转基因小鼠和基因突变小鼠微卫星不稳定性分析

目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。     

高频度微卫星不稳定性大肠癌"修饰型"微卫星变化与MLH1及KRAS基因突变密切相关

本研究通过方法学的改良和观察方式的创新试图阐明这种现象的原因.微卫星非传统的检测方法仅能实现微卫星定性检测,我所在的研究组开发了自动片段分析双荧光标识技术,提高了微卫星检测的感度和重复性,并实现了微卫星片段变化长度的定量.小于6碱基的微卫星变化被定义为修饰型微卫星不稳定,大于8碱基的变化被定义为跳跃型微卫星不稳定,它们的电泳谱截然不同.前者表现为在非肿瘤来源微卫星位点基础上的增加或减少,后者表现为距离非肿瘤微卫星片段远隔部位的新波形的出现.通过研究我们发现,在DNA错配修复缺陷细胞系及基因敲除大鼠自发肿瘤样本,仅有修饰型微卫星不稳定性检出;在人类DNA错配修复缺陷细胞系连续80次传代也没有检出跳跃型变化.跳跃型变化不能通过简单重复序列不稳定基础上的增加或减少的累加而获得.在76例散发大肠癌,我们检测了微卫星不稳定性,KRAS基因突变,并对高频度微卫星不稳定性病例的两个主要DNA错配修复基因MSH2和MLHl进行了全长测序.我们发现,在大肠癌,按频度的传统分类与按波形变化的分类有高度的一致性,高频度微卫星不稳定性病例均检测到跳跃型表现,低频度微卫星不稳定性都表现为修饰型变化.在12例高频度微卫星不稳定病例,有三例检出了跳跃型和修饰型同时存在微卫星不稳定的特殊表型,这3例均检出KRAS的突变,更有趣的是该3例病例也同时检出了DNA错配修复基因MLH1的变异.而在其他9例高频度微卫星不稳定病例,KRAS突变及MLH1、MSH2交变未检出.通过对突变谱的分析我们还发现,修饰型微卫星不稳定与KTAS基因12号密码子的转换型突变高度相关,而微卫星稳定的病例检出的KRAS基因12号密码子突变多为颠换型突变.修饰型微卫星不稳定表型检出的高频度转换突变可由DNA错配修复缺陷的分子背景解释.通过本研究,我们认为以波形为基础的微卫星不稳定新分型可能是解决目前微卫星研究领域矛盾的一个选项.一直公认为高频度微卫星不稳定性是"真正"的DNA错配修复缺陷表型,我们的研究提示实际上高频度微卫星的可能是多元的.修饰型微卫星不稳定与DNA错配修复缺陷直接关联,而跳跃型微卫星不稳定的原因尚未阐明.在高频度为微型不稳定中,携带修饰型变化的病例可以通过DNA错配修复系统缺陷来解释其病因.     

高频度微卫星不稳定性大肠癌“修饰型”微卫星变化与MLH1及KRAS基因突变密切相关

本研究通过方法学的改良和观察方式的创新试图阐明这种现象的原因。微卫星非传统的检测方法仅能实现微卫星定性检测,我所在的研究组开发了自动片段分析双荧光标识技术,提高了微卫星检测的感度和重复性。并实现了微卫星片段变化长度的定量。小于6碱基的微卫星变化被定义为修饰型微卫星不稳定,大于8碱基的变化被定义为跳跃型微卫星不稳定．它们的电泳谱截然不同。前者表现为在非肿瘤来源微卫星位点基础上的增加或减少,后者表现为距离非肿瘤微卫星片段远隔部位的新波形的出现。通过研究我们发现,在DNA错配修复缺陷细胞系及基因敲除大鼠自发肿瘤样本,仅有修饰型微卫星不稳定性检出;在人类DNA错配修复缺陷细胞系连续80次传代也没有检出跳跃型变化。跳跃型变化不能通过简单重复序列不稳定基础上的增加或减少的累加而获得。在76例散发大肠癌,我们检测了微卫星不稳定性,KRAS基因突变,并对高频度微卫星不稳定性病例的两个主要DNA错配修复基因MSH2和MLH1进行了全长测序。我们发现,在大肠癌,按频度的传统分类与按波形变化的分类有高度的一致性,高频度微卫星不稳定性病例均检测到跳跃型表现,低频度微卫星不稳定性都表现为修饰型变化。在12例高频度微卫星不稳定病例,有三例检出了跳跃型和修饰型同时存在微卫星不稳定的特殊表型,这3例均检出KRAS的突变,更有趣的是该3例病例也同时检出了DNA错配修复基因MLH1的变异。而在其他9例高频度微卫星不稳定病例,KRAS突变及MLH1、MSH2突变未检出。通过对突变谱的分析我们还发现,修饰型微卫星不稳定与KRAS基因12号密码子的转换型突变高度相关,而微卫星稳定的病例检出的KRAS基因12号密码子突变多为颠换型突变。修饰型微卫星不稳定表型检出的高频度转换?     

