Genetic changes in methotrexate-resistant mosquito cells.

A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.     

Large scale production and characterisations of dihydrofolate reductase from a methotrexate-resistant human lymphoid cell line

Dihydrofolate reductase has been purified from a methotrexate-resistant human lymphoid cell line (CCRF/CEM-R3) and up to 1 mg of enzyme has been obtained from 5 litres of culture. The enzyme has a molecular weight of 22000 ±500 as determined by gel filtration. The pH activity profile shows a single optimum at pH 7.7, where marked activation is observed by addition of 0.2 M NaCl. TheK _m for NADPH is 3μM and dihydrofolate 0.7μM. The binding constant for the inhibitor, methotrexate, is 29 pM     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures

Summary By using mercury(II)-chloride (HgCl₂) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl₂ and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10^–5 and 2.0 x 10^–6 for HgCl₂ and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5–40 g/ml) that had no observable effect on the survival of the mutagenized cultures.     

Elevated amounts of methotrexate-binding protein,different from normal dihydrofolate reductase,in a petunia MTXR-cell line

Summary A petunia cell line, 1ECB, was previously isolated by the stepwise selection procedure, for resistance to methotrexate (MTX), an antimetabolite for the enzyme dihydrofolate reductase (DHFR). Using ammonium sulfate precipitates of cell lysates of cell line 1ECB and its parental cell line (WT), it was found that the mutant has an increase of 400 fold in ³H-MTX binding capacity and a decrease in the affinity for MTX binding, at two orders of magnitude, in comparison with the WT. In addition, the DHFR specific activity in the mutant increased only moderately (5- to 10-fold), this activity is extremely sensitive to MTX inhibition as compared to the WT. It is evident that the MTX resistance of line 1ECB results mainly from overproduction of an MTX-binding protein which differs from the WT DHFR by four biochemical criteria. This protein may serve as a trap for the excess amounts of MTX to which the cells are exposed.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

The regulation of respiration in cell suspensions of Petunia hybrida, studied by inhibiting mitochondrial protein synthesis with chloramphenicol

When Petunia hybrida L. cv. Rosy Morn Fertile suspension cells were inoculated in fresh medium with chloramphenicol (CAP), the activity of cytochrome C oxidase (EC 1. 9. 3. 1), and the respiration via the cytochrome pathway of isolated mitochondria decreased, while in untreated cells these parameters more than doubled in 2–3 days. However, the in vivo respiratory activity of the cytochrome pathway of CAP-treated cells showed a similar course in time as that of untreated cells, even in the presence of an uncoupler, a large rise during the first 2–3 days followed by a decline. This leads to the conclusion that the respiration via the cytochrome pathway, even when measured in the presence of an uncoupler, is not the capacity of this pathway. Furthermore, the results suggest that, although new-synthesis of proteins occurs directly after in-oculation, a large overcapacity must be present of cytochrome pathway elements (at least of those that are mitochondrial encoded). CAP had little effect on the uninhibited respiration and the cyanide-resistant, alternative pathway of the Petunia cells. However, the engagement of the alternative pathway (in the presence or absence of uncoupler) was increased in CAP-treated cells, especially after day 3 of the batch cycle, possibly as an effect of higher sugar degradation in combination with substrate phosphorylation to compensate the loss of ATP-synthesizing ability of the cytochrome pathway. It will be discussed that in general one should be careful using the term 'capacity' for the respiratory pathways.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

Changes of electric potential in pistils of Petunia hybrida Hort. and Brassica napus L. during pollination

The pattern of electric signals accompanying compatible and incompatible pollination were studied in pistils of petunia (Petunia hybrida L.) and rape (Brassica napus L). Electric potential was recorded for 4–7 hours with non-polarizable Ag/AgCl electrodes implanted into the ovary and beneath the sigma. At the end of measurements, pistils were fixed and the growth of pollen tubes was analyzed under a fluorescent microscope. Action potentials appeared in both species. In rape the potential dropped by 10 mV for few minutes after pollination regardless of the compatibility of the cross. In this species, during compatible pollination action potentials with amplitudes of 15–20 mV were recorded up to one hour after pollination. They were followed by a long lasting decrease of the potential by 10 to 50 mV. Contrary, after the self-incompatible pollination, action potentials were rare and of lower amplitudes and the potential gradually raised in comparison to the initial level. During the first hour after the compatible pollination of Petunia hybrida series of action potentials with amplitudes reaching 10–20 mV were recorded. At the time corresponding to the pollen tubes entrance to the transmitting tissue of the style, action potentials reaching up to 40 mV were followed by a steady decrease of the potential. The electric signals traveled along the style with velocity of 25 mm/s. Incompatible pollination in petunia resulted only in minor oscillation and gradual increase of the potential up to 100 mV in comparison to the initial level. The present investigation demonstrated that each phase of pollen-stigma recognition events, germination and growth of pollen tubes within the style have its characteristic pattern of electric changes which was species specific and depended on compatibility of the cross.     

