Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene1

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3β-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA9 and GA6.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

A role for CD36 in the regulation of dendritic cell function

Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown that Plasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. Intact P. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-α, they fail to activate T cells and now secrete IL-10. We therefore propose that intact P. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.

Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor.

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.

A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast.

Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.

Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus.

The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.

Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB.

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.