SLAⅠ类基因研究新进展

SLAⅠ类基因编码细胞表面高度多态的糖蛋白, 负责将内源性抗原肽递呈给T细胞, 激发特异的免疫反应。文章介绍了近年来SLAⅠ类基因分子结构、组织中的表达、表达调节、基因分型、多态性及进化研究的新进展, 重点介绍了SLAⅠ类基因分型方法及多态性研究。SLAⅠ类基因分型方法主要包括血清学法、DNA序列测定、PCR-SSP、PCR-SSOP、微卫星法, 其中PCR-SSP法快速简便, 在SLAⅠ类基因分型研究中应用广泛。文章对SLAⅠ类基因在基因功能、肽疫苗、异种移植等方面的研究和应用前景进行了讨论。     

不同猪种肠毒素大肠杆菌（ETEC）F4受体的微卫星标记遗传效应分析

从猪13号染色体选取与E.coli F4受体基因连锁的4个微卫星座位研究中外猪种间的遗传差异性,并分析不同基因型与F4受体黏附表型的关系。结果表明,4个猪种在4个基因座均具有高度多态性,杂合度（H）在0.6117～0.7500之间,多态信息含量（PIC）达0.5749以上;同时中外猪种基因频率存在差异,微卫星座位连锁越紧密,差异性越大。微卫星S0222不同基因型间在沙子岭猪F4ab血清型黏附表型中差异显著;SW458座位AC基因型在沙子岭、大白两个品种中无黏附表型,可望作为对E.coliF4抗性基因的遗传标记。     

新疆维吾尔族和哈萨克族人群3个微卫星座位分析

采用基因扫描技术研究了新疆维吾尔族和哈萨克族群体D16S539、D13S317、D7S8203个微卫星位点上的等位基因及基因型分布情况,获得3个基因座的群体遗传学数据。结果表明3个基因位点在两民族中均具有遗传多态性,两民族样本在这3个微卫星位点基因频率分布大致相同。     

小麦农家品种红麦(苏1661)中一个主效抗条锈病基因的微卫星标记定位

应用分离体分组混合分析法（bulked segregant analysis,BSA）和微卫星标记多态性分析方法,对红麦（保存单位编号：苏1661;统一编号：ZM008712）中的一个主效抗条锈病基因YrHm进行了分子标记和定位研究。共用512对微卫星引物对抗、感基因池进行了多态性分析,经用包括230个单株的F2分离群体进行遗传连锁性检测,发现4个与YrHm基因连锁的微卫星标记Xgwm904、Xbarcl73、Xcfdl3和Xcfd42,均位于小麦染色体6D短臂上。经Mapmaker3．0b软件计算,这4个标记与目的基因间的遗传距离分别为7．3、25．1、47．7和62．1cM,均位于YrHm基因远离染色体顶端的一侧。用全套中国春小麦缺体一四体材料进行检测,进一步确认了这4个标记均位于小麦6D染色体。因此,将YrHm基因定位于小麦染色体臂6DS上。     

hMLH1基因启动子CpG岛甲基化致MSI与肿瘤的关系

通过人类错配修复基因( hMLHl)启动子CpG岛甲基化与微卫星不稳定性(MSI)的分析,探讨癌症发病的机制.错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制,而hMLH1的表达失活则可导致MSI的产生,促进癌症的发生.根据一系列研究得出结论,在肿瘤组织中hMLH1基因启动子CpG岛甲基化和微卫星不稳定(MSI)有显著相关性,并在癌症早期发生、发展过程中起重要作用.因此临床检测hMLH1基因启动子CpG岛甲基化及微卫星不稳定可能成为癌症鉴别诊断、评价预后、指导化疗的分子标志物之一.     

