3. Genetic conservation in seed banks

The safest and most economical and convenient way of preserving the genetic resources of the majority of crop plants is by long-term seed storage. The technology is well developed, but recent research resulting from a greater understanding of behaviour at very low water potentials is leading to further improvements     

治疗用武汉抗CD3单克隆抗体(WuT_3)的致突变性研究

观察了武汉抗ＣＤ３单克隆抗体（简称ＷｕＴ３）对组氨酸缺陷型鼠伤寒沙门氏菌ＴＡ９７、ＴＡ９８、ＴＡ１００及ＴＡ１０２菌株的回复突变作用。结果显示在５～５０００μｇ／皿的剂量范围内,ＷｕＴ３所致的诱发回复突变菌落数与自发回复突变菌落数之比ＭＲ（Ｒｔ／Ｒｃ）,无论加大鼠肝匀浆,辅酶Ⅱ及葡萄糖６－磷酸（Ｓ－９混合液）或不加Ｓ－９混合液,均不超过２。同时观察了ＷｕＴ３对小鼠骨髓细胞微核率及对人外周血淋巴细胞染色体畸变率的影响,结果显示小白鼠ｉｖＷｕＴ３,每日一次,连续２日,在２５～１００ｍｇ·ｋｇ－１范围内,ＷｕＴ３各剂量组的微核细胞率与溶剂对照组相比均无显著性差异（Ｐ＞０．０５）。而环磷酰胺（ＣＰ）阳性组与溶剂对照组相比有极显著性差异（Ｐ＜０．０１）。ＷｕＴ３在２５～２５０μｇ／瓶的剂量范围内,各剂量组的染色体畸变细胞率与溶剂对照组相比无显著性差异（Ｐ＞０．０５）,而ＣＰ组与溶剂对照组相比有极显著性差异（Ｐ＜０．０１）。三项试验结果均未发现ＷｕＴ３有致突变性作用     

PIK3CA基因突变的MassARRAY分子量阵列分析

目的：利用MassARRAY分子量阵列分析系统检测胃癌组织PIK3CA基因突变。方法：从胃癌石蜡包埋组织中提取基因组,PCR反应扩增目的基因片段,MassARRAY分子量阵列分析系统检测PIK3CA基因突变;焦磷酸测序验证检测结果。结果：中国西部地区144例胃癌组织样本中PIK3CA_E542K（1624G〉A）突变携带率为77．6％,PIK3C_LIE545K（1633G〉A）突变携带率为84％。MassARRAY分子量阵列分析系统检测结果与焦磷酸测序结果达到100％吻合。结论：建立了MassARRAY分子量阵列分析系统检测基因突变的方法,初步建立了中国西北地区汉族人群胃癌组织PIK3CA基因PIK3CA_E542K（1624G〉A）PIK3CA_E545K（1633G〉A）位点突变频数。     

PIK3CA 基因突变的MassARRAY 分子量阵列分析

目的:利用MassARRAY分子量阵列分析系统检测胃癌组织PIK3CA基因突变。方法:从胃癌石蜡包埋组织中提取基因组,PCR反应扩增目的基因片段,MassARRAY分子量阵列分析系统检测PIK3CA基因突变;焦磷酸测序验证检测结果。结果:中国西部地区144例胃癌组织样本中PIK3CA_E542K(1624G>A)突变携带率为77.6%,PIK3CA_E545K(1633G>A)突变携带率为84%。MassARRAY分子量阵列分析系统检测结果与焦磷酸测序结果达到100%吻合。结论:建立了MassARRAY分子量阵列分析系统检测基因突变的方法,初步建立了中国西北地区汉族人群胃癌组织PIK3CA基因PIK3CA_E542K(1624G>A)和PIK3CA_E545K(1633G>A)位点突变频数。     

