Gene amplification of c-myc and N-myc in small cell carcinoma of the lung

The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.     

Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage

A domain of DNA designated N-myc is amplified 20- to 140-fold in human neuroblastoma cell lines but not in cell lines from other tumor types. N-myc has now been found to be amplified in neuroblastoma tissue from 24 of 63 untreated patients (38 percent). The extent of amplification appears to be bimodal, with amplification of 100- to 300-fold in 12 cases and 3- to 10-fold in 10 others. Amplification was found in 0 of 15 patients with stage 1 or 2 disease, whereas 24 of 48 cases (50 percent) with stage 3 or 4 had evidence of N-myc amplification. These data indicate that N-myc amplification is a common event in untreated human neuroblastomas. Furthermore, N-myc amplification is highly correlated with advanced stages of disease (P less than 0.001) and with the ability to grow in vitro as an established cell line, both of which are associated with a poor prognosis.     

Identification and characterization of the protein encoded by the human N-myc oncogene

The human N-myc gene is related to the c-myc proto-oncogene, and has been shown to have transforming potential in vitro. Many studies have reported amplification of N-myc in human neuroblastoma and retinoblastoma cell lines. In primary tumors, amplification of the gene was found to correlate directly with behavior of the tumor. Specific restriction fragments of a partial complementary DNA clone of N-myc from LA-N-5 human neuroblastoma cells were placed into a bacterial expression vector for the purpose of producing antigens representative of the N-myc protein. Rabbits immunized with these antigens produced antisera that recognized a protein of 62-64 kilodaltons in neuroblastoma cells. By several criteria, this protein appears to be part of the same proto-oncogene family as the c-myc protein. Moreover, the antisera to fragments of this protein were capable of histochemically identifying malignant cells in clinical specimens.     

长期继代培养的转基因苹果外源基因遗传及表达稳定性分析

为研究长期继代培养的转基因苹果组培苗中外源基因的遗传及表达稳定性，以继代培养9年的7个转GFP(绿色荧光蛋白)基因苹果株系组培苗为试材，分析转基因株系对Kan(卡那霉素)的抗性、DNA水平、转录水平和转录后水平GFP基因的遗传及表达稳定性。结果表明，在7个苹果转基因株系组培苗中均可检测出GFP特异基因片段，并且均可在含有50 mg/L卡那霉素的培养基上正常生长；绝对定量qRT-PCR检测发现，各转化株系GFP基因拷贝数并不相同；利用相对定量qRT-PCR法检测发现7个株系中GFP基因 mRNA表达量有明显差异，同时荧光显微镜下观察各转化株系叶片，绿色荧光强度有明显差异，利用SPSS软件对7个转基因株系中GFP基因拷贝数与GFP mRNA表达量相关性分析结果呈无显著相关性。以上结果表明，外源基因可以在长期继代培养的转基因苹果组培苗中保持其遗传稳定性，但不同转化株系中外源基因的表达量有显著差异，其表达量与拷贝数无显著相关，可能与外源基因在植物基因组中的插入位点有关。     

Human lineage-specific amplification, selection, and neuronal expression of DUF1220 domains

Extreme gene duplication is a major source of evolutionary novelty. A genome-wide survey of gene copy number variation among human and great ape lineages revealed that the most striking human lineage-specific amplification was due to an unknown gene, MGC8902, which is predicted to encode multiple copies of a protein domain of unknown function (DUF1220). Sequences encoding these domains are virtually all primate-specific, show signs of positive selection, and are increasingly amplified generally as a function of a species' evolutionary proximity to humans, where the greatest number of copies (212) is found. DUF1220 domains are highly expressed in brain regions associated with higher cognitive function, and in brain show neuron-specific expression preferentially in cell bodies and dendrites.     

SYBR Green实时定量PCR检测转基因大豆中外源基因拷贝数

采用SYBR Green I real-time PCR方法检测转基因大豆中外源基因35S边界基因的拷贝数,以大豆凝集素基因(Lectin)作为内参照基因,以转基因大豆基因组DNA为内参照基因标准品,初始浓度为0.43μg·μL-1,进行5倍梯度稀释得到内参照基因CT值与起始模板量的相关性标准曲线:y=-2.9915x...     