CTLA-4基因微卫星多态性与粤西汉族人Graves病的相关性研究

目的:探讨细胞毒性T淋巴细胞相关抗原-4(cytotoxie T lymphocyte associated antigen-4,CTLA-4)基因微卫星多态性[CTLA-4(AT)n]与湛江沿海地区汉族人Graves病(GD)的相关性.方法:收取2006年门诊及住院患者中确诊为Graves病(Craves' disease,GD)102例和正常对照组100例,应用聚合酶链式反应-单链长度多态性分析法(PCR-SSLP)确定CTLA-4基因第4外显子3'末端AT重复序列的基因型.结果:CTLA-4基因3'末端微卫星位点有20个等位基因,GD组与正常对照组之间之间的基因频率差异无统计学意义(P>0.05).结论:CTLA-4(AT)n多态性可能与GD无相关性.     

使用林鹳微卫星引物对东方白鹳基因组DNA进行交叉扩增

利用林鹳11个微卫星位点的引物对东方白鹳进行交叉扩增。经过PCR体系的优化,在11个位点中有6个得到清晰的扩增条带,其余5位点得不到确切的扩增产物。对上述6个位点的扩增产物进行克隆测序分析,发现其中4个位点上的扩增产物含有微卫星重复序列,而另外两个位点中无重复单元。通过基因分型对上述4个微卫星位点进行多态性分析后发现其中的WSμ13,WSμ17位点分别为高度多态和中度多态位点,而另外两个位点则无多态性。同时还对影响交叉扩增结果成功率及微卫星位点多态性的因素进行了分析和总结。     

Mutator Mutations Enhance Tumorigenic Efficiency across Fitness Landscapes

Background

Tumorigenesis requires multiple genetic changes. Mutator mutations are mutations that increase genomic instability, and according to the mutator hypothesis, accelerate tumorigenesis by facilitating oncogenic mutations. Alternatively, repeated lineage selection and expansion without increased mutation frequency may explain observed cancer incidence. Mutator lineages also risk increased deleterious mutations, leading to extinction, thus providing another counterargument to the mutator hypothesis. Both selection and extinction involve changes in lineage fitness, which may be represented as “trajectories” through a “fitness landscape” defined by genetics and environment.

Methodology/Principal Findings

Here I systematically analyze the relative efficiency of tumorigenesis with and without mutator mutations by evaluating archetypal fitness trajectories using deterministic and stochastic mathematical models. I hypothesize that tumorigenic mechanisms occur clinically in proportion to their relative efficiency. This work quantifies the relative importance of mutator pathways as a function of experimentally measurable parameters, demonstrating that mutator pathways generally enhance efficiency of tumorigenesis. An optimal mutation rate for tumor evolution is derived, and shown to differ from that for species evolution.

Conclusions/Significance

The models address the major counterarguments to the mutator hypothesis, confirming that mutator mechanisms are generally more efficient routes to tumorigenesis than non-mutator mechanisms. Mutator mutations are more likely to occur early, and to occur when more oncogenic mutations are required to create a tumor. Mutator mutations likely occur in a minority of premalignant lesions, but these mutator premalignant lesions are disproportionately likely to develop into malignant tumors. Tumor heterogeneity due to mutator mutations may contribute to therapeutic resistance, and the degree of heterogeneity of tumors may need to be considered when therapeutic strategies are devised. The model explains and predicts important biological observations in bacterial and mouse systems, as well as clinical observations.     

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.     

Pro-tumorigenic function of autophagy in mammary oncogenesis

Autophagy is a highly conserved catabolic cellular process by which cells degrade intracellular constituents in lysosomes, and its dysfunctions have been associated with a variety of human diseases including cancer. Previous studies have linked autophagy to both tumor-suppressive and promoting functions in different contexts, although the pro-tumorigenic function of autophagy has not been examined directly in breast or other cancers in animal models with intact immune functions in vivo. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a component of the ULK1-Atg13-FIP200-Atg101 complex that is essential for the induction of mammalian autophagy. In our recent study, we show that conditional knockout (KO) of FIP200 in the well-characterized MMTV-PyMT mouse model of human breast cancer significantly suppresses mammary tumorigenesis and progression. Similar to a number of recent studies in Ras-transformed cells, our studies revealed the importance of autophagy in promoting tumorigenesis through regulation of tumor cell glycolysis and proliferation. In addition to the intrinsic defects in proliferation of FIP200-null tumor cells, we also showed that FIP200 deletion in mammary tumor cells triggers increased host anti-tumor immune surveillance, which also contributes to the decreased mammary tumorigenesis and progression. Our study provides the first direct demonstration of a pro-tumorigenic role of autophagy in oncogene-driven tumor models with intact immune functions in vivo. They also suggest FIP200 and other autophagy proteins as potential therapeutic targets for cancer treatment, and raise a number of questions for future studies on the potentially dual functions of autophagy in promoting and suppressing tumorigenesis under different conditions in vivo.     