EMA3C/SEMA3D基因错义突变对蛋白稳定性和受体亲合力的影响

目的：明确先天性巨结肠患者携带的5 个基因错义突变对Semaphorin 3（Sema3）蛋白自身稳定性和受 体亲合力的影响作用。方法：构建Sema3-Neuropilin-Plexin 配体-受体复合物蛋白质模型,对全部5个错义突变进行定位,通过计 算标准能量功能赋值（驻驻G）和复合物界面值（驻I_sc）预测突变对Sema3 的影响作用。将野生型和突变型AP-tagged Sema3 质粒分 别转染HEK293T 细胞,72 h后收集含有融合蛋白的细胞培养液上清并与分别表达Neuropilin 1（Nrp-1）或Neuropilin 2（Nrp-2）的 COS-7 细胞孵育,洗脱未结合的蛋白后加入碱性磷酸酶底物显色拍片,或提取细胞总蛋白,利用融合蛋白N- 末端含有的碱性磷 酸酶在底物PNPP 存在时可以发生颜色变化的特性,对与受体结合的野生型和突变型AP-Sema3 蛋白进行定量。结果：5 个错义突 变中的4 个都会不同程度地影响相应Semaphorin 3蛋白与其受体Neuropilin 的结合（与Nrp-1 的结合：SEMA3C S329G,V337M, SEMA3D H424Q,V457I,P615T 分别与野生型相比：1.12± 0.15,0.37± 0.03,0.56± 0.07,0.51± 0.05,0.66± 0.05;与Nrp-2 的结合： SEMA3C S329G,V337M,SEMA3D H424Q,V457I,P615T 分别与野生型相比：1.18 ± 0.09,0.37 ± 0.03,0.76 ± 0.01,0.65 ± 0.06,0.85± 0.03,n=3,单因素方差分析,差异有统计学意义),说明它们可能通过严重影响分子通路的信号转导而妨碍蛋白功能的 正常行使。结论：先天性巨结肠患者携带的基因错义突变可不同程度影响蛋白与其受体的结合,提示 Semaphorin 3这类经典的神经元轴突导向因子在功能失常的情况下可能参与先天性巨结肠的发生。     

广西一脊髓小脑共济失调3型家系SCA3/MJD基因突变和多态性的分析

常染色体显性脊髓小脑型共济失调(Autosomal dominant spinocerebellar ataxias, ADCAs)是一种神经系统退行性疾病, 具有高度的遗传异质性, 其中脊髓小脑型共济失调3型(Spinocerebellar ataxias type 3, SCA3)是一种常见的类型。文章通过PCR扩增广西一个脊髓小脑共济失调家系SCA3/MJD基因片段, 用毛细管电泳和测序方法检测了SCA3/MJD基因的CAG重复序列大小、传递特点以及SCA3/MJD基因的变异。结果显示：家系的所有4名患者和3名无症状携带者(Asymptomatic carrier)的SCA3/MJD基因第10外显子中存在异常扩增的CAG重复序列, 重复次数为64~71次; CAG重复次数在具有cgg等位基因的正常个体间传递时保持不变, 提示cgg等位基因不是正常个体两代间CAG重复序列稳定性的影响因素。SCA3/MJD基因中另有两个单碱基点突变, 一个是内含子区的杂合性突变(IVS9-113 T>C), 另一个是外显子区域的错义突变(220 G>A, 220 Glu>Gly)。这两个点突变为首次报道, 但尚不能明确这两个新的点突变对SCA3表型的影响。     

Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3''-phosphotransferase-II

Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.     

Spontaneous mutations in bacteria: chance or necessity?

Several investigators have recently reported that significant numbers ofappropriately adapted mutants can be induced in bacterial and yeast strains by exposing stationary phase cells to specific environmental challenges. The resulting mutants are said to be both selection-induced and demonstrably non-random in origin; if this interpretation is correct, it is in direct conflict with the conventional neo-Darwinian view, which is that spontaneous mutants are truly random in origin and arise without the intervention of any overtly adaptive forces. We believe that there are alternative ways of accounting for the appearance of many (and probably all) of the additional mutants which proponents of the adaptive mutation theory claim are observed only after they applied the appropriate selective pressure. Having reviewed the available evidence, we consider that most (if not all) of the sorts of mutants which are said to have been induced following exposure of stationary-phase cells to intense selective pressure are equally likely to have been generated during the operation of certain well-known, conventional (and essentially random) cellular DNA repair processes. Evidence in support of our view can be found in the mainstream literature on the origins of spontaneous mutations. We also note that some of the molecular models which have recently been proposed to explain the production of selection-induced mutations preferentially (or even only) in genes of adaptive significance may turn out to be of considerable interest in their own right, even although the mutants whose origins they were intended to explain may turn out to have arisen in a manner which is totally independent of the conditions used for their selection.     

