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A cloned human ribosomal protein gene functions in rodent cells
1Division of Biology, Kansas State University, Manhattan 66506.
Abstract:
Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.
Insights
Human ribosomal protein S14 (RPS14) DNA functions in hamster cells, producing mRNA. Gene expression appears regulated post-transcriptionally, not by copy number, suggesting complex control mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Investigating the functional expression of human ribosomal protein S14 (RPS14) in a heterologous cellular system.
- Understanding the regulatory mechanisms governing ribosomal protein gene expression.
Purpose of the Study:
- To determine if cloned human RPS14 DNA can be transcribed and translated in cultured Chinese hamster (CHO) cells.
- To assess the functional complementation of a drug-resistant mutation in CHO cells by human RPS14.
- To investigate the post-transcriptional regulation of human RPS14 expression in CHO cells.
Main Methods:
- Transfection of cloned human RPS14 DNA fragments into CHO cells.
- Transient and stable expression assays to analyze mRNA production and protein function.
- Selection of emetine-resistant CHO cells (Emr-2) for complementation studies.
- Analysis of RPS14 mRNA levels and steady-state protein levels in transformed cells.
Main Results:
- Human RPS14 was transcribed into functional mRNA in CHO cells, independent of a canonical TATA sequence.
- Exogenous human RPS14 complemented the emetine-resistant phenotype of CHO cells, confirming gene functionality.
- Human RPS14 mRNA levels were not tightly regulated by gene copy number in CHO cells.
- Steady-state RPS14 protein levels showed minimal fluctuation despite significant differences in mRNA abundance.
Conclusions:
- Human RPS14 gene is functional in rodent cells and can be expressed.
- Regulation of human RPS14 expression in CHO cells occurs, at least in part, post-transcriptionally.
- The findings suggest a complex regulatory network controlling ribosomal protein homeostasis.