In E. coli
also, the role of anti-sense
RNA in regulating gene expression has been demonstrated in case of two genes for outer membrane protein. These two genes are ompC
and ompF (Omp
= outer membrane protein). The total amount of these two proteins remains constant, although their relative quantities may differ according to requirement. It has been shown that a DNA fragment upstream of ompC
when introduced into omp F+
cells, inhibited the production of OmpF
protein. This DNA fragment coded for a small 174 base RNA and is termed mic RNA (mRNA interfering complementary RNA)
which was transcribed in a direction opposite to ompC
gene. Since no ribosome binding site is present on mic
RNA, it can not be used for protein synthesis. But it has extensive homology with 5' end of ompF
mRNA, and therefore, can form a hybrid with it. Since the sequences present in mic RNA include sequences complementary to ribosome binding site of ompF
mRNA, hybrid formation will inhibit the translation initiation. Such a mechanism is believed to result in coordinated regulation of the production of OmpC
Even in eukaryotes, it is suggested that anti-sense RNA
(or even anti-sense DNA) may be used to inhibit gene expression and the presence of complementary RNA (or DNA) sequence was shown to reduce gene expression. The extent of utilization of this mechanism for control of gene expression in cell is not known, but it is being definitely utilized now for artificial manipulation of gene expression.
Inhibition of the expression of cloned genes by antisense RNA/DNA technology has been achieved using recombinant-DNA technology through the construction of antisense expression vectors (for details consult Genetic Engineering and Biotechnology 1. Recombinant DNA and PCR (Cloning and Amplification of DNA)
). These genes whose expression was thus manipulated, included the following : (i) thymidine kinase (TK)
gene from herpes simplex virus (HSV), (ii) actin gene
(actin protein is a major component of cytoskeleton), (iii) genes for pigment synthesizing enzyme in petunias; (inhibition of this enzyme led to unusual pigmentation in flowers), (iv) genes for enzymes responsible for fruit ripening in tomatoes (inhibition of these genes will help in slow ripening) and (v) plant virus genes causing diseases. In all these cases, an antisense expression vector was constructed, so that the orientation of the gene of interest is reversed with reference to promoter sequence. These will lead to the synthesis of antisense RNA inhibiting the translation of mRNA. Using this technology inhibition of the expression of several genes as above was achieved successfully.
Not only antisense RNA, but artificially synthesized oligonucleotides (both DNA and RNA) have also been used to inhibit the translation of mRNA. This technology will be increasingly used in future for study of gene function and for treatment of diseases.