NMR structures of fusion peptide from influenza hemagglutinin H3 subtype and its mutants

The influenza fusion peptide located at the N‐terminus of the hemagglutinin HA2 subunit initiates the fusing process of the viral membrane with the host cell endosomal membrane. It had been reported that the structure of a 20‐residue H3 subtype fusion peptide (H3‐HAfp20) was significantly different with that of a H1 subtype 23‐residue one (H1‐HAfp23). The sequential difference between the 12th and 15th residues of H1 and H3 subtypes could not fully explain the conformational variation. The first and last three amino acids of H3‐HAfp23 involved in formation of hydrogen bonds may play an important role in fusion process. To confirm this hypothesis, we investigate the structures of H3‐HAfp23 peptide and its mutants, G1S and G1V, in dodecylphosphatidyl choline micelles by using heteronuclear NMR technology. The results demonstrate that, similar to H1‐HAfp23 but significantly different with H3‐HAfp20, H3‐HAfp23 also has tight helical hairpin structure with the N‐ and C‐terminuses linked together because of the hydrogen bonds between Gly¹ and the last three amino acids, Trp²¹―Tyr²²―Gly²³. Although the ‘hemifusion’ G1S and lethal G1V mutants have hairpin‐like helical structures, the distances between the N‐ and C‐terminuses are increased as shortage of the hydrogen bonds and the larger kink angle between the antiparallel helices. The paramagnetic ion titration experiments show that the terminuses are inserted into the dodecylphosphatidyl choline micelles used as solving media. These may imply that the tight helical hairpin structure, especially the closed conformation at terminus, plays an important role in fusion activity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Haploinsufficiency of TBX3 causes ulnar-mammary syndrome in a large Turkish family

We present a large Turkish family with autosomal dominant inherited ulnar-mammary syndrome in which 10 affected family members, spanning three generations, were diagnosed. The phenotypic expression of the disease was found to be highly variable among the affected family members showing posterior-limb deficiencies and/or duplications, mammary-gland hypoplasia, apocrine dysfunction, dental and genital abnormalities. Mutation analysis of the TBX3 gene showed a novel one base-pair insertion at position 89 (designated 88_89insA) in the coding region. The mutation leads to a shift of the open reading frame and causes a premature truncation of the protein (M30fsX110). The truncated protein lacks almost all functional important parts of TBX3, most likely leading to a complete loss of functional protein. Our findings indicate that ulnar-mammary syndrome shows a wide range of phenotypes even within the same family and provide further evidence that haploinsufficiency of TBX3 is the disease-causing mechanism.     

E3泛素连接酶接头蛋白Keap1的研究进展

Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用.蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号而被利用.野生型Keap1能够识别、结合多种底物...     

mhbR和mhbT基因移码突变对3-羟基苯甲酸代谢的影响

Klebsiella pneumoniae M5a1菌株能以3-羟基苯甲酸作为唯一碳源和能源生长,编码分解代谢3-羟基苯甲酸的基因被克隆至-8kb的DNA片段上(pBSI质粒),含有该质粒的大肠杆菌获得了以3-羟基苯甲酸作为唯一碳源和能源生长的能力。经测序并与GenBank中的已知基因产物进行氨基酸同源性比较后,发现其中的mhbR与转录调节基因和mhbT与底物转运基因具有较高同源性,推断可能具有相应的功能。含有mhbR的移码突变子或mhbT的移码突变子的大肠杆菌在底物3-羟基苯甲酸中培养时,其生长速度和代谢速度都较野生型明显缓慢。     

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus,Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1 ⁿ) was shown to segregate as a Mendelian unit alternative to the Pgm-1 ^a and Pgm-1 ^b alleles. Mice expressing the Pgm-1 ⁿ allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1 ⁿ mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.Financial support for this work was provided in part by Contract #263-78-C-0393 from the National Institute of Environmental Health Sciences to the Research Triangle Institute.     

COL4A3 mutations cause focal segmenta glomerulosclerosis

Focal segmental glomerulosclerosis （FSGS） is a histologically identifiable gtomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Atport＇s syndrome （ARAS） patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five （12.5%） FSGS families and one （2%） sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane （GBM） was only detected in the patient with electronic microscopy examination results available. Five ARAS patients （55.6%） had homozygous or compound-heterozygous mutations in COL4.43 or COL4A4. Serious changes in the G BM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new sub- group of FSGS patients resulting from heterozygous C01.4A3 mutations was identified. The mutations are relatively frequent in famiUes diagnosed with inherited forms of FSGS. Thus, we suggest screening for C01.4A3 mutations in familial FSGS patients.     

