中国荷斯坦牛ABCG2基因编码区多态性检测及生物信息学分析

目的：研究中国荷斯坦牛ABCG2基因编码区（CDS）多态性,并进行生物信息学分析。方法：以中国荷斯坦牛为材料,利用PCR-SSCP技术对ABCG2基因CDS多态性进行检测,然后预测蛋白质序列的改变,并用生物信息学软件对蛋白质序列突变前后的结构及性质进行分析。结果：在外显子9中存在一个A→G碱基突变,导致氨基酸由酪氨酸突变为半胱氨酸,将此突变命名为Y367C;在外显子14中存在一个G→A突变,导致氨基酸由精氨酸突变为谷氨酰胺,将此突变命名为R578Q。2个突变一个位于功能区与跨膜区之间,一个位于跨膜区。生物信息学分析发现,蛋白质二级结构增加了1个卷曲（C）和2个转角（T）,同时减少了3个β折叠（E）,且ABCG2蛋白的组成和一些性质也发生了改变。结论：检测到的2个单核苷酸多态性引起了ABCG2蛋白性质和二级结构的改变;为进一步研究ABCG2蛋白对产乳性状的影响奠定了基础。     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

大熊猫FOXL2基因的克隆、序列分析及表达

从大熊猫基因组中克隆了FOXL2基因,并对其进行序列分析及原核表达和真核表达.将FOXL2编码区序列克隆到原核表达载体pET-32a(+)中,转化大肠杆菌BL21,经IPTG诱导表达出FOXL2重组蛋白.成功构建了真核表达载体FOXL2-pcDNA3.1/V5-His C,并通过脂质体介导转染HEK293细胞,Western blot检测FOXL2蛋白表达.SDS-PAGE分析表明,FOXL2重组蛋白在诱导4h后表达量达到峰值,其大小约为58.9 kDa,Western blot分析结果显示重组蛋白能够被抗His单克隆抗体特异性识别.FOXL2基因的克隆及其表达为进一步进行FOXL2的活性检测以及应用研究奠定了基础.     

Staufen基因在结直肠癌组织中的表达

为研究甲基化差异相关基因Staufen在结直肠癌不同组织中的表达情况,采用RT-PCR和免疫组织化学等方法在结直肠癌病人中检测Staufen基因在腺癌、癌旁粘膜和相应远端切缘正常组织中的表达。研究发现,在mRNA水平上,远端切缘正常组织中Staufen基因的表达水平显著高于癌旁粘膜和腺癌（P<0.05）;从正常组织、癌旁粘膜到腺癌,Staufen基因的表达有降低的趋势;未发现性别、年龄、肿瘤部位（结肠/直肠）、分化程度、淋巴结转移等临床病理因素与Staufen基因的表达有关（P均>0.05）。正常组织与腺癌组织的Staufen蛋白的表达水平显著高于癌旁粘膜（P<0.05）,而正常组织与腺癌组织之间未发现有统计学差别（P>0.05）。结果表明,Staufen基因在癌旁粘膜和肿瘤中有低表达的趋势,该基因可能在结直肠癌的发生、发展中起作用。     

Expression Profile of the REG Gene Family in Colorectal Carcinoma

Regenerating (REG) gene family belongs to the calcium-dependent lectin gene superfamily and encodes small multifunctional secretory proteins, which might be involved in cell proliferation, differentiation, and carcinogenesis. To clarify REG expression profile in colorectal carcinoma (CRC), the authors examined the expression of REG Iα, Iβ, III, HIP/PAP, and REG IV by immunohistochemistry on tissue microarray. The expression of REG Iα, III, and HIP/PAP was more frequently observed in the CRCs than adjacent non-neoplastic mucosa (p < 0.001), whereas it was the converse for REG Iβ and IV (p < 0.001). The expression of REG Iα, Iβ, III, and HIP/PAP was negatively correlated with the depth of invasion of CRCs (p < 0.05). The REG Iβ and HIP/PAP were less expressed in CRCs with than without venous invasion (p < 0.05). The positive rates of REG Iα and HIP/PAP were significantly higher in CRCs without than with lymph node metastasis (p < 0.05). Mucinous carcinoma more frequently expressed REG IV protein than well- and moderately differentiated ones (p < 0.05). There was a positive relationship between REG Iα, Iβ, III, and HIP/PAP expression (p < 0.05). Survival analysis indicated the REG Iβ or HIP/PAP expression was positively linked to favorable prognosis of carcinoma patients (p < 0.05). This study indicated that aberrant REG expression might be closely linked to the pathogenesis, invasion, or lymph node metastasis of CRCs. REG Iβ and HIP/PAP could be considered reliable markers of favorable prognosis of CRC patients.     

