Adventitious changes in long-range gene expression caused by polymorphic structural variation and promoter competition

It is well established that all of the cis-acting sequences required for fully regulated human α-globin expression are contained within a region of ≈120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the α-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the α-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of α-globin genes, affects the pattern of NME4 expression by altering the competition for the shared α-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.     

白纹伊蚊基因表达定量PCR内参基因的选择

目的筛选白纹伊蚊基因表达定量PCR研究中适合的内参基因。方法采用实时荧光定量PCR技术,对β-ac-tin、BTF3a、rsp5、rsp27a、superoxide、rspL40六个看家基因的mRNA表达水平进行了探讨。结果除rsp27a基因扩增效率高于设定值被剃除外,余5个基因在不同组织中的表达稳定度为rspL40,BTF3a〉rsp5〉β-actin〉superoxide;吸血不同时相表达稳定度为rspL40,rsp5〉superoxide〉BTF3a〉β-actin。结论 rspL40,BTF3在不同组织中表达最稳定;rspL40,rsp5在吸血不同时相表达最稳定。     

人脂联素、球状脂联蛋白基因克隆及真核表达载体的构建与表达

目的构建高效表达球状脂联蛋白的真核表达载体，为研究脂联素、球状脂联蛋白在2型糖尿病及动脉粥样硬化中的作用奠定基础。方法克隆人脂联素基因，构建球状脂联蛋白基因的真核表达载体，转染人脐静脉内皮细胞株（HUVEC），Western blot检测上清中球状脂联蛋白的表达。结果测序结果表明克隆的人脂联素基因序列正确;构建的球状脂联蛋白基因真核表达载体能有效转染HUVEC，并在上清中检测到该基因的表达。结论完成人脂联素基因克隆，成功构建了人球状脂联蛋白基因真核表达载体，并在HUVEC中获得分泌表达，为研究球状脂联蛋白对2型糖尿病的干预作用提供了可能。     

Lineage-specific selection and the evolution of virulence in the Candida clade

Candida albicans is the most common cause of systemic fungal infections in humans and is considerably more virulent than its closest known relative, Candida dubliniensis. To investigate this difference, we constructed interspecies hybrids and quantified mRNA levels produced from each genome in the hybrid. This approach systematically identified expression differences in orthologous genes arising from cis-regulatory sequence changes that accumulated since the two species last shared a common ancestor, some 10 million y ago. We documented many orthologous gene-expression differences between the two species, and we pursued one striking observation: All 15 genes coding for the enzymes of glycolysis showed higher expression from the C. albicans genome than the C. dubliniensis genome in the interspecies hybrid. This pattern requires evolutionary changes to have occurred at each gene; the fact that they all act in the same direction strongly indicates lineage-specific natural selection as the underlying cause. To test whether these expression differences contribute to virulence, we created a C. dubliniensis strain in which all 15 glycolysis genes were produced at modestly elevated levels and found that this strain had significantly increased virulence in the standard mouse model of systemic infection. These results indicate that small expression differences across a deeply conserved set of metabolism enzymes can play a significant role in the evolution of virulence in fungal pathogens.

