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Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.

ABSTRACT: The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferox-amine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.

SUBMITTER: Ye Z

PROVIDER: S-EPMC102820 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.

Ye Z Z Connor J R JR

Nucleic acids research 20000401 8

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferox-amine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries w ...[more]

PMID: 10734200

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Project description:BACKGROUND: Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model. RESULTS: We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis. CONCLUSION: The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

Project description:AIM:To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS:pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BamHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coli strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS:The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of beta-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by GenBank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION:The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.

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Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.

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Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.

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