be 4.62±1.20, 7.81±2.89, 13.51±5.12, and 188.24±374.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b-thalassemia major(bT/bT,aa/aa) respectively. The a/(b+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Theγ/(b+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Following statistical analyses, the a/b and a/(b+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, b- thalassemia minor and b- thalassemia major). The γ/(b + γ) globin gene mRNA ratio was significantly different among all groups except for between infant and b- thalassemia major patients. Human b globin gene mRNA levels decrease progressively and dramatically from normal adults to b-thalassemia patients with b-thalassemia major having the lowest levels. On the other hand, the γglobin gene mRNA levels increase progressively from normal adult to b-thalassemia patients with b-thalassemia major having the highest levels. Infants have relatively lower levels of b but higher levels of γglobin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of a, b or γglobin genes varied but inter-related among different ages of normal individuals and different b-thalassemia genotypes.     

Alpha-thalassemia due to the deletion of nucleotides -2 and -3 preceding the AUG initiation codon affects translation efficiency both in vitro and in vivo.

We previously hypothesized that a 2 nucleotide deletion, causing a A-greater than C change at position -3 preceding the ATG initiation codon of alpha globin gene, reduced translation efficiency of alpha globin mRNA and was responsible for a form of alpha + thalassemia displayed by an Algerian patient. We presently show that this deletion leads to a 30-45% reduction in translation efficiency of synthetic alpha globin mRNA in rabbit reticulocyte lysate. In other experiments, we constructed alpha/G gamma hybrid globin genes in which the 3' end of normal or mutated alpha globin genes downstream to the ATG initiation codon was substituted by the 3' part of a G gamma globin gene. COS cells transfected with either of these 2 hybrid genes were shown to synthesize a similar amount of alpha/G gamma hybrid mRNAs but 50% less G gamma globin when transfected with the alpha/G gamma hybrid gene carrying the deletion. These results definitively establish that the 2 nucleotide deletion reduces translation efficiency by 30-50%. This contrasts with the 93% reduction induced by a similar A-greater than C change at position -3 in the different nucleotide context preceding the ATG codon of the rat preproinsulin gene.     

Role of gene order in developmental control of human gamma- and beta-globin gene expression.

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.     

Developmental regulation of fetal to adult globin gene switching in human fetal erythroid x mouse erythroleukemia cell hybrids

Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (gamma) to adult (beta) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in trans by preparing hybrid cells containing marked fragments of the gamma and beta genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequences present in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.