Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6543 - 7 The 3' untranslated region of a rice alpha-amylase gene functions as a sugar-dependent mRNA stability determinant; Chan MT et al.; In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs . The sugar repression of alpha-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation . We have shown previously that sugar repression of alpha-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability . The alpha-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells . To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of alphaAmy3 3' untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells . We found that the entire alphaAmy3 3' UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation . Analysis of reporter mRNA half-lives demonstrated that the entire alphaAmy3 3' UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA . Nuclear run-on transcription analysis further confirmed that the alphaAmy3 3' UTR and the two subdomains did not affect the transcription rate of promoter . The identification of sequence elements in the alpha-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms. J Biol Chem, 1998 Apr 24, 273(17), 10120 - 31 Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer; Lu CA et al.; Expression of alpha-amylase genes in both rice suspension cells and germinating embryos is repressed by sugars and the mechanism involves transcriptional regulation . The promoter of a rice alpha-amylase gene alphaAmy3 was analyzed by both loss- and gain-of-function studies and the major sugar response sequence (SRS) was located between 186 and 82 base pairs upstream of the transcription start site . The SRS conferred sugar responsiveness to a minimal promoter in an orientation-independent manner . It also converted a sugar-insensitive rice actin gene promoter into a sugar-sensitive promoter in a dose-dependent manner . Linker-scan mutation studies identified three essential motifs: the GC box, the G box, and the TATCCA element, within the SRS . Sequences containing either the GC box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression . Nuclear proteins from rice suspension cells binding to the TATCCA element in a sequence-specific and sugar-dependent manner were identified . The TATCCA element is also an important component of the gibberellin response complex of the alpha-amylase genes in germinating cereal grains, suggesting that the regulation of alpha-amylase gene expression by sugar and hormone signals may share common regulatory machinery. Plant Physiol, 1998 May, 117(1), 43 - 53 Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds van Hengel AJ, Guzzo F, van Kammen A, de Vries SC. Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11 . Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes . No expression was found in somatic embryos . In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds . No expression was found in zygotic embryos . These results support the hypothesis that the EP3 endochitinase has a "nursing" function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis. Plant Cell Physiol, 1998 Mar, 39(3), 275 - 84 Cloning and expression of the soybean chlH gene encoding a subunit of Mg-chelatase and localization of the Mg2+ concentration-dependent ChlH protein within the chloroplast; Nakayama M et al.; From the soybean cDNA library, we isolated and analyzed the chlH gene encoding a subunit of Mg-chelatase . The subunit was a polypeptide of 1,383 amino acids with a molecular mass of 153,491 Da, which shared 90% identity with the olive gene from Antirrhinum majus . The regulation of the expression of chlH was investigated in photomix-otrophic soybean suspension cells (SB-P) . The expression was light-inducible, and the induction was more rapid than those of chlI and cab2 . Furthermore, the levels of the transcripts and products of chlH appeared to be regulated by a circadian oscillation . The subchloroplastic localization of ChlH was investigated by immunoblot analyses with antiserum against recombinant ChlH . Depending on the concentration of Mg2+ in the lysis buffer, the localization of ChlH protein migrated between the stroma and the envelope membrane; ChlH was localized on the envelope membrane, a major site of chlorophyll biosynthesis, when the Mg2+ concentration of the lysis buffer was high (above 5 mM) . These results indicated that the activity of Mg-chelatase was regulated by modulation of the expression and subchloroplastic localization of ChlH protein. Plant Cell, 1998 Mar, 10(3), 435 - 50 Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp; Zhang S et al.; Two purified proteinaceous fungal elicitors, parasiticein (an alpha elicitin) and cryptogein (a beta elicitin), as well as a fungal cell wall-derived carbohydrate elicitor all rapidly activated a 48-kD kinase in tobacco suspension cells . The maximum activation of this kinase paralleled or preceded medium alkalization and activation of the defense gene phenylalanine ammonia-lyase (PAL) . In addition, the two elicitins, which also induced hypersensitive cell death, activated a 44- and a 40-kD kinase with delayed kinetics . By contrast, the cell wall-derived elicitor only weakly activated the 44-kD kinase and failed to activate the 40-kD kinase . The size and substrate preference of the 48-kD kinase are reminiscent of the recently purified and cloned salicylic acid-induced protein (SIP) kinase, which is a member of the mitogen-activated protein kinase family . Antibodies raised against a peptide corresponding to the unique N terminus of SIP kinase immunoreacted with the 48-kD kinase activated by all three elicitors from Phytophthora spp . In addition, the cell wall elicitor and the salicylic acid-activated 48-kD kinase copurified through several chromatography steps and comigrated on two-dimensional gels . Based on these results, all three fungal elicitors appear to activate the SIP kinase . In addition, inhibition of SIP kinase activation by kinase inhibitors correlated with the suppression of cell wall elicitor-induced medium alkalization and PAL gene activation, suggesting a regulatory function for the SIP kinase in these defense responses. Plant Cell, 1998 Jan, 10(1), 51 - 62 AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity; Kim EJ et al.; Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants . We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database . The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively . Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E . coli triple mutant . In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo . A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells . RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation . It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types. Biol Pharm Bull, 1998 Feb, 21(2), 163 - 6 The involvement of Ca2+-dependent protein kinase in the regeneration of rice cultured suspension cells; Karibe H et al.; Short-term cultured cells of rice (Oryza sativa) were found to be capable of regeneration, in contrast to those obtained from long-term cultures . For clarification of the mechanism of regeneration, it was first necessary to distinguish protein kinase activity in long-term and short-term cultured cells; this activity was found greater in the former than latter . The activity was dependent on calcium, not phospholipid, phorbol ester or calmodulin . The apparent Mr of both Ca2+-dependent protein kinases was 32 kDa according to gel phosphorylation . Phosphoserine was identified in serine residues in phosphorylated histone III-S by phosphoamino acid analysis . A Ca2+-dependent protein kinase having a relative Mr of 32 kDa is thus shown to be possibly essential to regeneration in rice cultured cells. J Virol, 1998 Mar, 72(3), 2323 - 34 Anchorage-independent transcription of the cyclin A gene induced by the E7 oncoprotein of human papillomavirus type 16; Schulze A et al.; To develop an experimental model for E7-mediated anchorage-independent growth, we studied the ability of E7-expressing NIH 3T3 subclones to enter S phase when they were cultured in suspension . We