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GENETIC CONTROL OF FETAL Y-GLOBIN IN ADULT RED CELLS

成年红细胞中胎儿 Y 珠蛋白的遗传控制

基本信息

批准号：
3153960
负责人：
JOHN G GILMAN
金额：
$ 8.64万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-20 至 1988-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3153960
关键词：
erythrocytes erythroleukemia genetic mapping human population genetics immunogenetics immunoglobulin genes linkage mapping molecular cloning molecular genetics mouse leukemia nucleic acid sequence

项目摘要

Fetal hemoglobin (Hb F) is present in fetuses and newborn, and a small amount (less than 1%) is found in normal adults. The two types of Gamma chains of Hb F (Ggamma and Agamma) are found in a 70:30 ratio in most newborn and 40:60 in most adults. However, about 20% of Black SS patients have a high (60:40) ratio of G-gamma to A-gamma chains. A specific haplotype for polymorphic restriction endonuclease sites in the Beta-globin gene cluster is found only in SS individuals with G-gamma greater than or equal 50% of total Gamma chain; this suggests close linkage of a high G-gamma genetic determinant to the Beta-globin gene cluster. Three observations suggest that this determinant is near (and possibly 5' to), the G-gamma and/or A-gamma genes: 1) A subhaplotype, + - + +, for sites in the Gamma genes and near the PSBeta gene, is associated with high G-gamma in Black SS, in the Saudi Arabian form of SS, and in low HbF G-gamma-B+-HPFH, although the full haplotypes (for 7 sites) differ. 2) In the newborn baby, the hybrid gene -Ggamma Agamma-produces Agamma polypeptide at levels expected for the G-gamma gene. 3) A mutation 5' to the Ggamma gene is hypothesized as the cause of high Ggamma levels in high Hb F Ggamma-B+-HPFH. This proposal presents the hypothesis that DNA sequence variation near Ggamma and/or Agamma genes changes their relative rates of transcription, as the result of altered interactions of specific DNA sequences with adult erythroid cell factors. To test this, comparisons will be made of the high G-gamma haplotype of Black SS with a low G-gamma haplotye, and two types of high Ggamma HPFH (high and low HB F Ggamma-Beta+-HPFH) with a low Ggamma HPFH (Agamma-B+-HPFH), for the following: 1) DNA sequence of Ggamma and Agamma genes, including flanking regions; this will also be done for three other high Ggamma haplotypes - two seen in Black Beta-thalassemia, and that of Saudi SS. 2) Levels of mature and precursor Ggamma and Agamma mRNA, in reticulocytes and bone marrow cells, and the extent of methylation and DNase I hypersensitivity of DNA of the latter. 3) mRNA expression of cloned Ggamma and Agamma genes in stably-transfected non-erythroid (L) cells and erythroid (mouse erythroleukemia) cells. 4) The effect of in vitro alterations of DNA sequence on gamma-gene expression of cloned DNA, to confirm the effects on expression of mutations found in aim #1. Data obtained in testing this hypothesis will provide important information on the nature of genetic determinants controlling adult G-gamma:A-gamma ratios and HB F levels.

胎儿血红蛋白（Hb F）存在于胎儿和新生儿中，在正常成人中发现的量（小于1%）。两种类型的Gamma Hb F链（G γ和A γ）的比例为70：30，新生儿和40：60在大多数成年人。然而，大约20%的黑人SS患者具有高的G-γ与A-γ链的比例（60：40）。特定 β-珠蛋白多态性限制性内切酶位点的单体型基因簇仅在G-γ大于或等于的SS个体中发现。等于总伽马链的50%;这表明高伽马链的密切联系。 β-珠蛋白基因簇的G-γ遗传决定因子。三观察表明该决定子接近（并且可能是5 ′ to）， G-γ和/或A-γ基因：1）一个亚单倍型，+ - + +， Gamma基因和PSBeta基因附近，与高G-γ相关在黑色SS中，在沙特阿拉伯形式的SS中，以及在低HbF中， G-γ-B+-HPFH，尽管全单倍型（7个位点）不同。 2)在新生儿，杂交基因-Ggamma Agamma-产生Agamma G-γ基因预期水平的多肽。 3)突变5'至 Ggamma基因被假设为高Ggamma水平的原因， Hb F Ggamma-B+-HPFH。这个提议提出了一个假设，即DNA序列变异在 G γ和/或A γ基因改变它们的相对转录速率，由于特定的DNA序列与成年人的相互作用发生了改变，红系细胞因子为了验证这一点，将比较高 G-γ单倍型的黑色SS与低G-γ单倍型，和两种类型的高G γ HPFH（高和低HB F G γ-β +-HPFH），具有低G γ HPFH（Agamma-B+-HPFH），用于以下：1）G γ的DNA序列和 γ基因，包括侧翼区;这也将对三个其他高G γ单倍型-两种见于黑色β地中海贫血，沙特阿拉伯SS。 2)成熟和前体Ggamma和Agamma水平网织红细胞和骨髓细胞中的mRNA，以及甲基化程度和后者DNA的DNase I超敏性。 3)mRNA表达在稳定转染的非红细胞系（L）中克隆G γ和A γ基因细胞和红系（小鼠红白血病）细胞。 4)影响在 DNA序列的体外改变对克隆DNA的γ-基因表达的影响，以确认对目标#1中发现的突变的表达的影响。在检验这一假设时获得的数据将提供重要的信息关于控制成人G-γ的遗传决定因素的性质：A-γ 比率和HB F水平。