Site-specific integration of H-ras in transformed rat embryo cells

A karyotypic analysis was performed on seven independently derived clones of primary rat embryo cells transformed by the ras oncogene plus the cooperating oncogene myc. The transfected oncogenes were sometimes present in amplified copy number, with heterogeneity in the levels of amplification. Some chromosomal features, such as aberrantly banding regions and double-minute chromosomes, typical of cells carrying amplified genes, were also seen in three of the seven cell lines. Underlying this heterogeneity there was an unexpected finding. All seven lines showed a common integration site for ras on the q arm of rat chromosome 3 (3q12), though some lines also had other sites of integration. In four of the lines integration of ras was accompanied by deletion of the p arm of chromosome 3 or its possible translocation to chromosome 12.     

利用PTVA法提高超氧化物歧化酶基因在毕赤酵母中的表达

为得到高表达超氧化物歧化酶毕赤酵母工程菌株,促进SOD的产业化生产,将来源于蜡样芽孢杆菌M22菌株的Mn-SOD-2基因转入毕赤酵母GS115中,构建了表达工程菌株YS 1～100。采用PTVA法(Posttransformational vector amplification)进行拷贝数扩增处理。建立了SOD重组菌株MMH(Minimal Methanol+Histidine)平板简易检测法,使用该法对转化子进行初筛,并用荧光定量PCR(Fluorescence quantitative PCR)法检测部分重组酵母工程菌中外源SOD基因的拷贝数。结果表明:试验中得到80株高抗Zeocin的菌株YSP 1～80,经MMH平板法筛选得到菌株YSP14、YSP30、YSP48和YSP54的SOD酶活性明显高于处理前;菌株YSP54的SOD基因的拷贝数达到8个拷贝,而处理前仅为1个拷贝;Native-Page分析与NBT酶活性测定显示菌株YSP54发酵上清液中的SOD酶活性达到150U/mL,为处理前的2倍。提高毕赤酵母中外源SOD基因的拷贝数可以相应提高SOD的酶活。     

A general method for the chromosomal amplification of genes in yeast

The yeast retrotransposon Ty can be used to insert multiple copies of a gene at new sites in the genome. The gene of interest is inserted into a GALI-Ty fusion construct; the entire "amplification cassette" is then introduced into yeast on a high copy number plasmid vector. Transposition of the Ty element carrying the gene occurs at multiple sites in the genome. Two genes, a bacterial neomycin phosphotransferase gene and the yeast TRPl gene, were amplified in this way. Although the amplified genes were about 1 kilobase in length, they were amplified to about the same extent as a 40-base pair segment. The benefit of this "shotgun" approach is that amplification can be achieved in one set of manipulations.     

鸭PRLR基因拷贝数的变异

本研究以台湾褐色菜鸭为试验动物,采用荧光定量PCR技术,使用2-ΔΔCt换算法对PRLR基因拷贝数进行定量,分析了其多态性与台湾褐色菜鸭生产性能的相关性.结果表明,PRLR和Ldh-B标准曲线的R2分别为0.991和0.990,扩增效率分别为111.68%和108.429%,斜率分别为-3.070和-3.135,PRLR和Ldh-B的扩增效率近似一致,可通过2-ΔΔCt方法对PRLR基因进行定量分析.在94羽台湾褐色菜鸭的样品中共检测到5种拷贝数(1、2、3、4、5)变异类型,拷贝数变异与台湾褐色菜鸭的蛋壳厚度和蛋形指数显著相关(P0.05),拷贝数为2的个体蛋壳厚度显著高于拷贝数为1和5的个体;拷贝数为3的个体蛋形指数显著低于拷贝数为1、2和4的个体;其余性状均无显著相关(P0.05).因此,PRLR基因拷贝变异区域可能影响蛋壳厚度和蛋形指数.     

马铃薯T-DNA插入拷贝数的检测及对农艺性状的影响研究

以转基因马铃薯株系为材料,采用Real-time PCR方法,以SYBR Green I为荧光染料,以马铃薯块茎贮藏蛋白基因(Patatin)作为内参基因,以植物表达载体上的潮霉素抗性基因(HPT)为筛选标记基因,建立目的基因CT值与起始模版的相关性标准曲线,通过实时荧光定量PCR分别获得每个样品中内参基因和外源基因的CT值,根据Pfaffl法计算获得了T-DNA在转基因马铃薯中的拷贝数,且与Southern blot方法进行了比较,并结合田间农艺性状,分析了外源基因拷贝数马铃薯株高及薯块产量的影响。结果表明,内参基因和外源基因标准曲线的相关系数分别为R2=0.996、R2=0.995和R2=0.990。在所测的23株转基因株系中,12株为单拷贝插入,6株为双拷贝,5株为多拷贝。Real-time PCR比Southern blot方法结果更准确、操作更简便快捷、成本更低。且插入拷贝数的多少与马铃薯田间农艺性状变化有一定的关系,发现2拷贝以上的T-DNA插入较容易引起部分性状的改变。     

Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.     

