Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus

~~Identification of antiviral- relevant genes in the cultured fish cells induced by UV-inactivated virus~~     

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50—400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.     

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2α kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2α kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2α kinases might play similar roles and that PoPKR is the predominant kinase.     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress geneFdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×10⁵ pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that anFdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

用差减cDNA文库筛选由热激导致的小鼠睾丸中差异表达的基因

采用抑制性差减杂交（Suppression Subtractive Hybridization, SSH）方法,构建正常和热激小鼠睾丸组织的差减 cDNA 文库,以期 筛选出小鼠生精过程中对热敏感的基因。分别从正常和热激小鼠睾丸组织提取总RNA,反转录成cDNA,以正常小鼠睾丸组织cDNA作为待检组织(tester),以热激小鼠睾丸组织cDNA作为驱动组织(driver), 经过2轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了2种组织间差异表达基因的差减cDNA文库。用半定量RT-PCR方法,进一步验证 了该文库中差减出的基因基本上为差异表达的基因。对随机挑选其中的932个克隆测序,对有效测序的565个基因序列与GenBank数据库中发布的序列进行同源性比较,大部分片段都可以检索到同源序列。 结果显示,cPGES/p23等13个基因热激后表达量上调;septin2等120个基因热激后表达量下调。其中cPGES/p23为本研究中首次发现的小鼠生精过程中对热敏感的基因。     

Detection of Grass Carp Hemorrhage Virus （GCHV） from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as “Red Spot Disease“, is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and eomparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplifica-tion. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain in-duces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines,including CAB, FHM, CIK, EPC, CCO and G(X), infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHVstrains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and thetwo strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-Vviruses have been replicated and amplified despite there being no cytopathogenic eifects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published re-cently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp frag-ment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is i-dentical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously,the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product,and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.     

通过差减文库筛选山羊羔肠道淋巴集结特异表达基因

羊羔肠道淋巴集合淋巴结（Peyer's patch，以下简称PP），是B淋巴细胞的主要来源地和发育、成熟场所，兼具中枢淋巴器官和周围淋巴器官的功能.采用抑制性差减杂交（suppression subtractive hybridization，SSH）方法，构建山羊羔肠道淋巴集结与其非PP肠壁组织的差减 cDNA 文库，以期找到在山羊羔肠道淋巴集结中特异表达的与免疫相关的基因. 分别从山羊羔PP与非PP肠壁组织提取总RNA，反转录成cDNA，以PP作为待检组织(tester)，非PP肠壁作为驱动组织(driver)，经过两轮杂交和抑制性PCR扩增，产物与T载体连接，经蓝白斑筛选，提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了两种组织间差异表达基因的差减cDNA文库. 对其中160个克隆测序，得到几个可能与免疫相关的基因，为进一步从中挑选和表达活性蛋白基因用于生产免疫增强剂奠定了基础.     

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

“Bainong 3217 × Mardler” BC₅F₄ wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.     

Identification of the candidate genes associated with cellular rejection in pig-to-human xenotransplantation

To identify the genes associated with cellular rejection in pig-to-human xenotransplantation, the suppression subtractive hybridization (SSH) was used in screening the up-regulated genes from a co-culture of human peripheral blood mononuclear cells (PBMCs) and porcine vascular endothelial cell line PIEC. The up-regulated cDNAs were cloned into pGEM-T Easy vector and then sequenced. Nucleic acid homology searches were performed using the BLAST program. A subtracted cDNA library including about 300 clones with the expected up-regulated genes was obtained. Twenty-four of these clones were analyzed by sequencing and homology comparison was made. These clones represent the genes of human perforin (PRF1), proteasome, lymphocyte specific interferon regulatory factor/interferon regulatory factor 4 (LSIRF/IRF 4), muscleblind-like (MBNL) protein and a porcine expressed sequence tag (EST) which has 81% homology with human oxidative-stress responsive 1 (OSR 1). These genes might be the candidate genes which are associated with cellular rejection in pig-to-human xenotransplantation.     

Molecular cloning, identification and characteristics of NYD-SP9: Gene coding protein kinase presumably involved in spermatogenesis

Using cDNA microarray hybridization from a human testicular cDNA library, one gene exhibiting ten-fold difference at expression level between adult and embryo human testes was cloned and named NYD-SP9, which was believed to be involved in spermatogenesis. Southern blot hybridization results showed that NYD-SP9 expressed highly in testis but low in ovary. Protein motif analysis of this cDNA sequence revealed a cluster of phosphorylation sites, indicating its potential involvement in signal pathways during spermatogenesis. Furthermore, one transmembrane helix was predicted in N-terminal region, indicating that putative NYD-SP6 may be served as a transmembrane protein. The proximity of these potential phosphorylation sites to each other indicates that there may be interaction among these sites to regulate spermatogenesis. These findings suggested that protein kinase NYD-SP9 might play a role in male germ cell differentiation.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

杜洛克与梅山猪大卵泡消减文库的构建

为了鉴定杜洛克猪相对于梅山猪在大卵泡中高表达的基因,本研究应用抑制性消减杂交技术成功构建了以梅山大卵泡c DNA为driver,杜洛克大卵泡c DNA为tester的消减c DNA文库。结果显示:以G3PDH和β-actin为指标检测文库的消减效率为25,从该文库中获取了350个有效的阳性克隆,PCR检测插入片段主要分布在150-750 bp。克隆测序得到74个有效的EST序列,GO分析表明主要与细胞信号、细胞结构、代谢、细胞分化、基因/蛋白质合成、细胞组织防护等功能相关。利用q PCR技术验证了ELTD1、Grb14、SNRPE、CSDE1、ALDH18A1、e IF4E、BMPR-IB等基因在梅山和杜洛克大卵泡中的表达模式。结果发现在两猪种的大卵泡间存在显著性差异。本研究有助于揭示影响猪卵泡发育和生殖数量控制的分子基础。     

大蕉冷诱导差减文库的构建与分析

以低温处理的大蕉幼苗cDNA为检测子,未处理的大蕉幼苗cDNA为驱赶子,利用抑制性差减杂交技术(suppression subtractive hybridization,SSH)构建了大蕉幼苗冷诱导差减cDNA文库,通过点杂交差异筛选cD-NA文库,得到约50个低温下表达增强的候选克隆,对其进行DNA测序和同源性比较,发现获得的ESTs在功能上主要涉及信号传导、表达调控、非生物胁迫防御、蛋白质加工和能量代谢等方面。该SSH文库的构建为克隆大蕉抗寒相关基因奠定了基础。     

Isolation and cloning of a novel cDNALDB1 encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.