A visible marker for antisense mRNA expression inplants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehydeaminotransferase antisense gene.

Abstract

Glutamate 1-semlaldehyde otra se [(S)-4-amino-5-oxopentanoate 4,5-amiomutse, EC 5.4.3.8] catalyzes the last step In the conversion of glutamate to 8-aminolevulinate of which eight molecules are needed to synthesize a chlorophyll molecule. Two fill-length cDNA clones that probably represent the homeologous Gsa genes of the two tobacco (Nicotiana tabacum) genomes have been isolated. The deduced amino acid sequences of the 468-residue-long precursor polypeptides differ by 10 amino adds. The cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative in an expression vector and was introduced by Agrobacteriummediated transformation into tobacco plants. Antisense (3), has been attributed to position effects arising from the random integration of antisense genes into the host chromosomes. Additionally, a lack of quantitative relationships between the steady-state level of antisense mRNA, the target RNA, and the amount of protein produced is commonly observed as well as a great variability of the antisense phenotype in response to temperature and light conditions. Cornelissen (13,14) has demonstrated with transformed tobacco cells and plants that the antisense gene can control the transcript level in the nucleus and the translation efficiency of the target mRNA in the cytoplasm independently. The target enzyme was phosphinotricine acetyltransferase produced constitutively with a cauliflower mosaic virus (CaMV) 35S promoter-driven bar gene. In a second transformation of these plants, an antisense bar gene was introduced, and the turnover of the bar mRNA in the cyto-

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…A few glutamate-1-semialdehyde aminotransferase cDNAs have been isolated (Grimm, 1990;Hofgen et al, 1994;Ilag et al, 1994;Matters and Beale, 1994). Their encoded products seem to be very similar to one another and appear to display also a high degree of sequence similarity to the hemL gene product of E. coli and other bacteria, which synthesize 5-aminolevulinic acid via the C, pathway (Grimm, 1990;Sangwan and O'Brian, 1993;Ilag et al, 1994;Matters and Beale, 1994).…”

“…Expression of antisense glutamate-1 -semialdehyde aminotransferase RNA has been shown to cause a remarkable variety of chlorophyll deficiencies in transgenic tobacco plants (Hofgen et al, 1994).…”

Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts

“…In eukaryotes, relatively few examples of antisense RNA-mediated gene regulation have been reported (3)(4)(5)(6)(7)(8). The regulation is thought to occur primarily in the nucleus (9,10) and rarely, if at all, at the translational level (9,11).…”

Function Search in a Large Transcription Factor Gene Family in Arabidopsis: Assessing the Potential of Reverse Genetics to Identify Insertional Mutations in R2R3 MYB Genes

“…In plants, unlike yeast, targeted gene disruption is laborious and inefficient (Kempin et al, 1997). Gene silencing by antisense or sense suppression is also a common approach to studying plant gene function (Kooter and Mol, 1993;Baulcombe, 1996), but the specificity and extent of gene disruption through such methods have not been extensively tested, so the analysis and interpretation of suppression sometimes have proved difficult (van der Krol et al, 1990;Höfgen et al, 1994).…”

“…The Plant Cell so the analysis and interpretation of suppression sometimes have proved difficult (van der Krol et al, 1990;Höfgen et al, 1994).…”