采用优化的数字PCR方法分析转基因小麦外源基因拷贝数

【目的】 探索基于数字PCR的小麦基因拷贝数分析技术,提高目标基因拷贝数的分析通量,促进小麦基因组研究及基因工程研究。【方法】 采用中国农业科学院作物科学研究所小麦抗逆分子育种课题组创制的高抗小麦黄花叶病转nib8小麦为试验材料,使用小麦D基因组中的单拷贝纯合内源基因PINb-D1b（籽粒硬度基因）为内参基因,根据转化的抗病基因nib8序列设计4对特异性引物及相应探针,通过试验确定探针和引物的最优浓度,优化体系的退火温度,寻找最合适的模板浓度。然后用数字PCR检测转nib8小麦中外源基因的拷贝数;同时用Real-time PCR和Southern blot 2种不同方法分析的拷贝数结果,验证上述采用数字PCR方法分析拷贝数的准确性;以PINb-D1b内参基因检测Wx012（蜡质基因）和SSII（淀粉合成基因）2个内参基因的拷贝数,以Wx012和SSII为内参基因检测转nib8小麦中外源基因的拷贝数,验证以PINb-D1b内参基因的检测结果准确性;在nib8的不同区段设计特异性引物来检测转nib8株系拷贝数分析结果是否有差异。最终,建立基于数字PCR技术的高通量检测小麦目标基因拷贝数的分析方法。【结果】 通过试验最终确定了基于数字PCR方法的小麦目标基因拷贝数的检测体系。试验确定引物和探针的最佳终浓度分别为500和250 nmol·L^-1,确定体系反应最佳退火温度为59℃,最佳DNA模板量为40 ng。以PINb-D1b作为内参基因检测转nib8小麦中nib8的拷贝数,拷贝数检测结果显示第12、16、17、23、29、30株系中nib8拷贝数分别为7个、1个、1个、1个、1个、7个;同时通过比较发现此结果与Real-time PCR以及Southern blot结果一致;在转基因株系中,以PINb-D1b为内参基因,对Wx012和SSII 2个基因进行拷贝数进行分析,同时发现采用这3种内参基因分析转nib8小麦中nib8基因拷贝数的结果一致,说明这3个内参基因都适合用于数字PCR方法;在nib8上游、中游和下游不同区段的设计引物分析拷贝数检测结果一致。【结论】 优化了基于数字PCR方法的小麦目标基因拷贝数分析方法和反应体系,确立了基于数字PCR方法的小麦目标基因拷贝数的检测体系,数字PCR分析结果稳定、可靠,检测通量明显提高,具有一定的应用前景。     

南方根结线虫诱导辣椒基因CaRKNIF2的克隆与分析

 【目的】明确辣椒(Capsicum annuum L. HDA149)中与南方根结线虫(Meloidogyne incognita)不亲和互作过程中抗性相关WRKY转录因子的基因结构及其表达模式。【方法】采用RACE结合RT-PCR方法克隆cDNA序列,并根据cDNA序列设计特异引物扩增DNA序列,Southern杂交确定基因拷贝数,实时荧光定量PCR方法分析该基因在线虫侵染后辣椒的不同组织和不同时间点的表达。【结果】从辣椒中克隆到一个WRKY转录因子基因CaRKNIF2(GenBank登录号：GQ253367),该基因编码区全长1 662 bp,编码553个推定的氨基酸,为单拷贝基因。CaRKNIF2基因编码区全长DNA序列2 530 bp,包含5个内含子和6个外显子。在线虫接种诱导处理时,该基因表达具有组织特异性,根尖组织中的表达量最高;接种3 h后在根尖组织中的表达量开始升高,12 h最高,此时相对表达量约为3.1倍。【结论】CaRKNIF2在线虫接种处理后的表达上调,表明该基因参与了抗性基因Me3介导的辣椒与根结线虫的不亲和互作,可能在此过程中具有重要功能。     