Microbial pathogens of humans typically have at least one close, nonpathogenic relative, and comparisons between the two provide a powerful entry point to identify and study disease-causing determinants. This approach has revealed numerous bacterial pathogenicity islands, clusters of genes required for a given strain or species to cause disease in humans. Because these gene clusters can be horizontally transferred, they can often be initially identified by comparing genome sequences of pathogenic and nonpathogenic strains. The situation with fungal pathogens is substantially different. Like their human hosts, these microbes are eukaryotes, and genes that work together are typically dispersed across different chromosomes rather than clustered; not surprisingly, horizontal transfer of groups of cofunctioning genes from one fungal species to another is rare. Thus, identifying and understanding how groups of genes work together to contribute to virulence remains a challenge in fungi.In this report, we consider two fungal pathogens that are closely related but differ in their virulence. Candida albicans is an opportunistic pathogen; it is a component of the normal human microbiota but is also the leading cause of systemic fungal infections in humans, which can have mortality rates as high as 40%. It is highly virulent when injected into the tail vein of mice, a standardized laboratory procedure that initiates a systemic blood stream infection. Candida dubliniensis is the closest known relative of C. albicans; it was first identified from the oral cavity of an HIV-infected patient and is typically found only as secondary infections. Although it is found throughout the world, C. dubliniensis is much less prevalent than C. albicans in the clinic; it is also less virulent in the mouse model of systemic infection, based on time-to-illness measurements (1–5).C. albicans and C. dubliniensis last shared a common ancestor nominally 10 million y ago, and their genomes are very similar in terms of gene content and synteny. A small number of individual genes are “missing” in one species compared to the other but there are also wide-scale differences in the expression of those genes conserved in both species (6–10). It has been proposed that both types of differences—in gene content and in regulation—contribute to the difference in pathogenicity, although neither idea has been directly demonstrated.In this report, we used an approach known as “allele-specific expression” to highlight patterns of gene-expression differences between C. albicans and C. dubliniensis and to identify those that bear the hallmarks of selection (11–15). Specifically, we created an interspecies hybrid between C. albicans (strain SC5314) and C. dubliniensis (strain CD36) by forcing them to mate with each other (16) (Fig. 1A). Both strains were originally isolated from human patients, and both have been studied extensively in the laboratory. In the hybrid, both parental genomes reside in the same cell, so any bias in mRNA levels must be due to cis-acting sequences specific to that genome. We show that ∼40% of orthologous gene pairs show statistically significant differences in their mRNA levels in the interspecies hybrid, although many of these differences are small in magnitude. We searched for pathways that showed a systematic up-regulation from one genome compared to the other, and were led to the genes for glycolysis, all 15 of which exhibited increased expression from the C. albicans genome compared with the C. dubliniensis genome. This pattern requires cis-acting changes to have accumulated at each of the 15 genes, all resulting in higher mRNA expression from the C. albicans alleles in the hybrid. The likelihood of this pattern emerging by chance is extremely low, and the observation strongly implies that this nonrandom pattern is the result of natural selection.Open in a separate windowFig. 1.RNA-seq of an interspecies hybrid systematically identifies gene-expression differences between C. albicans and C. dubliniensis that arose through cis-acting changes that accumulated since the two species shared a common ancestor. (A) C. albicans and C. dubliniensis (both diploid) were mated to a create tetraploid interspecies hybrid strain, using mating and auxotrophic marker complementation. We performed RNA-seq in the hybrid strain in the conditions listed, as well as the parent diploid strains in bovine plasma at 37 °C. We aligned RNA-seq reads to a concatenated C. albicans–C. dubliniensis genome and excluded reads mapping to both genomes or to multiple locations within a single genome (<5% of total reads). Systematic gene-expression differences of orthologous genes between the two species were assessed. Since both genomes were in the same trans-acting environment (that is, the hybrid strain), all measurable gene expression changes must be due to cis-acting changes. (B) Correlation between two replicate RNA-seq experiments of the interspecies hybrid grown in YPD at 30 °C. Each point represents the log₂ ratio of reads from the C. albicans genome versus reads from the C. dubliniensis genome in the interspecies hybrid, for each orthologous gene pair, with experimental replicates plotted on each axis. Points in the upper right quadrant represent genes with higher expression from the C. albicans genome than C. dubliniensis genome in the interspecies hybrid, and points in the lower left quadrant represent genes with higher expression from the C. dubliniensis genome than the C. albicans genome in that same hybrid, with Spearman correlation values shown in the lower right. We observed very high correlation between replicate experiments for all conditions tested (SI Appendix, Figs. S1 and S2).To test whether increased glycolysis gene expression could account for at least some of the virulence difference between C. albicans and C. dubliniensis, we created a C. dubliniensis strain in which the glycolysis genes were modestly overexpressed and found that this engineered strain showed enhanced virulence (compared with the parental strain) in the standard mouse model for disseminated candidiasis. This result indicates the importance of metabolic fitness in the establishment of fungal infections. More broadly, our results suggest that subtle changes in the expression of genes that are deeply conserved in all branches of life can play important roles in the evolution of specific pathogens.     

