Overexpression of an Arabidopsis cDNA Encoding a Sterol-C241-Methyltransferase in Tobacco Modifies the Ratio of 24-Methyl Cholesterol to Sitosterol and Is Associated with Growth Reduction

Higher plants synthesize 24-methyl sterols and 24-ethyl sterols in defined proportions. As a first step in investigating the physiological function of this balance, an Arabidopsis cDNA encoding an S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase, the typical plant enzyme responsible for the production of 24-ethyl sterols, was expressed in tobacco (Nicotiana tabacum L.) under the control of a constitutive promoter. Transgenic plants displayed a novel 24-alkyl-Δ5-sterol profile: the ratio of 24-methyl cholesterol to sitosterol, which is close to 1 in the wild type, decreased dramatically to values ranging from 0.01 to 0.31. In succeeding generations of transgenic tobacco, a high S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase enzyme activity and, consequently, a low ratio of 24-methyl cholesterol to sitosterol, was associated with reduced growth compared with the wild type. However, this new morphological phenotype appeared only below the threshold ratio of 24-methyl cholesterol to sitosterol of approximately 0.1. Because the size of cells was unchanged in small, transgenic plants, we hypothesize that a radical decrease of 24-methyl cholesterol and/or a concomitant increase of sitosterol would be responsible for a change in cell division through as-yet unknown mechanisms.

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA1 was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA53 and GA20, precursors of GA1, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA20, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA1 and GA20 contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA19, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA1 by suppressing GA 20-oxidase expression.

Overexpression of a Homeobox Gene, LeT6, Reveals Indeterminate Features in the Tomato Compound Leaf1

The cultivated tomato (Lycopersicon esculentum) has a unipinnate compound leaf. In the developing leaf primordium, major leaflet initiation is basipetal, and lobe formation and early vascular differentiation are acropetal. We show that engineered alterations in the expression of a tomato homeobox gene, LeT6, can cause dramatic changes in leaf morphology. The morphological states are variable and unstable and the phenotypes produced indicate that the tomato leaf has an inherent level of indeterminacy. This is manifested by the production of multiple orders of compounding in the leaf, by numerous shoot, inflorescence, and floral meristems on leaves, and by the conversion of rachis-petiolule junctions into “axillary” positions where floral buds can arise. Overexpression of a heterologous homeobox transgene, kn1, does not produce such phenotypic variability. This indicates that LeT6 may differ from the heterologous kn1 gene in the effects manifested on overexpression, and that 35S-LeT6 plants may be subject to alterations in expression of both the introduced and endogenous LeT6 genes. The expression patterns of LeT6 argue in favor of a fundamental role for LeT6 in morphogenesis of leaves in tomato and also suggest that variability in homeobox gene expression may account for some of the diversity in leaf form seen in nature.

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO2 Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen1

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 μL L−1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 μmol m−2 s−1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6oC the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.

Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Effect of ATP Sulfurylase Overexpression in Bright Yellow 2 Tobacco Cells1 : Regulation of ATP Sulfurylase and SO42− Transport Activities

To determine if the ATP sulfurylase reaction is a regulatory step for the SO42−-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO42− influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO42− (a toxic SO42− analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism.

Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.