Characterization of the transcription products of glyceraldehyde 3-phosphate-dehydrogenase gene in HeLa cells.

We have partially purified the messenger RNA coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from HeLa cells and obtained a cDNA clone containing part of its sequence. Using this clone to probe electrophoregrams of RNA transferred to nitrocellulose, we have investigated the characteristics of nuclear and cytoplasmic transcripts in these cells. In the cytoplasm, nature GAPDH mRNA was detected in Northern blots as an intense band, apparently unique, of approximately 1400 nucleotides. The half-life of this mRNA was determined both from the decay kinetics, after a chase with actinomycin D, and from the labeling kinetics during an accumulation experiment. Both kinds of experiments yielded a half-life value of about 8 h, while the accumulation experiment indicated that steady-state GAPDH mRNA amounted to about 1.6% of cytoplasmic poly(A)-rich RNA. Much longer species, likely to be restricted to the nucleus, were also detected in RNA extracted from total cells. At least three discrete species of 1600, 4000, 5800 and 6800 bases were observed above a trailing background extending up to about 8000 bases. This value is commensurate with a functional size of the GAPDH transcription unit in the order of 13000 bases, which we determined by measuring the size of the ultraviolet inactivation target. Until direct evidence can be obtained at the genomic level, the present results provide the first clue to the existence of introns, presumably at least four, in a GAPDH gene from a higher eucaryote.

Unusual abundance of vertebrate 3-phosphate dehydrogenase pseudogenes.

Only one gene coding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), a key enzyme in the control of glycolysis, is known to be functional in man, mouse, rat and chicken. The gene has been localized to chromosome 12 in human and chromosome 6 in mouse. Only a single mRNA species has been found in chicken and rat. However, analysis of genomic DNA blots of various species with a cloned GAPDH cDNA probe has revealed large differences in the level of reiteration, ranging from one to over 200 copies. On this basis, we have grouped these organisms into three classes according to the number of GAPDH-related sequences they contain; one class with a unique representation (chicken), another class of relatively low reiteration (10-30 copies in man, hare, guinea-pig and hamster) and a third class of high reiteration (greater than 200 copies in mouse and rat). The third class represents the first reported occurrence of such an extreme number of pseudogenes related to an enzyme-coding gene and suggests that a dramatic amplification event took place between 15 and 25 million years ago.

Extreme instability of myc mRNA in normal and transformed human cells.

To address the possibility that the expression of the myc gene might be regulated at a post-transcriptional level, we have investigated the half-life of myc mRNA in various cells. Our survey included normal human embryonic fibroblasts as well as transformed human cells of various origins: cervix carcinoma (HeLa), breast carcinoma (MCF7), Burkitt lymphoma (Daudi), and promyelocytic leukemia (HL60). All these cells revealed an extreme instability of myc mRNA (half-life, approximately equal to 10 min), suggesting that the control of myc mRNA degradation might be a general means (although not necessarily exclusive) of regulating both the level and the timing of myc gene expression. Inhibition of protein synthesis resulted in a dramatic stabilization of myc mRNA in HeLa, MCF7, and HL60 cells, suggesting that the controlling element might itself be, at least in these cells, a protein of rapid turnover. This finding opens the way to studying the mechanism of myc mRNA inactivation in these different cell types. However, protein synthesis inhibition had no effect on myc mRNA instability in other transformed (Daudi) cell lines as well as normal embryonic human fibroblasts. These different types of behavior suggest that the post-transcriptional control of myc gene expression might involve multiple factors that would be differently affected in various cell types.

Post-transcriptional regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression in rat tissues.

We have isolated and identified cDNA clones containing part of the coding sequence for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C. 1.2.1.12). By using one of these clones as a probe, we have shown that: i) the abundance of GAPDH mRNA is different in various tissues of the adult rat and in good correlation with the abundance of the enzyme; ii) the transcription rates are quite similar in all tissues tested. We therefore conclude that the tissue-specific differential GAPDH gene expression is regulated by adjusting the abundance of its mRNA at the post-transcriptional level.