Structural and functional analysis of a human 7 S K RNA gene.
Using purified RNA from HeLa cells, we have synthesized and cloned a cDNA encoding an almost entire 7 S K RNA. This cDNA probe was used to isolate 7 S K RNA gene sequences from a human genomic library by high-stringency colony hybridization. In order to differentiate between functional genes and related sequences, we have used a rapid in-vitro transcription assay of purified phage DNA. With this additional screening criterion applied to selected clones, we have obtained one recombinant phage that contained a complete 7 S K RNA gene and, immediately adjacent to its 3' end, a truncated pseudogene. The nucleotide sequence of both genes including the flanking regions has been determined. The functional integrity of the isolated 7 S K RNA gene was verified by in-vitro transcription studies with cell-free extracts and by fingerprinting of the specific transcripts with ribonuclease T1. Under optimal ionic conditions, the transcription efficiency in vitro of this 7 S K RNA gene was found to be comparable to that of a human 7 S K RNA in vitro depends on 5'-flanking sequences. The region up to position -67 was determined to be essential for efficient transcription in vitro of 7 S K RNA. While apparently a variety of 7 S K related sequences is distributed within the human genome, hybridization of 5'-flanking sequences to genomic DNA revealed that possibly not more than one copy of this gene is present per haploid genome.
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000235:genetics*; D020733:RNA, Small Cytoplasmic; D014158:Transcription, Genetic
D020733:RNA, Small Cytoplasmic; D012313:RNA; D004247:DNA