FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli. We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision. These mutants also retain their ability to autoregulate fis gene expression. Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction.
Site‐specific DNA inversion in phage Mu is catalysed by the phage‐encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34‐bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT‐rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.
Genetically modified potato (Solanum tuberosum L. cv. Desiree) and tobacco (Nicotiana tabacum cv. Samsun N.N.) plants were used to analyze the effects exerted by the chloroplastic (cp) fructose‐ 1,6‐bisphosphatase (FBPase) on the regulation of light energy discrimination at the level of photosystem II. The cp‐FBPase activity was progressively inhibited by an mRNA antisense to this FBPase. The chlorophyll fluorescence quenching parameters of these transgenic plants were compared to those of wild‐type and transgenic plants that were acclimated to low temperatures. In particular various lines of the transgenic potato and tobacco plants were exposed to a temperature treatment of 10 and 20°C for 10 days. Light intensities were kept low to reduce photoinhibition so that we could analyze exclusively the effects of a modification in the carbon fixation cycle on the chlorophyll fluorescence quenching parameters. The photon flux densities (PFDs) employed at the level of the middle leaves of all plants were set to two different values of 10 μmol m−2 s−1 and 50 μmol m−2 s−1. Subsequent to this 10‐day acclimation the chlorophyll‐fluorescence parameters of all plants were measured. Photoinhibition as expressed by the Fy/Fm ratio was minor in plants subjected to a PFD of 10 μmol m−2 s−1. Higher photon fluence rates of 50 μmol m−2 s−1 at temperatures of 10°C gave rise to a significant reduction in the Fy/Fm ratios obtained from the transgenic plants which were characterized by a restriction in cp‐FBPase capacity to 20% of normal activity. Furthermore, a progressive inhibition of the cp‐FBPase activity induced an amplified nonphotochemical quenching of chlorophyll fluorescence with in the genetically manipulated species (except at 10°C and 50 μmol m−2 s−1). The increase in nonphotochemical quenching depended upon light and temperature. Photochemical quenching of light quanta within the antisense plants declined relative to that in the wild type. To further characterize the mechanisms producing higher levels of nonphotochemical fluorescence quenching. we analyzed several of the xanthophyll cycle pigments. The deepoxidation state of the xanthophyll cycle pigments in potato plants increased with attenuating FBPase activities under all conditions. For tobacco plants, this elevation of the deepoxidation state was only observed at a PFD of 50 μmol m−2 s−1.
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