Construction and screening of suppression subtractive hybridization library of renal cell carcinoma

Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A+ RNA was isolated from RCC lines 786-O(tester) and renal cell(RC) lines HK-2 ( driver), respectiely. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit ( Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F’. All positive clones picked out were digested and some of which were sequenced. Results The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly,2 represented unknown genes and the other 48 derived from 36 known genes. Conclusion The quality of the SSH library of human RCC is reliable and is construction is the basis for further screening differentially expressed genes of RCC. 6 refs,4 figs, 1 tab.

Differentiated digital library evaluation in a hierarchical model stemming from its operational scope and complexity

Digital libraries are complex systems and this brings difficulties for their evaluation. This paper proposes a hierarchical model to solve this problem, and puts the entangled matters into a clear-layered structure. Firstly, digital libraries(DLs thereafter)are classified into 5 groups in ascending gradations, i.e. mini DLs, small DLs, medium DLs,large DLs, and huge DLs by their scope of operation. Then, according to the characteristics of DLs at different operational scope and level of sophistication, they are further grouped into unitary DLs, union DLs and hybrid DLs accordingly. Based on this simulated structure,a hierarchical model for digital library evaluation is introduced, which evaluates DLs differentiatingly within a hierarchical scheme by using varying criteria based on their specific level of operational complexity such as at the micro-level, medium-level, and/or at the macro-level. Based on our careful examination and analysis of the current literature about DL evaluation system, an experiment is conducted by using the DL evaluation model along with its criteria for unitary DLs at micro-level. The main contents resulting from this evaluation experimentation and also those evaluation indicators and relevant issues of major concerns for DLs at medium-level and macro-level are also to be presented at some length.

Gene Expression Profiles of Response to Water Stress at the Jointing Stage in Wheat

Drought is one of the most adverse environmental factors that impact on plant growth and reduce crop yields. To decipher the molecular mechanism of drought tolerance, we constructed a cDNA library using suppression subtractive hybridization （SSH） and dissected the gene expression profiles in seedling and jointing wheat plants after stress with PEG-6000. A total of 2 046 ESTs from the jointing stage library （J-Lib） were allocated to 961 contigs. Among the ESTs, 265 uni-genes in 12 categories were identified on the basis of sequence similarities and functional classifications. Most were known to be involved in protection, directly or indirectly, against water stress. To determine differences in gene expression profiles for water stress responses at the jointing and seedling stages, data from the J-Lib were compared with those from a 2- leaf seedling library （S-Lib） constructed by the same method. Significant differences were observed between the two libraries for function-known genes; signal transduction genes were far more active at jointing than at the seedling stage.

Molecular Basis of Unique Branching Phenotypes in Salvia splendens and the Role of PSY

The branching system of higher plants plays a very important role in plant morphogenesis,and the number of branches can directly affect crop yield and the ornamental value of plants.It is a complicated development process involving complex molecular mechanisms.The‘Cailinghong’variety of Salvia splendens is characterized by its great branching ability with the ability to grow into a spherical form naturally,without pinching.To gain insight into the molecular events during the branching development of S.splendens,suppressive subtractive hybridization(SSH)technology was used to screen differentially expressed genes between the erect plant type(strain 35)and the spherical plant type(‘Cailinghong’).In total,96 and 116 unigenes were annotated.Four and eight unigenes up-regulated in‘Cailinghong’and strain 35,respectively,were associated with plant hormone anabolism and signal transduction,suggesting that they participate in the branching process.One of these genes,phytoene synthase(PSY),is a precursor of the new plant hormone group strigolactones.Using the PSY fragment(192 bp)as a template,the cDNA sequence of PSY in S.splendens was cloned and named SsPSY.A relative expression analysis and transgenic test results indicated that SsPSY plays an important role in lateral branch development in‘Cailinghong’.These results provide new insight into the molecular mechanisms underlying branching in S.splendens.

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.Methods Polyadenylated RNA ［Poly (A)+ RNA］ was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.