StePSIM – a method for stepwise peak selection and identification of metabolites in 1 H NMR spectra

A method for stepwise selection of peaks in NMR spectra from multiple groups is described. This method is based on initial peak-finding among the spectra and uses jacknife classification performance as the basis for selection of peaks. The selection process is followed by the construction of correlation maps to identify sets of multiplets that are related to each of the selected peaks, aiding in the identification of metabolites that are responsible for differences among the groups. For illustrative purposes, this methodology is applied to a data set that contains 52 spectra from renal cell carcinoma and normal renal tissue samples. The new method is denoted as StePSIM, Stepwise Peak Selection and Identification of Metabolites. Research partially supported by NCI 1 R21 CA89671-01A1 and NIH NCRR 02584     

A kinetic model describes metabolic response to perturbations and distribution of flux control in the benzenoid network of Petunia hybrida

In recent years there has been much interest in the genetic enhancement of plant metabolism; however, attempts at genetic modification are often unsuccessful due to an incomplete understanding of network dynamics and their regulatory properties. Kinetic modeling of plant metabolic networks can provide predictive information on network control and response to genetic perturbations, which allow estimation of flux at any concentration of intermediate or enzyme in the system. In this research, a kinetic model of the benzenoid network was developed to simulate whole network responses to different concentrations of supplied phenylalanine (Phe) in petunia flowers and capture flux redistributions caused by genetic manipulations. Kinetic parameters were obtained by network decomposition and non‐linear least squares optimization of data from petunia flowers supplied with either 75 or 150 mm ²H₅‐Phe. A single set of kinetic parameters simultaneously accommodated labeling and pool size data obtained for all endogenous and emitted volatiles at the two concentrations of supplied ²H₅‐Phe. The generated kinetic model was validated using flowers from transgenic petunia plants in which benzyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT) was down‐regulated via RNAi. The determined in vivo kinetic parameters were used for metabolic control analysis, in which flux control coefficients were calculated for fluxes around the key branch point at Phe and revealed that phenylacetaldehyde synthase activity is the primary controlling factor for the phenylacetaldehyde branch of the benzenoid network. In contrast, control of flux through the β‐oxidative and non‐β‐oxidative pathways is highly distributed.     

Fish cell lines: Two new cell lines derived from explants of trunk musculature ofCynoscion arenarius

Summary Two cell lines were established from explants of trunk musculature of healthy, males sand seatrout,Cynoscion arenarius. One of the lines, designated CyA-1, has been carried through 150 subcultures during 6 yr. The other, designated CyA-2, has been carried through 100 subcultures during 2 yr. Both lines grow well in L15 medium adjusted to 0.150M NaCl and supplemented with 10% fetal bovine serum. Optimal growth occurs at temperatures between 24 and 30°C. The species of origin of both lines was confirmed by a cytotoxic antibody dye exclusion test. The karyotype of CyA-1 has not yet stabilized, showing a modal chromosome number of 120 at Passage 9, 89 at Passage 63, and 79 at Passage 100. The karyotype of CyA-2 is rather stable, with a modal chromosome number of 47 at Passage 1 and 49 at Passage 100. Chromosome morphology of CyA-2 is homogeneous (small, acrocentric), whereas the chromosomes of CyA-1 show considerable size variation (with small chromosomes possibly formed from fragmentation of original structures). Both lines were found to be free of bacterial or fungal contamination. Both lines supported replication of lymphocystis virus strains isolated fromBairdiella chrysura (the silver perch) and fromMicropogon undulatus (the Atlantic croaker) but were refractory to 11 other viruses (4 from fish, 1 from amphibians, and 6 from mammals). This study was supported in part by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58.     

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Summary Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.