Conrad P. Lichtenstein

What makes an mRNA anti-sensi-itive?

Antisense RNA has been used for some time as a versatile tool for silencing gene expression. There is ample evidence for gene regulation by endogenous antisense transcripts in prokaryotes and increasing insight into the molecular mechanisms underlying such regulation. The introduction of antisense gene constructs into eukaryotes has now become routine but the mechanisms by which gene expression is inhibited are barely understood. In recent years, several examples of endogenous eukaryotic antisense transcripts have been discovered, some of which probably serve regulatory functions. Here we will discuss a model to explain mechanisms of antisense-mediated gene silencing.

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants.

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose [gamma] irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.

Expression of TGMV antisense RNA in transgenic tobacco inhibits replication of BCTV but not ACMV geminiviruses.

Transgenic tobacco plants expressing an antisense RNA targeted against tomato golden mosaic virus (TGMV) show reduced/no symptoms and viral DNA accumulation upon TGMV infection [5]. The targeted region includes the AL1 gene, encoding an essential viral replication protein. This DNA sequence is conserved in various other geminiviruses, suggesting they too might show inhibition of replication in these plants. We infected leaf material with African cassava mosaic virus (ACMV) and beet curly top virus (BTCV) and saw a 4-fold reduction of BCTV, but not ACMV, DNA accumulation, compared to controls. The equivalent regions of BCTV and ACMV show similar overall homology to the TGMV target (63% and 64% respectively), but within this, BCTV displays a 280 nucleotide region of high homology (82%). In contrast, for ACMV, the homology is more dispersed. This indicates that a critical stretch of good complementarity is needed to block expression of the target mRNA, that is effective even within along antisense transcript. These studies indicate the potential for developing a multifunctional antisense cassette.

Prospects for engineering virus resistance in plants with antisense RNA

Abstract The use of antisense RNA to manipulate plant metabolism has already been shown to be successful in altering pigment production in flowers and manipulating the ripening and softening processes in fruit. The major losses in crop yields due to viral pathogens have led to investigations of many strategies for developing virus-resistant plants. By inhibiting viral replication, the use of antisense may permit the engineering of such resistant plants.

A sensitive and simple assay for neomycin phosphotransferase II activity in transgenic tissue.

A new assay for the detection of the enzyme neomycin phosphotransferase II (NPTII) in crude cell extracts is described. The method is based on the chromatographic separation of the compounds resulting from the reaction of NPTII with kanamycin (Km) and [gamma-32P]ATP; the labelled Km.phosphate is subsequently detected by autoradiography. Chromatography is carried out on polyethyleneimine cellulose plates. This assay has been tested with bacterial, plant and animal crude extracts and comparisons with the assays in current use have been made. Our assay has several advantages: it is simple, rapid, sensitive and safe.

Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7

The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E. Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7. Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed. Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins. The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7. The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7.