自身免疫性肝炎小鼠模型差异表达基因的筛查及柴胡皂苷D对部分差异表达基因表达的影响

目的对自身免疫性肝炎(AIH)小鼠模型的差异表达基因进行基因芯片筛查,并观察柴胡皂苷D(SS-d)对部分差异表达基因表达的影响,探讨AIH的发病机制及SS-d对该病的治疗作用机制。方法健康、雌性SPF级C57BL/6小鼠40只[体质量(20±2)g],分为基因芯片组(n=8)和SS-d治疗组(n=32)。基因芯片组小鼠又分为正常对照组(n=4)和模型组(n=4),正常对照组常规饲养,模型组小鼠按15 mg/kg剂量给予尾静脉注射刀豆蛋白A(Con A),12 h后处死并提取肝组织,按Agilent芯片说明书进行差异表达基因的筛选,采用荧光定量PCR技术对部分差异表达基因进行验证。SS-d治疗组小鼠随机分为正常组(常规饲养)、模型组(按15 mg/kg剂量给予尾静脉注射Con A)、SS-d低剂量组和SS-d高剂量组(分别按2.5 mg/kg和5.0 mg/kg剂量给予腹腔注射SS-d预处理,8 h后按15 mg/kg剂量给予尾静脉注射Con A)(每组8只)。12 h后收集各组小鼠静脉血,ELISA法检测血清ALT、AST。无菌条件下提取小鼠肝组织,部分多聚甲醛固定后切片,并进行HE染色;部分肝组织用于提取RNA,采用荧光定量PCR技术检测部分差异表达基因(IFNγ、IL-4、IL-5、IL-13和IL-17)的mRNA水平变化。多组间比较采用单因素方差分析,进一步两两比较采用LSD-t法检验;两组间比较采用t检验。结果基因芯片组小鼠共筛查差异表达基因11512个,其中上调5189个,下调6323个,显著富集于138条信号通路;荧光定量PCR验证结果显示,与正常对照组比较,模型组小鼠IFNγmRNA和IL-17 mRNA的表达升高(P值均<0.01),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达下调(P值均<0.01),与芯片筛查结果一致。在SS-d治疗组中,与正常对照组比较,模型组小鼠血清ALT、AST水平均升高(P值均<0.01);肝组织内可见大量淋巴细胞浸润;IFNγmRNA和IL-17 mRNA的表达水平明显升高(P值均<0.05),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平明显降低(P值均<0.05)。与模型组比较,SS-d低剂量组和SS-d高剂量组小鼠血清ALT、AST水平均降低(P值均<0.05),肝组织内淋巴细胞浸润程度减轻;IFNγmRNA和IL-17 mRNA的表达水平均降低(P值均<0.05),IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平均升高(P值均<0.05),上述基因的含量变化差异在SS-d高剂量组与SS-d低剂量组之间具有统计学意义(P<0.05)。结论AIH的发生发展系大量基因异常表达的共同作用结果。其中IFNγ、IL-4、IL-5、IL-13、IL-17与AIH的发病密切相关,同时也是SS-d发挥免疫调节及肝损伤保护作用的生物学靶点。     