C~3:Consensus Cancer Driver Gene Caller

Next-generation sequencing has allowed identification of millions of somatic mutations in human cancer cells.A key challenge in interpreting cancer genomes is to distinguish drivers of cancer development among available genetic mutations.To address this issue,we present the first webbased application,consensus cancer driver gene caller(C~3),to identify the consensus driver genes using six different complementary strategies,i.e.,frequency-based,machine learning-based,functional bias-based,clustering-based,statistics model-based,and network-based strategies.This application allows users to specify customized operations when calling driver genes,and provides solid statistical evaluations and interpretable visualizations on the integration results.C~3 is implemented in Python and is freely available for public use at http://drivergene.rwebox.com/c~3.     

NOTCH3基因c.520T>G突变致CADASIL一家系报告及文献复习

目的:研究探讨一CADASIL家系的临床特征及基因突变情况。方法:收集同一家系中3例CADASIL患者的临床资料,并对3例患者及先证者之兄进行全外显子测序(Whole Exome Sequencing, WES)。结果:该家系中3例患者临床表现多样,女性患者均有头痛病史,先证者及先证者之姐中年起病,先证者临床表现缺乏特异性,主要表现为头昏,认知功能检查正常,心理评估示轻度焦虑抑郁状态。先证者之姐主要表现为假性球麻痹及锥体束受损,认知功能检查示重度痴呆。先证者之女自4岁起诊断为癫痫-失神发作,认知功能检查示轻度认知功能障碍。影像学显示该家系3例患者均有脑白质病变,且随着年龄增大呈进行性发展,WES显示3例患者均存在NOTCH3基因第4外显子区域杂合突变:c.520T>G,导致氨基酸改变p.Cys174Gly。结论:NOTCH3基因c.520T>G所致该家系的临床表现具有多样性,且该家系中下一代起病较早,临床表现可与父代具有较大异质性,影像学表现可在青少年时期出现,并呈现进行加重的趋势。WES显示该家系中NOTCH3基因突变为第4外显子的杂合突变,该位点突变致CADASIL为国内首次报道。     

A resolution of the mutation load paradox in humans

Current information on the rate of mutation and the fraction of sites in the genome that are subject to selection suggests that each human has received, on average, at least two new harmful mutations from its parents. These mutations were subsequently removed by natural selection through reduced survival or fertility. It has been argued that the mutation load, the proportional reduction in population mean fitness relative to the fitness of an idealized mutation-free individual, allows a theoretical prediction of the proportion of individuals in the population that fail to reproduce as a consequence of these harmful mutations. Application of this theory to humans implies that at least 88% of individuals should fail to reproduce and that each female would need to have more than 16 offspring to maintain population size. This prediction is clearly at odds with the low reproductive excess of human populations. Here, we derive expressions for the fraction of individuals that fail to reproduce as a consequence of recurrent deleterious mutation () for a model in which selection occurs via differences in relative fitness, such as would occur through competition between individuals. We show that is much smaller than the value predicted by comparing fitness to that of a mutation-free genotype. Under the relative fitness model, we show that depends jointly on U and the selective effects of new deleterious mutations and that a species could tolerate 10's or even 100's of new deleterious mutations per genome each generation.     

Smith—Fineman—Myers综合征与GRIA3基因的连锁和突变分析

探讨GRIA3基因与中国山东Smith-Fineman-Myers综合征的连锁关系,并分析SFMS患者GRIA3基因突变。采用PCR结合变性聚丙烯酰胺凝胶电泳方法,分析GRIA3基因内多态位点与致病基因之间的连锁关系。采用PCR扩增结合PCR产物直接测序方法检测GRIA3基因开放性阅读框架区域基因突变。GRIA3基因内多态位点分析表明,GRIA3基因与中国山东SFMS家系致病基因紧密连锁,但在该基因开放性阅读框架区域内并未检测到导致疾病的基因突变。中国山东SFMS家系患者不是由于GRIA3基因编码区域基因突变所致。