Reverse genetics

Abstract: Flavonoids represent a large class of secondary plant metabolites, of which anthocyanins are the most conspicuous class, dueto the wide range of colors resulting from their synthesis. Anthocyanins are important to many diverse functions within plants. Synthesis of anthocyanins in petals is undoubtedly intended to attract pollinators, whereas anthocyanin synthesis in seeds and fruits may aid in seed dispersal. Anthocyanins and other flavonoids can also be important as feeding deterrents and as protection against damage from UV irradiation. The existence of such a diverse range of functions and types of anthocyanins raises questions about how these compounds are synthesized and how their synthesis is regulated. The study of the genetics of anthocyanin synthesis began last century with Mendel’s work on flower color in peas. Since that time, there have been periods of intensive study into the genetics and biochemistry of pigment production in a number of different species. In the early studies, genetic loci were correlated with easily observable color changes. After the structures of anthocyanins and other flavonoids were determined, it was possible to correlate single genes with particular structural alterations of anthocyanins or with the presence or absence of particular flavonoids. Mutations in anthocyanin genes have been studied for many years because they are easily identified and because they generally have no deleterious effect on plant growth and development. In most cases, mutations affecting different steps of the anthocyanin biosynthesis pathway were isolated and characterized well before their function was identified or the corresponding gene was isolated. More recently, many genes involved in the biosynthesis of anthocyanin pigments have been isolated and characterized using recombinant DNA technologies. Three species have been particularly important for elucidating the anthocyanin biosynthetic pathway and for isolating genes controlling the biosynthesis of flavonoids: maize (Zea mays), snapdragon (Anfirrhinum majus), and petunia (Wtunia hybrida). Petunia has more recently become the organism of choice for isolating flavonoid biosynthetic genes and studying their interactions and regulation. At least 35 genes are known to affect flower color in petuniawiering and de Vlaming, 1984). Because this field of research has been reviewed fairly extensively in the past (Dooner et al., 1991; van Tunen and MOI, 1991; Gerats and Martin, 1992), in this review we concentrate on the more recent developments in gene isolation and characterization. A review of the genetics of flavonoid biosynthesis in other species was recently covered by Forkmann (1993). The characterization of genetically defined mutations has enabled the order of many reactions in anthocyanin synthesis and their modification to be elucidated. Some reactions have been postulated only on the basis of genetic studies and have not yet been demonstrated in vitro. Chemico-genetic studies have been very important in determining the enzymatic steps involved in anthocyanin biosynthesis and modification. The generation of transposon-tagged mutations and the subsequent cloning of the transposons provided a relatively straightforward means of isolating many genes from maize (Wienand et al., 1990) and snapdragon (Martin et al., 1991). However, a number of genes in the pathway have not been amenable to transposon tagging. Anthocyanin biosynthetic genes have been isolated using a range of methodologies, including protein purification, transposon tagging, differential screening, and polymerase chain reaction (PCR) amplification. Functions of isolated anthocyanin genes can be confirmed by restriction fragment length polymorphism (RFLP) mapping, complementation, or expression in heterologous systems. Reverse genetics has also been used recently to identify gene function; this requires a welldefined pathway to correlate phenotype with gene function. Once a gene has been isolated from one species, it is usually a straightforward task to isolate the homologous gene from other species by using the original clone as a molecular probe.

Abstract: We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 103 plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 104–5 × 107 pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.