Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.     

A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed.     

大剂量~(60)Co照射后造血干细胞潜在的残留损伤

本文对受到临界全致死剂量——8.5Gy~(60)Co照射后的小鼠造血干细胞(HSC)的自我更新力进行了测试,并对照射后4个月,造血功能业已恢复的小鼠HSC的造血重建功能进行了研究。结果表明:应用骨髓连续移植实验所测得的照后3个月中骨髓CFU_s的自我更新潜能明显衰退了。用照射后造血恢复小鼠的CFU_s给全致死剂量照射的受体进行移植治疗,发现其移植效力比正常的显著减弱。在受体存活30天时,CFU_s的再生速率只有正常的1/17。通过对性染色体追踪观察的资料分析,此种小鼠的??造血细胞在受体中难以形成长期稳定的嵌合体。以上事实反映出,照射后小鼠的HSC的造血重建功能大大削弱了,揭示出残存干细胞质量上的缺陷。对于这种潜在的残留损伤的机制,从“干细胞增殖力耗竭学说”和“基因自我修整假说”的角度进行了讨论。     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.     

伸展素在黄瓜胞壁中的转化及其与羟脯氨酸和异联酪氨酸的关系

用~(14)C-Pro和~3H_2-Tyr离体暗培养黄瓜子叶,发现细胞质、SECW和RCW中的Hyp/Pro和Idt/Tyr都随时间呈线性增加。这两种比值后两者高于前者,而两种比值增加速度之比在胞质部分最大、RCW中最小。表明在胞质和胞壁中都有Pro羟化和Tyr异联化过程,但羟化作用主要发生在胞质中,异联化主要在RCW中。 理化处理(高渗溶液和CHM)和放射性示踪证明胞质中存在HRGP库;它被分泌到胞壁后,先以离子键与壁结合,后转变为共价键与壁结合的伸展素。