found that expression of E7 prevents the inhibition of cyclin E-associated kinase and also triggers activation of cyclin A gene expression in suspension cells . A point mutation in the amino terminus of E7 prevented E7-driven rescue of cyclin E-associated kinase activity in suspension cells; however, cells with this mutation retained some ability to activate cyclin A gene expression and promote S-phase entry . Activation of cyclin A gene expression by E7 was correlated with an increased binding of free E2F to a regulatory element in the cyclin A promoter which mediates both repression of cyclin A upon loss of adhesion and its reactivation by E7 . Surprisingly, expression of E7 led to a nuclear accumulation of one species of free E2F, namely, an E2F-4-DP-1 heterodimer, that is exclusively cytoplasmic in the absence of E7 . Taken together, the data reported here indicate that several different E7-dependent changes of cellular-growth-regulating pathways can cooperate to allow adhesion-independent entry into S phase. Plant Mol Biol, 1997 Nov, 35(5), 539 - 50 Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter; Bao X et al.; In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits . To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter . Fifteen introns were identified . The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity) . BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis . GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf . Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression . A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron . In addition, several conserved regulatory elements were identified in the BC promoter . Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs. J Cell Physiol, 1997 Dec, 173(3), 361 - 70 Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin; Chun J et al.; HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX) . The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin . Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations . There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread . Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content . The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation . In addition, PKC epsilon, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin . Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment . This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading . PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction . In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole . The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading . One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. Photochem Photobiol, 1997 Oct, 66(4), 464 - 70 Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells; Takeda J et al.; The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPAL1) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined . The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPAL1 promoter is categorizable as one of the UVB light responses . The same assay system was applied to variously truncated gDcPAL1 promoters and to CaMV35S promoter fusion with various parts 5'-upstream of the gDcPAL1 promoter . The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant response . However, gDcPAL1 promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e . they responded to 260 nm light as much as to 280 nm light . The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light . Therefore we assumed that the gDcPAL1 promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the down-stream below -190 (UVB and C responsive) . The overall UVB response of the gDcPAL1 full-length promoter is explained as the result of interaction of these three components. Leukemia, 1997 Sep, 11(9), 1453 - 8 Mechanical agitation induces gene expression of NOR-1 and its closely related orphan nuclear receptors in leukemic cell lines; Bandoh S et al.; NOR-1, NGFI-B and Nurr1 are closely related transcription factors that constitute a distinct subfamily within the nuclear receptor superfamily . Genes for these proteins are immediate-early genes, and are inducible in diverse cell types by various stimuli . In the present study, we investigated the effect of mechanical agitation on the gene expression of these transcription factors in cultured suspension cells by the quantitative reverse transcription-polymerase chain reaction . We found that mechanical agitation transiently induced NOR-1, NGFI-B and Nurr1 mRNAs in several leukemic cell lines in a dose-dependent manner . This induction was most pronounced in the HL-60 promyelocytic leukemia cell line, but also occurred to a lesser extent in other cell lines including KG-1, THP-1 and U937 cells . The induction was attenuated by serum or albumin, which are shear stress protectants for suspension culture cells . These reagents did not suppress forskolin-induced NOR-1 gene expression . These findings suggest the involvement of fluid shear stress in agitation-induced immediate-early gene expression . Since even moderate agitation could cause the induction, investigators should be cautious when evaluating the expression of immediate-early genes in some leukemic cell lines. Plant J, 1997 Aug, 12(2), 313 - 22 Identification of the peroxisomal targeting signal for cottonseed catalase; Mullen RT et al.; Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined . The most common peroxisome targeting signal (PTS) is a C-terminal tripeptide composed of a conserved SKL motif (type 1 PTS) . The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY-2 suspension cells serving as an in vivo import system . To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA)epitope-tagged at its N-terminus . Bombardment with HA-Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY-2 cells . The C-terminal tripeptide of Ccat, PSI, is necessary for import . Evidence for this were mislocalizations to the cytosol of PSI-truncated Ccat and AGV-substituted (for PSI) Ccat . PSI-COOH, however, was not sufficient to re-route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI-COOH was sufficient . Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT-KPSI) decreased import efficiency . However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g . CAT-NVKPSI) . Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non-conserved residues (e.g . -AGVNVRPSI for -SRLNVRPSI) . These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI-COOH) whose residues are functionally dependent on a strict context of adjacent C-terminal amino acid residues. J Cell Sci, 1997 Aug, 110 ( Pt 16), 1947 - 56 Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice; Holwell TA et al.; Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner . Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated . A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences . Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline . The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts . The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion . Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus . Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force . Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound . Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture . These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury. Placenta, 1997 Sep, 18(7), 577 - 85 In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium; Morrish DW et al.; Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation . After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection . EGF was required for mature syncytial formation . Compared to log-phase proliferating HeLa cells, uptake of {3H}thymidine incorporation was low and quickly decreased to negligible levels . Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture . Proto-oncogene changes were similar in attached and suspension cells . Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations . EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF . The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression . However, EGF is required for extensive syncytial development. FEBS Lett, 1997 Aug 11, 413(1), 181 - 4 Isolation and characterization of replication protein A (RP-A) from tobacco cells; Garcia-Maya MM et al.; Replication protein A (RP-A) was isolated from tobacco suspension cells and purified to near homogeneity by a procedure involving isolation of protoplasts, preparation of nuclei, nuclear lysis, binding to a column of single-stranded (ss) DNA cellulose and elution at different salt concentrations . The purified protein contained three subunits with molecular masses of 70, 34 and 14 kDa, and was free from nuclease activity . Tobacco RP-A had a high affinity for ssDNA . Binding competition experiments indicated only a weak affinity for double-stranded DNA and no detectable affinity for ssRNA . Photochemical cross-linking experiments indicated that the 70 kDa subunit has the ssDNA-binding activity . Tobacco RP-A was able to stimulate the activity of a tobacco alpha-like DNA polymerase about 4-fold . This is the first isolation of RP-A from a plant and the procedure may be generally applicable to other plant species. Plant Cell, 1997 May, 9(5), 809 - 24 Salicylic acid activates a 48-kD MAP kinase in tobacco; Zhang S et al.; The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors . Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate . This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase) . Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not . Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation . The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells . The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity . Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene . Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses. Biologicals, 1997 Mar, 25(1), 65 - 73 Phenotypic features of BHK-21 cells used for production of foot-and-mouth disease vaccine; Amadori M et al.; BHK-21 c13 monolayer and suspension cells were investigated with regard to some phenotypic features which could bear on the quality of foot-and-mouth disease virus (FMDV) antigen produced in them . Despite good viability, suspension cells differed from monolayer cells in fundamental features of susceptibility to FMDV . Most important, FMD virus particles grown in suspension cells at high passage levels were shown to be largely degraded following inactivation with an aziridine compound . Suspension cells were characterised by a downregulation in the surface expression of both alpha 5 and alpha V integrin chains . According to the results of binding assays, both integrins could act as FMDV receptors on BHK-21 c13 cells . Reduced surface expression of integrins was correlated with disappearance of actin stress fibres, which would play a role in regular encapsidation of viral RNA and hence in stability of virus particles . With regard to FMD vaccine production, practical suggestions are put forward to evaluate the quality of BHK-21 c13 cells and FMDV Ag, which must prove stable during downstream processing. Plant J, 1997 Mar, 11(3), 613 - 21 The green fluorescent protein as a marker to visualize plant mitochondria in vivo; Kohler RH et al.; To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated . The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue . For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary . Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP . In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm . It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts . In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye . This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development . Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria. Plant Physiol, 1997 Mar, 113(3), 853 - 62 Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes; Hwang CF et al.; Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes . We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y . Lin, C.-F . Hwang, J.B . Brown, C.-L . Cheng {1994} Plant Physiol 106: 477-484) . Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis . In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity . To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction . The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate . A 12-bp sequence is conserved between the NP1 site and the two NP2 sites . This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes . Gel mobility shift experiments indicate that these three regions bind to similar proteins . The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves. Plant J, 1997 Feb, 11(2), 263 - 76 Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice; Meijer AH et al.; This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors . Oshox1 maps to chromosome 10 and belongs to a family of related rice genes . Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein . This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms . Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants . Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator . In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor . Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression . Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain. Plant J, 1997 Feb, 11(2), 181 - 90 Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin; Sauter M; Progress through the eukaryotic cell cycle is regulated by cyclin-dependent cdc2 protein kinases . In rice (Oryza sativa L), two cdc2 protein kinases, cdc2Os-1 and cdc2Os-2, and two cyclins, cycOs1 and cycOs2, have been described . In this study, we report on the cell-cycle phase-specific expression of these genes . Using partially synchronized suspension cells from rice, we found that cdc2Os-1 was expressed constitutively throughout the cell cycle . The cdc2Os-2 transcript level was elevated in G1 and S phase . The cycOs1 and cycOs2 transcripts increased steadily through G2 and dropped off rapidly in mitosis as is typical for mitotic cyclins . We hypothesize that the cdc2Os-2 gene product acts in G1/S and that the growth-promoting hormone gibberellin (GA) that induces expression of cdc2Os-2, cycOs1 and cycOs2 in the intercalary meristem of deepwater rice internodes accelerates G1/S phase progression through increased expression of cdc2Os-2 and G2/M phase progression through increased expression of the mitotic cyclins cycOs1 and cycOs2 . The R2 gene from rice has 55% sequence identity to the cdc2-activating kinase (CAK) family of protein kinases which have been shown to phosphorylate and thereby activate cdc2 protein kinases in animals and yeast . In partially synchronized suspension cells, R2 mRNA levels were elevated in G1 and S phase . In GA-treated rice internodes, R2 transcript levels were elevated in the meristem and part of the elongation zone . These results are consistent with a role for R2 in regulating G1/S phase progression. Planta, 1997, 201(3), 349 - 58 Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L . cells; Smertenko A et al.; Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L . was analysed using a panel of specific antibodies . Polyglutamylated, tyrosinated, nontyrosinated, acetylated and delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-tubulin subunits . Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts . They were, however, located differently in the various microtubule structures . The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and delta 2-tubulin provided dot-like staining of interphase microtubules . Additionally, immunoreactivity of antibodies against acetylated and delta 2-tubulins was strong in the pole regions of mitotic spindles . High-resolution isoelectric focusing revealed 22 tubulin charge variants in N . tabacum suspension cells . Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit . Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-tubulin reacted with only one . The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Plant Mol Biol, 1997 Jan, 33(2), 359 - 66 Molecular cloning, characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach; Saito K et al.; Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis . A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves . Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA . It contains an N-terminal extension of ca . 60 amino acids in addition to the sequences from other organisms . The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA . The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282 . The enzymatic activity of PSA was demonstrated in vitro in the extracts of E . coli over-expressing the cDNA . Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome . RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition. FEBS Lett, 1996 Dec 2, 398(2-3), 248 - 52 Arrest of mitochondrial biogenesis in copper-treated sycamore cells; Padua M et al.; Sycamore suspension cells (Acer pseudoplatanus L.) were grown in the presence of sublethal concentrations of copper (50 microM) . During the first 5-6 days of treatment, growth was not affected, but cell respiration (coupled and uncoupled) declined to approximately 60% of its normal value . This decline of respiration was attributed to a progressive diminution of the number of mitochondria in copper-treated cells, based on the demonstration of the concomitant decline of (1) cardiolipin (diphosphatidylglycerol) and cytochrome aa3 (cytochrome oxidase), two specific markers of mitochondrial inner membrane, and (2) fumarase activity, a specific marker of mitochondrial matrix space . In addition, the mitochondria extracted from copper-treated cells presented the same properties as those from control cells, concerning substrate oxidation, cardiolipin and cytochrome aa3 contents, and fumarase activity . These results strongly suggest that copper triggered an arrest of mitochondrial biogenesis, which preceded cell division arrest. Plant J, 1996 Dec, 10(6), 1177 - 86 A plant in vitro system for the nuclear import of proteins; Merkle T et al.; This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei . In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored . It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent . Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system . Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-gamma-S . In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid . Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0-4 degrees C) . The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors . However, a block of nuclear import with GTP-gamma-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins . These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other. Gene Ther, 1996 Nov, 3(11), 1010 - 7 An electron microscopy study into the mechanism of gene transfer with lipopolyamines; Labat-Moleur F et al.; Cationic amphiphiles have been shown to mediate gene transfer to eukaryotic cells, although the nature and fate of the lipid-DNA complexes is still a matter of debate . Negative staining transmission electron microscopy (TEM) of the complexes in physiological medium, as well as thin-section TEM of transfected cells has been used to visualize the particles and the possible pathways leading to transgene expression . Lipopolyamines form a network of tubular micelles into which plasmid DNA is intertwined and condensed; the cationic particles contain hundreds of plasmid molecules and are heterogeneous with respect to size (0.1-0.5 microgram) and shape . Adherent cells (293M, 3T3, MRC5, primary leptomeningeal cells) take them up readily within minutes by spontaneous endocytosis . Among suspension cells, lymphocytes only incidentally show cytoplasmic inclusions and monocytes degrade the particles by phagocytosis . The marked decrease in transfection efficiency generally observed between adherent and nonadherent cells is thus due to reduce cell binding . This suggests that cationic particles bind to membrane components responsible for Ca2+-mediated cell anchoring to the extracellular matrix . Cation/anion-mediated endocytosis leads to endosomes that are entirely filled with the particles . Consequently, two escape mechanisms may operate: disruption of the lamellar envelope in close contact with tubular micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture . Even for moderately transfected MRC5 cells, 10(2)-10(3) particles are found either free or in cytoplasmic vacuoles 24 h after transfection, highlighting a very inefficient nuclear translocation process . Such high numbers are also the clue to the small concentration window between transfection and cytotoxicity that is often observed with nonviral vectors . Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exchange in the cytoplasm or in the nucleus . The still lower efficiency of free plasmid translocation to the nucleus suggests an active role for the cationic lipid during this step . Although the last stages of the transfection mechanism remain unclear, the present work shows that the major barrier which hampers in vitro gene delivery with cationic vectors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors. Plant Physiol, 1996 Nov, 112(3), 931 - 8 Immunolocalization of mannitol dehydrogenase in celery plants and cells; Zamski E et al.; Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme . MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles . Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction . Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts . The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression . Additionally, a developmental component may regulate the distribution of MTD within celery plants. Blood, 1996 Nov 1, 88(9), 3465 - 73 Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins; Hudson KM et al.; Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines . We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes . Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin . The K562, HEL, and CHRF-288-11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements . Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment . Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization . A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot . Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage. J Biol Chem, 1996 Oct 25, 271(43), 26998 - 7004 Carbohydrate starvation stimulates differential expression of rice alpha-amylase genes that is modulated through complicated transcriptional and posttranscriptional processes; Sheu JJ et al.; Expression of alpha-amylase genes in cultured rice suspension cells is induced by sucrose starvation . To study the mechanism of sugar metabolite regulation on the expression of individual alpha-amylase genes, DNA fragments specific to each of eight rice alpha-amylase genes were synthesized and used as gene-specific probes . Comparison of the relative abundance of mRNA revealed that expression of the eight alpha-amylase genes in rice cells was differentially regulated by sucrose starvation . Accumulation of all the alpha-amylase mRNAs increased in response to sucrose starvation; however, levels of the alphaAmy3 and alphaAmy8 mRNAs were distinctly higher and constituted 90% of total alpha-amylase mRNAs . RNA gel blot and nuclear run-on transcription analyses demonstrated a positive correlation between the increased transcription rates and the elevated steady-state levels of alpha-amylase mRNAs induced by sucrose starvation . The half-lives of alphaAmy3, alphaAmy7, and alphaAmy8 were prolonged by sucrose-starvation; however, the stability of the three mRNAs seems controlled by different mechanisms . The translation inhibitors cycloheximide and anisomycin preferentially blocked the sucrose-suppressed expression of alphaAmy3 but not that of alphaAmy7 and alphaAmy8 . These inhibitors also enhanced the sucrose starvation-induced accumulation of alphaAmy3 mRNA but not that of alphaAmy7 or alphaAmy8 mRNAs . Cycloheximide did not significantly alter the transcription rates of alpha-amylase genes, suggesting that labile proteins may selectively stabilize the alphaAmy7 and alphaAmy8 mRNAs but destabilize the alphaAmy3 mRNA. J Biol Chem, 1996 Oct 18, 271(42), 25742 - 5 Histone H1 enhances the DNA binding activity of the transcription factor EmBP-1; Schultz TF et al.; Previous work indicated that nuclear extracts isolated