Sequencing,identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals

Background

There are over a half a million copies of L1 retroelements in the human genome which are responsible for as much as 0.5% of new human genetic diseases. Most new L1 inserts arise from young source elements that are polymorphic in the human genome. Highly active polymorphic “hot” L1 source elements have been shown to be capable of extremely high levels of mobilization and result in numerous instances of disease. Additionally, hot polymorphic L1s have been described to be highly active within numerous cancer genomes. These hot L1s result in mutagenesis by insertion of new L1 copies elsewhere in the genome, but also have been shown to generate additional full length L1 insertions which are also hot and able to further retrotranspose. Through this mechanism, hot L1s may amplify within a tumor and result in a continued cycle of mutagenesis.

Results and conclusions

We have developed a method to detect full-length, polymorphic L1 elements using a targeted next generation sequencing approach, Sequencing Identification and Mapping of Primed L1 Elements (SIMPLE). SIMPLE has 94% sensitivity and detects nearly all full-length L1 elements in a genome. SIMPLE will allow researchers to identify hot mutagenic full-length L1s as potential drivers of genome instability. Using SIMPLE we find that the typical individual has approximately 100 non-reference, polymorphic L1 elements in their genome. These elements are at relatively low population frequencies relative to previously identified polymorphic L1 elements and demonstrate the tremendous diversity in potentially active L1 elements in the human population.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1374-y) contains supplementary material, which is available to authorized users.     

Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci.

The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.     

How can we distinguish between mutational "hot spots" and "old sites" in human mtDNA samples?

New research into variation in mutation rates across nucleotide positions in human mitochondrial DNA (mtDNA) calls into question population genetics models that assume a constant mutation rate for all sites in a sequence, particularly for hypervariable control region segments I and II. Related to this research is discovering the extent to which highly polymorphic sites are really mutational "hot spots" rather than "old" sites rooted early in the phylogenetic tree. This issue is addressed through the analysis of linkage disequilibrium patterns in the mtDNAs of 10 human populations. Hot spots can be expected to show little or no disequilibrium since they can be interpreted as having randomly expressed patterns. In fact, the results suggest that many highly polymorphic sites are not old sites, but instead are hot spots. Suspected hot spots are listed and compared with hypervariable sites given by Wakeley (1993) and Hasegawa et al. (1993).     

Mutator activity of a short Okazaki fragment mutant of Escherichia coli.

A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected. The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity.     

The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas

Background

The AID/APOBECs are deaminases that act on cytosines in a diverse set of pathways and some of them have been linked to the onset of genetic alterations in cancer. Among them, APOBEC1 is the only family member to physiologically target RNA, as the catalytic subunit in the Apolipoprotein B mRNA editing complex. APOBEC1 has been linked to cancer development in mice but its oncogenic mechanisms are not yet well understood.

Results

We analyze whether expression of APOBEC1 induces a mutator phenotype in vertebrate cells, likely through direct targeting of genomic DNA. We show its ability to increase the inactivation of a stably inserted reporter gene in a chicken cell line that lacks any other AID/APOBEC proteins, and to increase the number of imatinib-resistant clones in a human cellular model for chronic myeloid leukemia through induction of mutations in the BCR-ABL1 fusion gene. Moreover, we find the presence of an AID/APOBEC mutational signature in esophageal adenocarcinomas, a type of tumor where APOBEC1 is expressed, that mimics the one preferred by APOBEC1 in vitro.

Conclusions

Our findings suggest that the ability of APOBEC1 to trigger genetic alterations represents a major layer in its oncogenic potential. Such APOBEC1-induced mutator phenotypes could play a role in the onset of esophageal adenocarcinomas. APOBEC1 could be involved in cancer promotion at the very early stages of carcinogenesis, as it is highly expressed in Barrett''s esophagus, a condition often associated with esophageal adenocarcinoma.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0417-z) contains supplementary material, which is available to authorized users.