GENETIC CONTROL OF FETAL Y-GLOBIN IN ADULT RED CELLS

胎儿血红蛋白(HbF)升高可改善成人疾病 例如，镰状细胞性贫血(SS)和β-地中海贫血。 这个实验室已经证明了与 体内γ基因表达增加，在-158(C到T)和 -161(G到A)的G伽马基因和-202(C到T)的A伽马。 A伽马基因-222至-225处的4个碱基对缺失为 地中海地区与A-γ表达降低有关 单倍型II型β-地中海贫血。 贝宁贝塔斯单倍型通常与低Hb F相关， 但初步数据显示，高Hb F决定因素与 某些家庭。文献数据表明，亚洲的BS (沙特)单倍型也可能与不明原因的高Hb- F行列式。这项提案的一部分旨在澄清 SS和β-HBs单倍型与Hb F表达的关系 萨尔。我们将检验相关的假设，即某些事件 贝宁和亚洲贝塔的单倍型紧密相连，但 不明原因的高Hb F突变。对于选定的情况(高Hb F 贝宁和亚洲贝塔)，扩增的基因组DNA将在 G伽马和A伽马推进器。如果没有发现突变，那么 伽马增强剂将在Bodine&Ley表达中进行测试 如果活跃度升高，则对其进行排序。我们还将测试 假设4个碱基对缺失是一个主要因素 应用寡核苷酸探针筛查贝塔地贫的严重程度 轻、重度病例。 该提案的第二部分将检验以下假设： -158、-161、-202突变和4个碱基缺失通过改变 对转录调节蛋白的亲和力。我们的模型表明 几个序列基序上可能的蛋白质结合位点， 包括“TC”(198至-192)、“富含G-C”(-208至-200)、SphI(- 226到-219)和“增强器核心”(-270到-264)。突变启动子 将被放置在5‘端到一个“标记”的伽马珠蛋白基因的存在 以及缺乏伽马增强剂，并在K562中进行了表达测试 红白血病细胞用信使核糖核酸测定。删除5‘至-200和 -158个突变将测试5‘序列的重要性为提升 这些突变的表达。蛋白质与蛋白质结合的比较 在正常和突变启动子片段中的这些基序将被完成 凝胶滞留试验。正常和突变之间的竞争 寡聚体将显示突变对蛋白质结合的影响。 足迹和甲基化干扰数据将精确地 定义结合部位，检验假设-158和-161 突变会影响与已知基序的结合，并有助于表征 部分纯化的K562蛋白与骨髓红系 细胞。这些数据将有助于绘制结构图- 红细胞中丙种球蛋白转录的功能关系 人类成年人的。

Elevated fetal hemoglobin (Hb F) ameliorates diseases of adult hemoglobin, such as sickle cell anemia (SS) and beta-thalassemia. This laboratory has demonstrated mutations associated with increased expression of gamma genes in vivo, at -158 (C to T ) and -161 (G to A) of the G gamma gene and -202 ( C to T) of A gamma. A 4 base pair deletion at -222 to -225 of the A gamma gene is associated with decreased A gamma expression in Mediterranean haplotype II betao-thalassemia. The Benin betaS haplotype is generally associated with low Hb F, but preliminary data show high Hb F determinants linked to it in certain families. Literature data suggest that the Asian BS (Saudi) haplotype may also be associated with unidentified high Hb- F determinants. Part of this proposal aims to clarify the relationship between haplotype and Hb F expression in SS and beta- thal. We will test the related hypotheses that certain occurrences of the Benin and Asian betaS haplotypes have closely-linked but unidentified high Hb F mutations. For selected cases (high Hb F Benin and Asian betaS), amplified genomic DNA will be sequenced in the G gamma and A gamma promoters. If no mutations are found, then the gamma enhancer will be tested in the Bodine & Ley expression system and sequenced if activity is elevated. We will also test the hypothesis that the 4 bp deletion is a major factor the severity of betao -thal by using oligonucleotide probes to screen mild and severe cases. The second part of this proposal will test the hypothesis that the -158, -161, -202 mutations and the 4 bp deletion act by altering affinity for transcription-regulating proteins. Our model suggests possible protein-binding sites at several sequence motifs, including "TC" (198 to -192), "G-C rich" (-208 to -200), SphI (- 226 to -219) and "enhancer core" (-270 to -264). Mutant promoters will be placed 5' to a "marked" gamma globin gene in the presence and absence of gamma enhancer, and expression tested in K562 erythroleukemia cells by mRNA assays. Deletions 5' to the -200 and -158 mutations will test the importance of 5' sequences to elevated expression of those mutations. Comparison of protein binding to these motifs in normal and mutant promoter fragments will be done by gel retardation assays. Competition between normal and mutant oligomers will show the effects of mutations on protein binding. Footprinting and methylation interference data will precisely define binding sites, test the hypothesis that the -158 and -161 mutations affect binding to known motifs, and aid in characterizing partially purified proteins of K562 and bone marrow erythroid cells. These data will contribute to the mapping of structure- function relationships of gamma globin transcription in red cells of human adults.