To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon mem-brane and the over-expressed genes were then screened by hybridizing the filter with radioac-tively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank da-tabase. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular en-dothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohis-tochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.
AI Junkui1, ZHANG Zhiwen2, XIN Dianqi1, ZHU Hongjian1, YAN Quanjian3, XIN Zhongcheng1, NA Yanqun1 & GUO Yinglu1 1. Institute of Urology, Peking University & Department of Urology, First Hospital, Peking University, Beijing 100034, China
EMSA and footprinting analyses have revealed that the -489—-414 bp and the -390—-345 bp (designated DC and PC respectively) upstream of the Aspergillus niger T21 glaA gene were bound by one protein factor in the A. niger T21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. niger T21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA re-vealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. niger T21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.
