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Abstract
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.