Human ribosomal proteins (r-proteins) are encoded by a complex family of vital, constitutively expressed housekeeping genes whose structure and regulation differ significantly from more thoroughly studied tissue specific genetic markers. A plan to study these genes' structure, chromosomal organization, transcriptional regulation and molecular evolution is described. Experiments proposed rely on recombinant DNA technology, nucleic acid sequencing, the polymerase chain reaction, strategies to characterize DNA:protein interactions, and methods for assessing expression of cloned r-protein genes in cultured cells as well as cell-free reactions.
Three specific aims are indicated: 1) Human r- protein genes will be isolated as molecular clones using recombinant DNA and PCR technology together with existing cDNA probes. Active genes will be distinguished from processed pseudogenes by functional and structural criteria and mapped to specific chromosomal sites. In addition, complete nucleic acid sequences and intron-exon architectures will be determined for the active genes cloned. 2) Human r-protein gene (RPS14 and RPS17) transcriptional promoters will be characterized with respect to their cisactive DNA sequence motifs and transactive nuclear transcription factors. and 3) Homologous eukaryotic r-protein genes from multiple vertebrate phyla will be compared by structural as well as functional criteria. Previous studies indicate that, despite remarkable conservation of protein-coding sequences, r-protein homologs derived from diverse eukaryotic phyla differ dramatically with regard to the number, location and nature of their intervening sequences. During the proposed project period, more closely related vertebrates species' r-protein S14 homologs will be compared to gain insight into genetic processes which affect evolution of gene architecture. All three specific goals are designed to yield new insights into the structure, genomic organization and transcriptional regulation of a vital mammalian housekeeping gene family.
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