Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Molecular and statistical approaches to the detection and correction of errors in genotype databases.

Errors in genotyping data have been shown to have a significant effect on the estimation of recombination fractions in high-resolution genetic maps. Previous estimates of errors in existing databases have been limited to the analysis of relatively few markers and have suggested rates in the range 0.5%-1.5%. The present study capitalizes on the fact that within the Centre d'Etude du Polymorphisme Humain (CEPH) collection of reference families, 21 individuals are members of more than one family, with separate DNA samples provided by CEPH for each appearance of these individuals. By comparing the genotypes of these individuals in each of the families in which they occur, an estimated error rate of 1.4% was calculated for all loci in the version 4.0 CEPH database. Removing those individuals who were clearly identified by CEPH as appearing in more than one family resulted in a 3.0% error rate for the remaining samples, suggesting that some error checking of the identified repeated individuals may occur prior to data submission. An error rate of 3.0% for version 4.0 data was also obtained for four chromosome 5 markers that were retyped through the entire CEPH collection. The effects of these errors on a multipoint map were significant, with a total sex-averaged length of 36.09 cM with the errors, and 19.47 cM with the errors corrected. Several statistical approaches to detect and allow for errors during linkage analysis are presented. One method, which identified families containing possible errors on the basis of the impact on the maximum lod score, showed particular promise, especially when combined with the limited retyping of the identified families. The impact of the demonstrated error rate in an established genotype database on high-resolution mapping is significant, raising the question of the overall value of incorporating such existing data into new genetic maps.     

山西糜子核心种质分子身份证构建

为快速鉴定糜子(Panicum miliaceum)资源, 建立分子标记检测平台, 以272份山西糜子核心种质为研究材料, 利用85对简单重复序列(SSR)引物, 应用ID Analysis 4.0软件, 构建DNA分子身份证。结果表明, 对85对SSR引物进行筛选, 发现20对引物组合(RYW67、RYW53、RYW37、RYW65、RYW62、RYW77、RYW5、RYW49、RYW84、RYW19、RYW11、RYW40、RYW54、RYW28、RYW31、RYW7、RYW16、RYW8、RYW9和RYW18)可区分272份材料。共检测到等位变异(Na) 60个, 平均每个位点检出3个; Shannon多样性指数(I)为0.957 8 (RYW16)-1.096 7 (RYW5), 平均值为1.055 2; 多态性信息含量(PIC)为0.604 4 (RYW77)-0.753 0 (RYW37), 平均值为0.692 1。利用20对引物构建山西糜子核心种质的字符串、条形码和二维码DNA分子身份证, 可为种质身份标识和溯源提供实践路径。     

白细胞介素2-绿脓杆菌外毒素融合基因的克隆及高效表达

利用聚合酶链式反应和寡核苷酸介导的定向诱变技术构建了白细胞介素2-绿脓杆菌外毒素IL2-PE40、IL2-PE40KDEL、IL2-PE66~(4Glu)和IL2-PE66~(4Glu)KDEL融合基因的原核表达重组质粒,并实现了高效表达,表达产物占菌体总可溶蛋白的20％～30％。此外,由于这一表达质粒在IL-2cDNA与PE基因连接处引入了唯一的SmaⅠ位点,其5＇、3＇端分别含有唯一的EcoRⅠ、PstⅠ位点,因此可方便地用其它基因替换IL-2或PE基因而获得相应融合蛋白的表达质粒。     

Characterization of Calpain-Mediated Proteolysis of GluR1 Subunits of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate Receptors in Rat Brain

Abstract: Previous results have indicated that GluR1 subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [³H]AMPA or 6-cyano-7-[³H]nitroquinoxaline-2,3-dione ([³H]CNQX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 m M resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [³H]AMPA and [³H]CNQX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Activation of CD74 inhibits migration of human mesenchymal stem cells

Therapeutic administration of mesenchymal stem cells (MSCs) by systemic delivery utilizes the innate ability of the cells to home to damaged tissues, but it can be an inefficient process due to a limited knowledge of cellular cues that regulate migration and homing. Our lab recently discovered that a potent pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), inhibits MSC migration. Because MIF may act on multiple cellular targets, an activating antibody (CD74Ab) was employed in this study to examine the effect of one MIF receptor, CD74 (major histocompatibility complex class II-associated invariant chain), on MSC motility. CD74 activation inhibits in a dose-dependent manner up to 90% of in vitro migration of MSCs at 40 μg/ml CD74Ab (p?<?0.001), with consistent effects observed among three MSC donor preparations. A blocking peptide from the C-terminus of CD74 eliminates the effect of CD74Ab on MSCs. This suggests that MIF may act on MSCs, at least in part, through CD74. Late-passage MSCs exhibit less chemokinesis than those at passage 2. However, MSCs remain responsive to CD74 activation during ex vivo expansion: MSC migration is inhibited ～2-fold in the presence of 5 µg/ml CD74Ab at passage 9 vs. ～3-fold at passage 2 (p?<?0.001). Consistent with this result, there were no significant differences in CD74 expression at all tested passages or after CD74Ab exposure. Targeting CD74 to regulate migration and homing potentially may be a useful strategy to improve the efficacy of a variety of MSC therapies, including those that require ex vivo expansion.