from embryogenic rice suspension cells treated with the phytohormone abscisic acid (ABA) have enhanced binding activity to an ABA response element (Em1a) in the promoter of the Em gene from wheat . We identified an activity in wheat and maize nuclear extracts that enhances binding of the recombinant transcription factor EmBP-1 to Em1a by 80-fold . Fractionation of nuclear extracts led us to identify histone H1 and HMGb (but not HMGc or -d) as two factors that can enhance the ability of EmBP-1 to bind to Em1a and account for at least a part of this activity of nuclear extracts . Our results, which indicate for the first time that histone H1 possesses this type of activity, lend further support to the model that positively charged proteins can drastically affect the DNA binding activity of specific transcription factors . Furthermore, our study points to these chromosomal proteins as potential targets of an ABA-mediated modification (e.g . acetylation) that could affect the regulation of Em gene expression. Plant Cell Physiol, 1996 Sep, 37(6), 748 - 53 Phosphorylation of a protein (pp56) is related to the regeneration of rice cultured suspension cells; Komatsu S et al.; Short-term cultured suspension cells of rice (Oryza sativa L.) are capable of regeneration, but not in long-term culture . For clarification of the mechanism of regeneration, protein phosphorylation in short-term and long-term cultured suspension cells was compared by two dimensional-polyacrylamide gel electrophoresis . A 56 kDa protein having an isoelectric point of 4.5 was phosphorylated in vitro in short-term cultured suspension cells, but was not phosphorylated after regeneration . This protein in long-term cultured suspension cells remained phosphorylated after transfer to the regeneration medium . However, using an antibody raised against this protein from short-term cultured suspension cells, it was always detected in long-term and short-term cultured suspension cells after transfer to the regeneration medium . The partial amino acid sequence of the HPLC-purified protein showed homology to a calcium-binding protein from maize . The phosphorylation of the 56 kDa protein (pp56) appears to be associated with the regeneration of cultured rice cells. Plant Physiol, 1996 Sep, 112(1), 343 - 51 Purification and characterization of pyrophosphate-dependent phosphofructokinase from phosphate-starved Brassica nigra suspension cells; Theodorou ME et al.; Previously, we reported that inorganic phosphate (Pi) deprivation of Brassica nigra suspension cells or seedlings leads to a progressive increase in the alpha: beta-subunit ratio of the inorganic pyrophosphate (PPi)-dependent phosphofructokinase (PFP) and that this coincides with a marked enhancement in the enzyme's activity and sensitivity to its allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2) . To further investigate the effect of Pi nutrition on B . nigra PFP, the enzyme was purified and characterized from Pi-starved B . nigra suspension cell cultures . Polyacrylamide gel electrophoresis, immunoblot, and gel-filtration analyses of the final preparation indicated that this enzyme exists as a heterooctamer of approximately 500 kD and is composed of a 1:1 ratio of immunologically distinct alpha (66 kD) and beta (60 kD) subunits . The enzyme's alpha subunit was susceptible to partial proteolysis during purification, but this was prevented by the presence of chymostatin and leupeptin . In the presence and absence of 5 microM Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and 7.6, respectively . Maximal activation of the forward and reverse reactions by Fru-2,6-P2 occurred at pH 6.8 . The potent inhibition of the forward activity by Pi (concentration of inhibitor producing 50% inhibition of enzyme activity {I50} = 1.3 mM) was attributed to a marked Pi-dependent reduction in Fru-2,6-P2 binding . The reverse reaction was substrate-inhibited by Pi (I50 = 13 mM) and product-inhibited by PPi (I50 = 0.9 mM) . The kinetic data are consistent with the hypothesis that PFP may function to bypass the ATP-dependent PFP in Pi-starved B . nigra . The importance of the Pi nutritional status to the regulation and predicted physiological function of PFP is emphasized. Mol Plant Microbe Interact, 1996 Sep, 9(7), 594 - 9 Mutational analysis of the putative nicking motif in the replication-associated protein (AC1) of bean golden mosaic geminivirus; Hoogstraten RA et al.; Geminiviruses are circular single-stranded DNA viruses that replicate by a rolling circle mechanism involving the viral-encoded AC1 protein . DNA nicking is necessary both for initiating replication of the covalently closed double-stranded DNA templates and for releasing unit-length monomers . The effects of mutations in a putative nicking motif (K101 A Y I D K106; E . V . Koonin and T . V . Ilyina, J . Gen . Virol . 73:2763-2766, 1992) of the AC1-derived protein for bean golden mosaic geminivirus isolate GA (BGMV-GA) were studied . The amino acids equivalent to Y103 and K106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses . Phaseolus vulgaris plant infectivity assays showed that the mutants K101-->H, K101-->A, and D105-->T produced symptoms, but mutants Y103-->A, Y103-->F, K106-->R, and K106-->H did not . A mutant with a stop codon in the N terminus of the AC4 open reading frame (ORF) produced the same symptoms as the wild-type BGMV-GA . Only those that were infectious replicated in NT-1 tobacco suspension cells . These results indicate that the Y103 and K106 residues are essential for replication, and that this putative DNA-nicking motif of the AC1 ORF may be functional in the rolling circle mechanism of replication for geminiviruses . The potential role of these mutants in the design of antiviral strategies is discussed. Plant J, 1996 Aug, 10(2), 251 - 9 Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus; Chan J et al.; A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised . The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents . Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules . Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins . Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble . By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol . In the electron microscope, these arrays are seen to be composed of mainly single microtubules . Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs . Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules . Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained . Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints') . Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs. J Cell Biol, 1996 Jun, 133(6), 1251 - 63 Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates; Aubert S et al.; Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process . Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids . In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant . In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated . P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy . Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM . Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy . The biochemical marker P-Cho was used to investigate the factors controlling autophagy . P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources . Both compounds entered the cells and sustained normal rates of respiration . No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy . On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles . These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates. Anal Biochem, 1996 May 1, 236(2), 322 - 6 A chemiluminescence-based method for the detection and quantification of antigen-antibody interactions on the surface of eukaryotic cells; Lischke A et al.; Enhanced chemiluminescence was applied to detect the binding of monoclonal antibodies to surface antigens on intact cells . The fast and simple assay is performed in the microtiter scale and thus allows for the simultaneous processing of a large number of samples with a sensitivity comparable to conventionally used techniques such as cytometry or Western blot analysis . In two model experiments, we demonstrate (a) the detection of a heterologously expressed cytokine receptor subunit on the surface of suspension cells and (b) the screening of hybridoma clones for the production of antibodies specifically recognizing surface antigens on a tumor cell line . Moreover, the assay is shown to be suitable for the determination of antibody affinities and of antibody binding sites per cell. Biotechnol Prog, 1996 May-Jun, 12(3), 398 - 402 Confocal laser scanning microscopy examination of cell distribution in macroporous microcarriers; Bancel S et al.; Macroporous microcarriers are often used to cultivate animal cells . The pores in the interior of the beads provide surface and space for cell growth . It is not clear how anchorage-dependent and suspension cells populated these microcarriers during cultivation . Confocal laser scanning microscopy was employed to perform time lapse observation of the cells in the interior . The structure of the bead was stained with fluorescein isothiocyanate for visualization, while the cells were stained with dialkyl indocarbocyanines for tracking over time . It was observed that mouse fibroblastic cells CRE BaG2 did not move extensively after initial attachment . Some cell divisions were observed during the course of the experiments, and essentially all cells remained viable throughout . Few hybridoma cells were deposited into the pores in the interior of the microcarriers . The results suggest that the occupancy of the internal volume by cells after prolonged cultivation is largely due to the growth of cells that are deposited in the interior as opposed to the migration of cells from the external surface into the interior . This method of observing cell behavior in a three-dimensional structure may find applications in other three-dimensional cell culture systems . The animation of time lapse sections is available on the worldwide web at approximately wshu_grp/acre/microcarrier.html. Planta, 1996, 200(1), 2 - 12 Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase; Zhang K et al.; In excised pith parenchyma from Nicotiana tabacum L . cv . Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34cdc2-like protein, recoverable in the p13suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive . In suspension-cultured N . plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34cdc2-like H1 histone kinase . Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells . Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13suc1-purified enzyme from pith and suspension cells cultured without cytokinin . Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis . Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals. Plant Cell, 1996 Jan, 8(1), 119 - 132 A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants; Liu B et al.; The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development . However, relatively little is known about plant KLPs . We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide . Such KLPs typically move materials toward the minus end of microtubules . An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts . During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division . Staining was concentrated near the midzone during metaphase and was retained there during anaphase . The phragmoplast was also stained . Similar localization patterns were seen in tobacco BY-2 cells . The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP) . A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass . When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor . Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody . Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast . A similar protein is also present in other species. Plant Mol Biol, 1995 Dec, 29(6), 1267 - 77 Repair mechanisms of UV-induced DNA damage in soybean chloroplasts; Cannon GC et al.; In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR) . The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation. J Biotechnol, 1995 Dec 1, 43(2), 103 - 10 Dextran as protectant against damage caused by sparging for hybridoma cells in a bubble column; van der Pol LA et al.; The effect of the addition of dextran as a protective polymer against sparging was examined with hybridoma suspension cells in a bubble column under standardized conditions . The protective effect of high concentrations of high molecular weight dextran showed a correlation with the bulk viscosity of the medium . A distinct protective effect occurs at viscosities greater than 20 x 10(-3) Pa s-1 . In contrast, low molecular weight dextrans that cause a minor increase in viscosity, also provide no protection against sparging . There is no strict correlation between surface tension and the protective effect of dextran against sparging . Oxygen transfer is strongly reduced by high concentrations of high molecular weight dextran . Therefore, addition of dextran as protective polymer against sparging for large-scale production processes with animal cells in stirred reactors does not seem feasible. Nucleic Acids Res, 1995 Nov 11, 23(21), 4246 - 54 Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract; Baumstark T et al.; The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular viroids was studied in a nuclear extract from potato suspension cells . The nuclear extract was prepared according to a modified procedure for a plant transcription extract . The transcript of the potato spindle tuber viroid (PSTVd) consists of a monomeric molecule with 17 additional nucleotides, thus doubling most of the central conserved region of viroids of the PSTVd-class . The transcript can assume four different secondary structures, which either co-exist as conformers in solution or can be kept as metastable structures after different treatments by temperature and/or ionic strength . The structures were analysed by thermodynamic calculations and temperature-gradient gel electrophoresis and were confirmed by oligonucleotide mapping . Only the so-called extended middle structure was processed to exact viroid circles . In this structure the 5'- and 3'-ends are branching out from the rod-like viroid structure at the loop starting with nucleotide 87 . The other structures were processed only if they could be rearranged into the active structure. Plant Mol Biol, 1995 Nov, 29(3), 413 - 29 Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103-1 and Nt103-35; Droog F et al.; We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes . All active deletion constructs tested showed expression of the reporter gene beta-glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots . Auxin treatment greatly enhanced the level of expression . The Nt103-1 promoter region -370/-276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of -370 . The region -651/-370 contains sequence information necessary for uninduced expression . The Nt103-35 promoter manifested its auxin-responsiveness within the -504/-310 region . Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position -560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site . A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element . The as103 element is present in both promoters and positioned around -360, so within the region determined to be indispensable for the response to auxin . A third factor was found binding to the -276/-190 region of both promoters . Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes . The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response. Virology, 1995 Aug 1, 211(1), 1 - 9 Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of bean golden mosaic geminivirus; Hanson SF et al.; Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome . AC1 is the only ORF required for geminiviral replication . A putative NTP-binding motif, EGX4GKTX32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263) . We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies . Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site . A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain . Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from 1190 to R . Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication . The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed. Plant Mol Biol, 1995 Aug, 28(5), 885 - 900 Stress responses in alfalfa (Medicago sativa L.) XIX . Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response; Fahrendorf T et al.; We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.) . These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals . Transcripts encoding both enzymes are expressed at high levels in roots and nodules . Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins . Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation . The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state . These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism . Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity. Plant Mol Biol, 1995 Aug, 28(5), 859 - 70 Isolation of cDNA clones of genes with altered expression levels in phosphate-starved Brassica nigra suspension cells; Malboobi MA et al.; Differential gene expression at the transcriptional level was examined as an initial step in the investigation of the P(i) starvation response of Brassica nigra suspension cells . Total RNA was extracted from 7-day old cells grown in media containing either no P(i), 1.25 mM or 10 mM Pi . In vitro translation was carried out using their respective poly(A)+ RNA isolates and the resultant polypeptides were separated on a high-resolution SDS-PAGE gel . Scanning densitometry identified four polypeptides (ca . 31.7, 32.3, 52.5 and 64.8 kDa) present only in the P(i)-starved samples . Screening by differential hybridization was performed on a cDNA library constructed from mRNA isolated from P(i)-starved cells . Probes prepared from mRNA from P(i)-deficient and P(i)-sufficient cells identified a number of clones representing mRNA species that were preferentially transcribed under P(i) deficiency . These phosphate starvation-responsive (psr) clones were placed into eleven groups as determined by cross-hybridization . Northern blots showed that the corresponding genes are inducible in both mild and severe P(i) starvation conditions . Preliminary sequencing identified one of the clones as being homologous to beta-glucosidases from several plant species . The possible role of beta-glucosidase during Pi starvation and the identities of the other psr genes are discussed. J Cell Physiol, 1995 Aug, 164(2), 334 - 43 THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-beta 1 via increased expression of urokinase and the urokinase receptor; Falcone DJ et al.; Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen . The regulation of these receptors is important for several macrophage functions . In these studies, the effect of transforming growth factor-beta 1 (TGF-beta 1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined . TGF-beta 1 induction of uPA expression by THP-1 cells was differentiation dependent . Suspension and adherent cultures expressed similar constitutive levels of uPA . Exposure of adherent cells to TGF-beta 1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA . In contrast, uPA expression by suspension cultures was unresponsive to TGF-beta 1 . The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-beta 1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors . TGF-beta 1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity . Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-beta 1 . The increase in membrane-uPA activity expressed by TGF-beta 1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-beta 1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity . Furthermore, immunoreactive uPA receptor was increased in TGF-beta 1-treated cells . Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-beta 1-treated cells . However, no change in immunoreactive membrane-bound plasmin(ogen) was observed . In addition, binding of 125I-Lys-plasminogen to THP-1 cells was not affected by TGF-beta 1 treatment . We conclude that TGF-beta 1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression. Immunopharmacology, 1995 Jun, 30(1), 89 - 101 High susceptibility of U937-derived subclones to infection with human immunodeficiency virus type 1 is correlated with virus-induced cell differentiation and superoxide generation; Kameoka M et al.; The promonocytic human leukemic cell line U937, when infected with lymphotropic human immunodeficiency virus type 1 (HIV-1), becomes a continuous virus producer . A total of 46 U937-derived subclones in suspension was isolated and classified into three (2 high, 42 middle, and 2 low) types based on their susceptibility to the infection . By analyzing subclones before infection, we found that the high-type subclones expressed LFA-1 antigens at a relatively low level . In addition, the ability of these subclones to induce adherence after exposure to phorbol 12-myristate 13-acetate (PMA) was reduced . In contrast, a transition by HIV-1 infection to adherent macrophage-like cells was induced only in the high-type, but not in the low-type subclones . The high-type adherent cells obtained by HIV-1 infection were followed by further lineage to become retrodifferentiated suspension cells showing reduced syncytia formation ability . Superoxide was generated in the high-type subclones, without PMA-mediated differentiation, from the early stage of infection before HIV-1 replication, as well as during undifferentiated, differentiated and retrodifferentiated stages . In contrast, it was only transiently generated at acute phase of HIV-1 replication in low-type subclones . Long-term culture of the low-type subclones decreased the expression of major structural viral protein Gag and also virus production . Thus, the mechanism by which PMA differentiates U937 cells is not the same as that induced by HIV-1 infection . The latter mechanism results in high susceptibility to infection . The HIV-1 phenotypes of finally obtained persistently infected cells were also affected by the cell stages at the time of infection. Mol Biol Cell, 1995 Jun, 6(6), 637 - 47 Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase; Hildebrand JD et al.; Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions . pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals . Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk . In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin . The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK . The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin. Mol Biotechnol, 1995 Jun, 3(3), 181 - 90 Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen; Saunders JA et al.; The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells . Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices . The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco . Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse . The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse . A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources. J Biol Chem, 1995 Mar 3, 270(9), 4368 - 74 The plant inorganic pyrophosphatase does not transport K+ in vacuole membrane vesicles multilabeled with fluorescent probes for H+, K+, and membrane potential; Ros R et al.; It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J . M., Poole, R . J., Rea, P . A., and Sanders, D . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 11701-11705) . Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M . (1994) J . Biol . Chem . 