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。     

Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15

Aims:  This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15.
Methods and Results:  The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli , and rHepII was purified using two simple ion–exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC.
Conclusions:  The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l⁻¹ sodium phosphate buffer with 75 mmol l⁻¹ NaCl (pH 7·4) at 32°C, and the activity is stable at 4°C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure.
Significance and Impact of the Study:  This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum ; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.     

A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Inheritance and expression of introduced DNA in transgenic onion plants (Allium cepa)

Transgenic onion plants (Allium cepa) containing the Cauliflower mosaic virus 35s promoter (CaMV35s) and gfp gene construct encoding the visual green fluorescent reporter protein from pBINm gfp ER and the CaMV35s‐bar gene construct encoding resistance to the herbicide phosphinothricin from pCAMBlA3301 were produced by Agrobacterium‐mediated transformation. These plants weregrown to maturity and selfed in order to determine the expression and inheritance of the transgenes. CaMV35s regulation in onion, as observed by GFP expression, was essentially constitutive, and profiles of regulation were typical of those observed in dicotyledonous plants. Inhibition of CaMV35s regulated gene expression was only observed in one transformant. Both the expression of GFP and tolerance to phosphinothricin appeared to be inherited in a Mendelian fashion. Levels of expression in F1 offspring varied, presumably due to environmental and genetic factors. However, it appeared that copy number did strongly influence GFP protein production and expression. In the majority of plants there were no obvious detrimental phenotypic effects caused by the transgene, the integration event, or Somaclonal variation due to the need to perform tissue culture.     

The complete sequence and expression patterns of the atrial myosin heavy chain in the developing chick

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.     

苏云金芽胞杆菌营养期杀虫蛋白基因的克隆及表达分析

选择本实验室分离的苏云金芽胞杆菌李氏亚种 (subsp. Leesis) 菌株YBT833、鲇泽亚种(subsp.Aizawai) 菌株YBT-1416和库斯塔克亚种(subsp. Kurstaki)菌株YBT1535为出发菌株,以营养期杀虫蛋白基因PCR扩增的特异片段为探针,进行总DNA酶切片段的Southern杂交定位。结果显示3株菌株的营养期杀虫蛋白基因,均位于经XbaI完全消化的4～5kb大小的DNA 片段上。将该区域DNA片段回收后克隆到pUC19载体,建立了3个较基因组文库小的亚基因组文库。通过菌落原位杂交筛选和酶切鉴定分别得到3个相应的营养期杀虫蛋白基因vip83、vip14和vip15,并对其测序。DNA序列比较发现基因vip83与已知营养期杀虫蛋白基因存在5个差异碱基。将vip83、vip14基因亚克隆到苏云金芽胞杆菌大肠杆菌穿梭载体pHT315, 分别得到重组质粒pBMB8901和pBMB8902。将它们电转化到vip^-的B.t.受体菌BMB171和4Q7,获得了相应的工程菌BMB8901-171,BMB8902-171,BMB8901-4Q7和BMB8902-4Q7。SDS-PAGE电泳检测均有88kD大小的蛋白表达。生物测定结果亦表明了,营养期杀虫蛋白Vip83和Vip14对鳞翅目棉铃虫、小菜蛾和甜菜夜蛾的三龄幼虫均有一定的杀虫活性;其中对小菜蛾的毒力最高,LC₅₀值分别为28.6,31.6,45.4和37.6μL/mL。该结果为构建高效广谱工程菌提供了实际材料和理论依据。      

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi fromtheParacoccidioides brasiliensis complex. Previous studies havedemonstrated that the severity of disease is associated with a T-helper 2 immuneresponse characterised by high interleukin (IL)-4 production. In the present study weanalysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area.The production of IL-4 by peripheral blood mononuclear cells after antigen orphytohaemagglutinin stimulation was determined by ELISA. A significant correlationwas observed between the RP2/RP2 intron-3 genotype and infection withParacoccidioides sp. (p = 0.011), whereas the RP1/RP1 genotypewas correlated with resistance. No significant correlation was observed forthe IL-4 promoter polymorphism. Furthermore, the low IL-4expression observed in the control group compared with patients was associated withthe RP1/RP1 genotype. These results suggest that IL-4polymorphismsmight be associated with the ability of the host to control Paracoccidioidessp. infection. The relevance of this polymorphism is supported by theobservation that patients with disease produce high levels of IL-4 following mitogenor antigen stimulation. The IL-4 gene is located in the cytokinecluster region of chromosome 5 where other polymorphisms have also beendescribed.