生物信息学在发现新基因方面的应用

自生物信息学作为一门交叉学科诞生以来,其在计算机、农业和生命科学等各方面发挥了重要的作用,在后基因组时代,更是成为发现新基因的重要手段。对生物信息学的概况做了回顾与展望,并简述了生物信息学近年在发现新基因方面所取得的成果。     

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

家蚕多聚泛素基因的电子克隆、序列分析及表达谱预测

具选择性蛋白质降解功能的泛素在昆虫生长发育过程中起着重要的调控作用。本研究采用电子克隆的方法钓取家蚕基因组中多聚泛素基因序列,命名为Bm-polyUB(GenBank登录号:AADK01019318),并进行序列分析和电子表达谱预测。序列分析表明,该编码区长3426bp,编码1141个氨基酸残基的15聚体,预测分子量和等电点分别为127.97kD和7.33,二级结构中α-螺旋、延伸带、β-转角和无规则卷曲各占17.09%、32.78%、14.37%和35.76%,亚细胞定位于细胞质、细胞核和线粒体各占43.5%、34.8%和21.7%,无前导肽、信号肽和跨膜区;多重序列比对显示,15个泛素单体基因序列间同源性和遗传距离分别介于89.0%～100.0%和0.000～0.120,其中Bm-UB3与Bm-UB10、Bm-UB5与Bm-UB7及Bm-UB10与Bm-UB14的序列相同;遗传多样性分析可见,共检出33个多态性位点,共生成12个单倍型,单倍型多样性(Hd=0.962)、平均核苷酸差异数(K=7.495)、核苷酸多样性(Pi=0.03287)、密码子有效值(ENC=41.62361)、偏爱指标(CBI=0.4960)和χ2检验计算(χ2=0.713)显示泛素单体间遗传多样性较丰富且呈现较强的密码子偏爱性;电子表达谱预测结果表明Bm-polyUB基因在家蚕中高表达的组织和发育阶段所占比例显著高于低表达。本文的研究结果可为进一步开展该基因进化机制、表达特性和生理功能等方面的实验研究提供基础资料。     

MGMT Promoter Methylation Correlates with an Overall Survival Benefit in Chinese High-Grade Glioblastoma Patients Treated with Radiotherapy and Alkylating Agent-Based Chemotherapy: A Single-Institution Study

Promoter methylation of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene has been considered a prognostic marker and has become more important in the treatment of glioblastoma. However, reports on the correlation between MGMT and clinical outcomes in Chinese glioblastoma patients are very scarce. In this study, quantitative methylation data were obtained by the pyrosequencing of tumor tissues from 128 GBM patients. The median overall survival (OS) was 13.1 months, with a 1-year survival of 45.3%. The pyrosequencing data were reproducible based on archived samples yielding data for all glioblastomas. MGMT promoter methylation was detected in 75/128 cases (58.6%), whereas 53/128 (41.4%) cases were unmethylated. Further survival analysis also revealed that methylation was an independent prognostic factor associated with prolonged OS but not with progression-free survival (PFS) (p = 0.029 and p = 0.112, respectively); the hazard radios were 0.63 (95% CI: 0.42–0.96) and 0.72 (95% CI: 0.48–1.09), respectively. These data indicated that MGMT methylation has prognostic significance in patients with newly diagnosed high-grade glioblastoma undergoing alkylating agent-based chemotherapy after surgical resection.     

Oxalate production at different initial Pb²+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium

The production of oxalate at different initial Pb²⁺ concentrations during solid-state fermentation of straw with Phanerochaete chrysosporium was investigated. It was found that the maximal peak value of oxalate concentration (22.84 mM) was detected at the initial Pb²⁺ concentration of 200 mg kg⁻¹ dry straw, while the minimum (15.89 mM) at the concentration of 600 mg Pb²⁺ kg⁻¹ dry straw, and at moderate concentration of Pb²⁺ the capability of oxalic acid secretion was enhanced. In addition, it was also found that more oxalic acid accumulation went together with better Pb²⁺ passivation effect and higher manganese peroxidase (MnP) activity. The present findings will improve the understandings of the interactions of heavy metals with white-rot fungi and the role of oxalate in lignin degradation system, which could provide useful references for more efficient treatment of Pb-contaminated lignocellulosic waste.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.     

Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant

Studies on some plant species have shown that increasing the growth temperature gradually or pretreating with high temperature can lead to obvious photosynthetic acclimation to high temperature. To test whether this acclimation arises from heat adaptation of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activation mediated by Rubisco activase (RCA), gene expression of RCA large isoform (RCA_L) and RCA small isoform (RCA_S) in rice was determined using a 4‐day heat stress treatment [40/30°C (day/night)] followed by a 3‐day recovery under control conditions [30/22°C (day/night)]. The heat stress significantly induced the expression of RCA_L as determined by both mRNA and protein levels. Correlative analysis indicated that RCA_S protein content was extremely significantly related to Rubisco initial activity and net photosynthetic rate (Pn) under both heat stress and normal conditions. Immunoblot analysis of the Rubisco–RCA complex revealed that the ratio of RCA_L to Rubisco increased markedly in heat‐acclimated rice leaves. Furthermore, transgenic rice plants expressing enhanced amounts of RCA_L exhibited higher thermotolerance in Pn and Rubisco initial activity and grew better at high temperature than wild‐type (WT) plants and transgenic rice plants expressing enhanced amounts of RCA_S. Under normal conditions, the transgenic rice plants expressing enhanced amounts of RCA_S showed higher Pn and produced more biomass than transgenic rice plants expressing enhanced amounts of RCA_L and wild‐type plants. Together, these suggest that the heat‐induced RCA_L may play an important role in photosynthetic acclimation to moderate heat stress in vivo, while RCA_S plays a major role in maintaining Rubisco initial activity under normal conditions.