亚洲璃眼蜱成蜱不同发育期miR-451与MIF表达谱的动态分析

【目的】microRNA （miRNA）作为一类内源性的非编码RNA,仅有18-25 nt,其广泛存在于动植物细胞中,可诱导生物基因沉默,参与细胞生长、发育、基因转录和翻译等诸多生命活动的调控过程。以亚洲璃眼蜱为研究对象,对miR-451及其靶基因（巨噬细胞游走因子, MIF）在相互作用关系进行分析。【方法】参考miR-451成熟体序列（AAA CCG UUA CCA UUA CUG AGU UU）来自研究单元亚洲璃眼蜱高通量测序所获得的结果。根据该成熟体序列设计stem-loop及PCR引物,RT-PCR扩增获得蜱源性miR-451序列;并对不同物种来源的miR-451序列特征进行分析。基因合成方法获得抑制miR-451的dsRNA序列,用于亚洲璃眼蜱饥饿成蜱体内注射。参考美洲钝眼蜱MIF基因（登录号：AF289543.2）,设计亚洲璃眼蜱的特异性实时荧光定量引物,以β-actin作为内参基因,采用SYBR Green real-time RT-PCR方法检测注射dsRNA后亚洲璃眼蜱饥饿成蜱不同发育时间点MIF基因的表达水平,以及miR-451的表达谱特征。【结果】琼脂糖凝胶电泳检测miR-451的PCR产物条带与预期大小一致,为72 nt。不同物种来源的miR-451具有较高的保守性,特别是种子序列极度保守,仅在短尾负鼠miR-451成熟体的19位点处存在A→U的突变。miR-451的RNA干扰实验证实,0-30 h miR-451表达逐渐上调,6 h达到最高值,其拷贝数为1.0×108。随后表达丰度逐渐降低。30 h其表达水平与PBS对照组相同。48-60 h miR-451再次出现一个表达上调微小变化过程。而miR-451抑制后的MIF直到30 h才有表达的上调,42 h达到峰值（拷贝量仅为9.0×103）,此后其表达规模受到抑制,48 h时与PBS对照组水平一致。84 h后表达才逐渐上调,直到90 h达到峰值,并呈现正态分布趋势。【结论】利用RT-PCR获得亚洲璃眼蜱成蜱阶段miR-451序列,生物信息学方法分析了miR-451序列在不同物种间具有较高保守性。miRNA的保守性决定其靶标基因的特异性,因此,以上结果提示miR-451在动物细胞中可能扮演重要的生物学功能,是MIF功能发挥的一个重要调控因子。MIF作为miR-451靶标基因,其功能的实现可能受该miRNA的调控。基于此类推测qPCR及RNA干扰分析表明,miR-451参与了MIF的表达调控。且是一种负调控作用。当miR-451表达量升高时,MIF表达量明显下调。此研究首次证实miR-451在亚洲璃眼蜱成蜱发育阶段的表达是一种普遍现象,且对MIF存在负调控作用。这一研究为后期miR-451参与蜱的免疫应答及miR-451与靶标基因的相互作用机制提供了参考。同时证明,miRNA参与基因功能的调控具有一定的特异性和时序性。     

