Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene.Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3 half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5 half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes.Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.     

Expression in yeast of antisense RNA to ADE1 mRNA

The utility of antisense RNA as a means of regulating gene expression in yeast has been explored by inserting into a high copy number yeast expression vector an ADE1 gene fragment in such an orientation so as to produce antisense RNA in vivo which could hybridize to natural ADE1 mRNA. Northern blotting analysis of total cellular RNA extracted from transformed yeast cells confirmed the presence of high levels of antisense RNA to ADE1 mRNA within cells. However the high level of expression of antisense RNA did not result in production of Ade- cells.     

Monitoring antisense oligodeoxynucleotide activity in hematopoietic cells

Traditionally, methods designed to impair translation through direct interactions with target messenger RNA (mRNA) have been designated as "antisense" strategies because of their reliance on the formation of reverse complementary (antisense) Watson-Crick base pairs between the targeting oligodeoxynucleotide (ODN) and the mRNA whose function is to be disrupted. Proof of putative "antisense effects," and other mechanistic studies, would be greatly facilitated by the ability to directly demonstrate hybridization between an antisense (AS) ODN and its mRNA target in vivo. In addition, evidence of AS activity by demonstrating reduced levels of RNA or protein or by showing cleaved target molecules would lend proof of the concept. In this article we discuss how AS ODN may be used to down-regulate target gene expression with an emphasis on those targets chosen for our investigations, and we summarize the methods employed for this type of study.     

Organ-specific modulation of gene expression in transgenic plants using antisene RNA

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS gene dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense. RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially down mutations in essential genes where only a short 5 sequence of the mRNA is required. They also suggest that the position effect on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.     

Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation.

Cell differentiation is associated either with a complete loss of proliferative potential or with a change in growth requirements. Neoplastic transformation may result from the activation of oncogenes that support growth or from inactivation or loss of tumor suppressor genes, which are thought to regulate differentiation. To examine the relationship between tumor suppressor genes and cell differentiation, we chose the gene "deleted in colorectal cancer" (DCC) and studied its role in a pheochromocytoma cell line, PC-12, using antisense RNA as well as antisense oligonucleotides to DCC. When exposed to nerve growth factor for several days, PC-12 cells develop long dendrites. This morphological change follows the transient expression of immediate early genes and is associated with an up-regulation of DCC. Interestingly, if the up-regulation of DCC was counteracted using an antisense RNA technique, the morphological changes were prevented, but the other parameters of the nerve growth factor response were unaffected. Moreover, when DCC expression was inhibited by antisense oligonucleotides to DCC in nerve growth factor-differentiated cells, the neuron-like phenotype was reversed. Our results demonstrate that the gene DCC is involved in a distal segment of neural differentiation and provide the first direct evidence that a tumor suppressor gene plays a role in cell differentiation.     

Stimulatory effect of an indirectly attached RNA helicase-recruiting sequence on the suppression of gene expression by antisense oligonucleotides

Antisense oligonucleotides (ODNs) are powerful tools with which to determine the consequences of the reduced expression of a selected target gene, and they may have important therapeutic applications. Methods for predicting optimum antisense sites are not always effective because various factors, such as RNA-binding proteins, influence the secondary and tertiary structures of RNAs in vivo. To overcome this obstacle, we have attempted to engineer an antisense system that can unravel secondary and tertiary RNA structures. To create such an antisense system, we connected the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, to an antisense sequence so that helicase-binding hybrid antisense ODN would be produced in cells. We postulated that this modification would enhance antisense activity in vivo, with more frequent hybridization of the antisense ODN with its targeting site. Western blotting analysis demonstrated that a hybrid antisense ODN targeted to the bcl-2 gene suppressed the expression of this gene more effectively than did the antisense ODN alone. Our results suggest that the effects of antisense ODNs can be enhanced when their actions are combined with those of RNA helicases.     

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.     

Antisense RNA in transgenic plants

The data on the effects of antisense RNA in plants is reviewed. Results of expression of the genes for selective markers, antisense reporter genes, functioning and viral genes are analyzed. The molecular mechanisms for inhibiting effects of antisense RNA and the potential use of the phenomenon in the plants biotechnology are discussed. The formation of long duplexes between the antisense RNA and messenger RNA are supposed to be irrelevant to suppression of gene expression in plants by the antisense RNA.     

In vitro and in vivo action of antisense RNA

The transient or permanent expression of antisense RNA represents one option to apply antisense techniques in biotechnology and medical research. Despite the increasing importance and use of antisense nucleic acids as well as their significant antisense-specific phenotypic effects in vivo, there is an obvious lack of explanation for the mechanism of their action. By studying naturally occurring antisense RNA and analyzing their mechanism of action we attempt to learn more about the design, the use, and the critical parameters of artificial antisense RNA. Attempts to derive models from biochemical and structural studies for the interactions between antisense RNAs and their targets will be discussed.     

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to introns

The objective of the experiments described in this paper was to test the potential of antisense RNAs complementary to the internal portion of an intron to inhibit the splicing process and to determine the mechanism of such inhibition. The results obtained indicate that RNA fragments complementary to the internal portion of an intron can effectively inhibit the splicing of pre-mRNA. Inhibition was observed only with antisense RNA complementary to pre-mRNA suggesting that the inhibitory effect was due to the formation of a hybrid with the corresponding portion of the pre-mRNA's intron. The observed inhibition was not due to interference with possible intron elements essential for the splicing process, for the deletion of the sequences complementary to inhibitory antisense RNA from the corresponding pre-mRNA molecule did not affect the efficiency of a splicing reaction, and the addition of antisense RNA to pre-mRNA mutants carrying such deletions did not result in any inhibition. Our results indicate that the observed inhibition is a function of the length of the antisense RNA expressed as a fraction of an intron with which it interacts when antisense RNA is modified by incorporation of a "hinge" element, it loses its inhibitory potential suggesting that the inhibitory effect is probably due to limitation of conformational flexibility of an intron.     

Antisense inhibition of Xbrachyury impairs mesoderm formation in Xenopus embryos

Expression of the Xbrachyury (Xbra) gene was inhibited by antisense RNA synthesized in situ from an expression vector read by RNA polymerase III, injected into the fertilized egg or the 2-cell stage embryo of Xenopus laevis. Antisense-treated embryos had markedly reduced levels of Xbra mRNA and protein, and showed deficiencies in mesodermal derivatives and axis formation. In particular, organization of the posterior axis was affected, but often the anterior axis was also reduced. Some embryos failed to form mesoderm altogether and remained amorphous. The antisense effect is dose-dependent and may be "rescued" by overexpression of Xbra. In Xbra-deficient embryos, expression of several mesodermal genes (Xvent, pintallavis, Xlim, Xwnt-8 and noggin) was reduced to varying degrees, whereas goosecoid levels remained normal. The modified expression levels were partly normalized when Xbra deficiency was rescued. The observation that antisense inhibition yields slightly different phenotypes from dominant-negative inhibition suggests the recommendation of using several surrogate genetic approaches to determine the functional role of a gene in Xenopus development.     

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.