ABSTRACT
Serial analysis of gene expression (SAGE) allows for a quantitative, representative, and comprehensive profile of gene expression. We have utilized SAGE technology to contrast the differential gene expression profile in rat embryo fibroblast cells producing temperature-sensitive p53 tumor suppressor protein at permissive or non-permissive temperatures. Analysis of approximately 15,000 genes revealed that the expression of 14 genes (P < 0.001, > or = 0.03% abundance) was dependent on functional p53 protein, whereas the expression of three genes was significantly higher in cells producing non-functional p53 protein. Those genes whose expression was increased by functional p53 include RAS, U6 snRNA, cyclin G, EGR-1, and several novel genes. The expression of actin, tubulin, and HSP70 genes was elevated at the non-permissive temperature for p53 function. Interestingly, the expression of several genes was dependent on a non-temperature-sensitive mutant p53 suggesting altered transcription profiles dependent on specific p53 mutant proteins. These results demonstrate the utility of SAGE for rapidly and reproducibly evaluating global transcriptional responses within different cell populations.
Subject(s)
Cloning, Molecular/methods , Gene Expression Regulation , Genes, p53 , Transcription, Genetic , Animals , Cell Division/genetics , Cell Line , Embryo, Mammalian , Fibroblasts/cytology , Fibroblasts/metabolism , Rats , Reproducibility of Results , TemperatureABSTRACT
Transcriptionally regulated growth-response genes play a pivotal role in the determination of the fate of a cell. p53 is known to transcriptionally regulate genes important in regulating cell growth potential. Using differential reverse transcription-PCR analysis of rat embryo fibroblast cells containing a temperature-sensitive p53 allele, we were able to isolate several transcripts up-regulated specifically in cells harboring functional p53 protein. Two of these genes, SM20 and microsomal epoxide hydrolase (mEH), are previously described genes. Two previously uncharacterized cDNAs, cell growth regulatory (CGR) genes CGR11 and CGR19, were isolated. The predicted amino acid sequence of these novel proteins contain known motifs; EF-hand domains (CGR11) and a ring-finger domain (CGR19), suggestive of function. CGR11 and CGR19 appear to be primary response genes expressed to moderate levels in functional p53 cells. Both CGR11 and CGR19 are able to inhibit the growth of several cell lines.
Subject(s)
Calcium-Binding Proteins/genetics , Cell Division/genetics , Gene Expression Regulation , Genes, Regulator , Growth Inhibitors/genetics , Proteins/genetics , Tumor Suppressor Protein p53/physiology , Amino Acid Sequence , Animals , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/chemistry , DNA, Complementary/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Genes, p53 , Growth Inhibitors/biosynthesis , Growth Inhibitors/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Biosynthesis , Protein Structure, Tertiary , Proteins/chemistry , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Rats , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Subtraction Technique , TemperatureABSTRACT
The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.