Broad Shifts in Gene Expression during Early Postnatal Life Are Associated with Shifts in Histone Methylation Patterns

During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration.     

电激转化GFP基因在小麦合子和早期原胚中的高频表达

用电激法将GFP基因成功转入分离的小麦(Triticum aestivum L.)合子及早期原胚中.在电场强度150 V/cm、电容25 μF、线性DNA浓度200 μg/mL、电激缓冲液pH 7.2的条件下,得到GFP基因在早期原胚中的高频瞬间表达(46.7%).与开花后5 d胚胎的最适电场强度相比,合子和早期原胚因较幼嫩而最适电场强度较低.电激后的一些早期原胚可以在KM8p培养基中培养并继续分裂,分裂后的细胞中也能观察到GFP基因的表达.此外,电激后的合子及2细胞、4细胞和8细胞原胚中没有看到基因表达的嵌合现象.     

电激转化GFP基因在小麦合子和早期原胚中的高频表达

用电激法将GFP基因成功转入分离的小麦（Triticum aestivumL.)合子及早期原胚中。在电场强度150V/cm,电容25μF。线性DNA浓度200μg/mL,电激缓冲液pH7.2的条件下,得到GFP基因在早期原胚中的高频瞬间表达（46.7%)。与开花后5d胚胎的最适电场强度相比,合子和早期原胚因较幼嫩而最适电场强度较低。电激后的一些早期原胚可以在KM8p培养基中培养并继续分裂,分裂后的细胞中也能观察到GFP基因的表达。此外,电激后的合子及2细胞。4细胞和8细胞原胚中没有看到基因表达的嵌合现象。     

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.     

Low Sequence Variation in the Gene Encoding the Human β-Myosin Heavy Chain

Over 40 different mutations in the cardiac myosin heavy chain gene (MYH7) have been associated with familial hypertrophic cardiomyopathy (FHC), but no study has analyzed variation at this locus within the normal human population. Here we determine the extent and distribution of nucleotide variation in the 5808-bp MYH7 coding sequence in 25 normal individuals without FHC. We identified six single-nucleotide polymorphisms, none of which changes the encoded amino acid. At one of these sites, the frequencies of both alleles are equal; at the other five sites, the frequency of the rarer allele varies from 0.02 to 0.08. The nucleotide diversity (π) calculated from these data is 1.73×10⁻⁴±0.49×10⁻⁴, which is lower than the nucleotide diversity found in most other human autosomal genes. Substitution analysis of homologous genes between human and rodent also indicates that the MYH7 sequence has evolved at a very slow rate. The rate of both synonymous and nonsynonymous substitutions, especially in the portion of the sequence that encodes the α-helical myosin rod, is extremely low. The low level of even silent sequence variation in MYH7 in comparisons between human sequences and between human and rodent sequences may be a consequence of strong selective pressure against mutations that cause cardiomyopathy.     

亚麻中雄性不育基因同源序列MS2-F的克隆和表达分析

用同源序列克隆法从亚麻中克隆了雄性不育基因同源序列MS2-F cDNA(登陆号:EU363493).该cDNA全长1 91lbp,包含一个1 608 bp的ORF,编码535个氨基酸.推导的蛋白质序列中包含2个雄性不育保守区:NAD结合区域和雄性不育C-末端区域.该基因与油菜和拟南芥雄性不育基因的一致性分别为59.65%和59.16%,为花蕾特异表达基因,推测在亚麻花粉发育过程中与脂酰辅酶A还原酶有相似功能.MS2-F cDNA对应的gDNA大小为2 696 bp(登陆号:EU365361),含有8个内含子和9个外显子.