与胎儿珠蛋白基因持续表达有关的(A_γδβ)°—地贫纯合子及杂合子的分子鉴别

我们分子鉴别了一个缺失型中国(A_γδβ)°-地贫家系。先证者为这一缺失的纯合子,具有中度贫血症状。家系的另五个成员均为这一缺失的杂合子,其胎儿血红蛋白(HbF)为16—21％,接近或达到HPFH杂合子的HbF水平,并且几乎不表现贫血症状。限制性内切酶图谱分析证明了β-珠蛋白基因簇内的DNA顺序缺失,缺失的5′端点位于Aγ基因IVSⅡ内,3′端点在β-珠蛋白基因下游区远端,距HPFH-2的3′缺失端点上游区约11kb。缺失的总长度约为80kb。本文讨论了这一缺失导致胎儿血红蛋白在成人中持续活跃表达的可能机制。     

用直接测定基因组DNA序列的方法检测β-地中海贫血基因突变

本文报道一种结合聚合酶链反应(PCR)技术直接测定基因组DNA中单考贝基因片段序列的方法,以及利用这种方法测定两例β-地贫纯合子的β珠蛋白基因序到结果。测定出基因点突变,一例为编码子17(A→T)突变纯合子,另一例为编码子69(G→A)突变纯合子。针对上述两个点突变合成寡核苷酸片段,末端标记~(82)P后为探针进行斑点杂交的结果与测序结果一致。     

Formation of substrate and transition-state analogue complexes in crystals of phosphoglucomutase after removing the crystallization salt.

Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.