番茄Trihelix家族SIP1亚家族基因(SlGT-33)克隆表达分析及功能研究

【目的】对番茄三螺旋（Trihelix）家族SIP1亚家族基因（SlGT-33）进行克隆表达及功能研究,为深入解析SIP1亚家族成员调控植物生长发育机制及选育矮化番茄品种提供理论依据。【方法】以番茄品种AC++为材料,PCR扩增SlGT-33基因,对其进行序列分析,采用实时荧光定量PCR（qRT-PCR）检测SlGT-33基因在不同组织及外源激素和非生物胁迫下的表达模式,并利用RNAi技术鉴定SlGT-33基因的生物学功能。【结果】扩增获得的SlGT-33基因（GenBank登录号Solyc12g043090）开放阅读框（ORF）为1125 bp,编码374个氨基酸残基,与已知同源基因序列（GenBank登录号XP004252336.1）仅缺少3个碱基。SlGT-33与SlGT-17（GenBank登录号Solyc05g018350）位于同一分支,虽然二者的氨基酸序列相似性最高,但仅为45.8%。SlGT-33基因在茎和成熟叶中的相对表达量较高,显著高于其他组织（P<0.05,下同）;经IAA、GA₃和MeJA处理后,SlGT-33基因的相对表达量均与对照无显著差异（P>0.05,下同）;SlGT-33基因经ABA处理2～24 h时相对表达量均显著高于对照。SlGT-33基因在高盐胁迫和机械损伤下的相对表达量与对照无显著差异;高温胁迫和低温胁迫下SlGT-33基因表达整体上均呈逐渐降低的变化趋势;在脱水胁迫下SlGT-33基因表达整体上均呈逐渐升高的变化趋势。通过农杆菌介导转化法获得6个SlGT-33-RNAi沉默株系,沉默效率为64%～83%。生长70 d的SlGT-33-RNAi沉默株系RNAi-4和RNAi-5幼苗株高和节间长度均为野生型的60%左右,且复叶结构尺寸明显变小,花序提前形成。茎尖组织中顶端分生组织关键转录因子基因KNOX2和WUS基因在SlGT-33-RNAi沉默株系的相对表达量均极显著（P<0.01）或显著高于野生型,腺苷酸异戊烯转移酶（CTK合成的关键酶）编码基因IPT2和茎尖生长点调控基因PHAN基因在2个SlGT-33-RNAi沉默株系的相对表达量均显著低于野生型。顶端分生组织关键转录因子基因KNOX1基因在2个SlGT-33-RNAi沉默株系的相对表达量与野生型无显著差异。SlGT-33-RNAi沉默株系茎尖组织的细胞分裂素（CTK）含量显著低于野生型。【结论】SlGT-33基因属于环境敏感型基因,其表达受ABA和脱水胁迫诱导,但受极端温度的抑制。抑制SlGT-33基因表达会导致了番茄植株矮化和生殖生长加速,其作用机制与顶端分生组织的CTK合成受抑制及茎尖组织调控基因KNOX2、PHAN和WUS的异常表达密切相关。     

转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用

[目的]探讨转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用。[方法]将PCR扩增得到的人β-珠蛋白MAR插入到真核表达载体pCATG的下游,构建转基因表达盒3’端含MAR的表达载体。酶切鉴定正确后,转染CHO细胞,G418筛选出稳定转化的细胞株,ELISA分析转基因的表达水平,半定量PCR分析转基因相对拷贝数。[结果]结果表明,表达盒3’端含MAR序列能使转基因表达水平降低,其基因拷贝数则有一定程度的增加。转基因下游β-珠蛋白MAR在一定程度上能抑制外源基因的表达水平;外源基因表达量与基因拷贝数不成正比,未呈现出“拷贝数依赖性”。[结论]该研究结果为进一步研究MAR的调控机制奠定基础。     

八倍体小黑麦体细胞耐盐变异体发生机制初步研究

对同一植株不同分蘖进行幼穗、花药培养,分别诱导双、单倍体愈伤组织。继代选出生长好的无性系用于耐盐变异体筛选,测定各无性系出现耐盐变异体的频率。计算单、双倍体无性系的变异体频率之比值（H/D）。据变异体发生机制的不同会导致单、双倍体愈伤组织出现变异体频率的不同,将几种可能机制的理论H/D值与实测H/D值比较分析,发现八倍体小黑麦体细胞耐盐变异体的发生机制是具有显性作用的基因扩增,而非严格意义上的基因显性突变。     

猪增生性肠炎黏膜免疫相关细胞数量研究

为了揭示猪增生性肠炎黏膜免疫相关细胞数量的变化情况.采用组织学方法,比较PCR检验阳性猪(获得胞内劳森氏菌特异性扩增产物)与阴性猪(无胞内劳森氏菌特异性扩增产物)的十二指肠、空肠、回肠黏膜淋巴细胞、杯状细胞、肥大细胞的数量和分布变化.结果表明,阳性猪十二指肠、空肠黏膜上皮内每100个柱状细胞间淋巴细胞的数量同比阴性猪稍...     

Individual tumors of multifocal EB virus-induced malignant lymphomas in tamarins arise from different B-cell clones

Cotton-top tamarins were inoculated with sufficient Epstein-Barr virus to induce multiple tumors in each animal within 14 to 21 days. The tumors consisted of large-cell lymphomas that contained multiple copies of the Epstein-Barr virus genome and generated Epstein-Barr virus-carrying cell lines showing no detectable consistent chromosomal abnormality. Hybridization of tumor DNA with immunoglobulin gene probes revealed that each lymphoma was oligo- or monoclonal in origin and that individual tumors from the same animal arose from different B-cell clones. Thus the virus induced multiple transformation events in tamarins in vivo to cause malignant tumors resembling the Epstein-Barr virus-associated lymphomas of patients with organ transplants.