SORL1基因表达与阿尔茨海默病关系的研究

目的研究中国汉族人迟发散发型阿尔茨海默病(sporadic Alzheimer’s disease,SAD)外周血SORL1 mRNA的表达,为临床诊断提供依据。方法选择迟发SAD患者23例(SAD组),根据WHO疾病分类第10修订版临床诊断标准为迟发SAD,另选健康体检者12例(对照组)。分离2组外周血白细胞,提取总RNA,反转录合成cDNA,采用实时荧光定量PCR检测,以SORL1和磷酸甘油醛脱氢酶的mRNA含量比值作为评价SORL1 mRNA表达水平的指标,并计算出待测样本中SORL1 mRNA表达的准确含量。结果 SAD组外周血SORL1 mRNA表达明显高于对照组,差异有统计学意义(1.17±0.33 vs 0.39±0.29,P0.01),与性别、年龄无明显相关性。结论汉族人外周血SORL1 mRNA表达水平与迟发SAD密切相关;通过实时荧光定量检测外周血SORL1 mRNA表达,对迟发SAD早期诊断可起到辅助的指导作用。     

Higher expression of somatic repair genes in long-lived ant queens than workers

Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair.     

肝癌组织相关基因表达与肝癌术后生存的关系

目的 探讨肝癌组织内多个肝癌相关基因表达情况及其与术后生存预后的关系。方法对72例手术切除肝癌患者的追踪随访资料进行生存分析,分析各临床和病理指标与生存预后的关系。对手术切除的肝癌标本以半定量RT-PCR方法测定癌组织中多个基因的表达情况。结果术前肝功能分级、血清AFP、AFU、癌组织分化程度、有无癌栓与预后相关（P〈0．05）。肝癌组织内COL1A2、Geminin、IGFBP3、SPINK2表达与患者的癌组织分化程度相关。结论肝癌组织内COLlA2、Gemlnin、IGFBP3和SPINK2基因表达为肝癌术后生存的主要相关分子生物学因素。     

胃癌p53基因表达与转移及预后的研究

目的观察胃癌组织ｐ５３基因的超表达及其与预后的关系。方法用抗人Ｐ５３基因蛋白单克隆抗体Ｓ＿Ｐ免疫组织化学方法，观察１２８例胃癌组织ｐ５３表达状况，并对ｐ５３表达与胃癌淋巴结转移状态和术后５年生存率进行比较分析。结果胃癌组织１２８例的ｐ５３表达阳性率为４３８％（５６／１２８）；ｐ５３表达阳性和阴性组的胃癌局部和远处淋巴结转移率分别为６７９％（３８／５６）和５１４％（３７／７２），两者经统计学处理无显著性差异（Ｐ＞００５）。获得随访９８例，胃癌术后５年生存率的随访结果显示，ｐ５３阳性和阴性组分别为３８１％（１６／４２）和３０１％（１７／５６），两组间无统计学意义（Ｐ＞００５）。结论胃癌的发生与ｐ５３基因突变关系密切，并可用免疫组化检测，但Ｐ５３基因蛋白在胃癌组织中的超表达，似不能作为判断胃癌预后的参考指标，应进一步探讨     

Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta signalling pathways

Microarray studies have revealed the differential expression of several genes in mantle cell lymphoma (MCL), but it is unknown which of these differences are dependent on the transformed MCL cell itself or on the tumour microenvironment. To investigate which genes and signalling pathways are aberrantly expressed in MCL cells we used oligonucleotide microarrays to perform gene expression profiling of both purified leukaemic MCL cells and their normal counterparts, the naive B cells. A total of 106 genes were differentially expressed at least threefold in MCL cells compared with naive B cells; 63 upregulated and 43 downregulated. To validate the microarray results in a larger set of samples, we selected 10 differentially expressed genes and quantified their expression by real-time polymerase chain reaction in peripheral blood of MCL patients (n=21), purified MCL cells (n=6) and naive B cells (n=4), obtaining fully concordant results. A computer-assisted approach was used to procure specific molecular signalling pathways that were aberrantly expressed in MCL cells. Several genes related to apoptosis and to the PI3K/AKT, WNT and tumour growth factor beta signalling pathways were altered in MCL cells when compared with naive B cells. These pathways may play a significant role in the pathogenesis of MCL and deserve further investigation as candidates for new therapeutic targets.