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification . We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential . This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+ . Proton pumping occurred in response to pyrophosphate addition in the absence of K+ . No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated . The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin . No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase . We conclude that PPase in native vacuolar membrane vesicles does not transport K+. Chin J Biotechnol, 1995, 11(3), 207 - 11 Protoplast culture and plant regeneration from the suspension cells of Gynostemma pentaphyllum (Thumb) Mak; Zhang H et al.; From the suspension cultures of Gynostemma pentaphyllum (Thumb) Mak . protoplasts were isolated and cultured in a different medium with liquid and nurse culture method . Cell division was observed within 4 days and microcalli were formed within 4 weeks . On an MS solid medium with 1 mg/L of KT and 0.5 mg/L of IAA, protoplast-derived calli differentiated into embryos . Stems and leaves were formed on the MS medium with 1 mg/L of 6-BA and 0.5 mg/L of IAA . Finally, complete plantlets were obtained on a hormone-free MS solid medium. Cell Motil Cytoskeleton, 1995, 31(2), 113 - 29 Experimental manipulation of gamma-tubulin distribution in Arabidopsis using anti-microtubule drugs; Liu B et al.; gamma-Tubulin-specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner . During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate . gamma-Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers . At higher drug concentrations, gamma-tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast . During UV-induced recovery from colchicine, gamma-tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei . With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles . In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal . As with CIPC, gamma-tubulin is concentrated at focal arrays . Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts . These results support a preferential association between gamma-tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts . Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but gamma-tubulin may also serve another function, such as in structural stabilization. Brain Res Bull, 1995, 38(3), 215 - 20 Rat pineal cell aggregates: ultrastructural and functional characteristics; Shacoori V et al.; The aggregates were obtained by constant gyratory shaking of suspension cells freshly isolated from adult rat pineal glands . Their sizes ranged from 60 to 120 microns . Within 4-5 days, the aggregates formed by pinealocytes, astrocytes, and other unidentified cells became organized in a tissue-like configuration . There was no proliferation of the fibroblast cells . Ultrastructural characteristics of the aggregates were revealed by the presence of granular lysosomes, which are typical of pinealocytes, and are actively involved in the secretion . Functional characteristics were studied in static incubation . The aggregates secreted melatonin and other indole amines in culture medium . Basal melatonin release was detected until Day 24 of culture . This secretion was stimulated 230% with Isoproterenol (beta-adrenergic agonist), 725% with Epinephrine (alpha- and beta-adrenergic agonists), and 140% with Vasoactive Intestinal Peptide after 5 days in culture, then > 1200% with Forskolin 9 days later (14-day-old aggregates) . The results indicate that three-dimensional aggregates obtained from isolated pineal gland cells were the functional multicellular structures with in vivo characteristics. Plant Mol Biol, 1994 Dec, 26(6), 1701 - 10 Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco; van Engelen FA et al.; To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants . A model monoclonal antibody was used that binds to a fungal cutinase . Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2' promoters in a single T-DNA . The chimeric genes were cloned both in tandem and in a divergent orientation . The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA . Immunoblotting showed assembly to a full-size antibody . In addition, a F(ab')2-like fragment was observed, which is probably formed by proteolytic processing . Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells . The construct with divergent promoters showed a better performance than the construct with promoters in tandem . It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35% . The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization. Plant J, 1994 Nov, 6(5), 625 - 36 Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient; Chen MH et al.; A rice suspension cell culture system has been established to study how sugar depletion regulates alpha-amylase expression, carbohydrate metabolism, and other physiological and cellular changes . It is shown here that a group of 44 kDa alpha-amylases are constitutively expressed whether or not the cells are starved of sucrose . However, expression of a new group of alpha-amylases of 46 kDa is dramatically induced when cells are starved of sucrose . Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells . Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts . Immunocytochemical studies revealed that alpha-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles . The presence of putative signal sequences in the N-termini of nine rice alpha-amylases suggests hitherto unidentified pathways for import of alpha-amylases into amyloplasts . The studies show that differential alpha-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium . As the starved suspension cells exhibit some sugar-regulated characteristics of alpha-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of alpha-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants. Mol Cell Biol, 1994 Sep, 14(9), 6125 - 34 Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids; Gourdeau H et al.; The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization . Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3 . Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death . Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex) . Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive . The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids . p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment . bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids . We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans . Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression . Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway. Zhongguo Zhong Yao Za Zhi, 1994 Sep, 19(9), 529 - 31, 573 {Studies on immobilization of suspension cells of peltate yam (Dioscorea zingiberensis C.H . Wright)}; Ren JW et al.; The suspension cells of D . zingiberensis were immobilized with 3% sodium alginate, and then cultured in MS+2, 4-D1.0 + 6-BA 0.1 at 25 degrees C for a long period of time . The culture fluid free from cells was extracted and analyzed by TLC . The result showed that the immobilized cells could secrete the main component of D . zingiberensis--diosgenin, but not consecutively. J Virol, 1994 Sep, 68(9), 5925 - 32 Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2; Bai M et al.; The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2 . The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence . Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection . The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers . In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels . Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2 . Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection . These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2. EMBO J, 1994 Jul 1, 13(13), 2970 - 5 Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell; Thuleau P et al.; Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development . However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells . Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels . It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells . By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels . These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions . Ca(2+)-permeable channels showed slow and reversible inactivation . The single-channel conductance was 13 pS in 40 mM CaCl2 . These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation . The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells. Mol Cell Biol, 1994 Jul, 14(7), 4350 - 9 Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element; Ueda T et al.; By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter . The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization . A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells . In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region . The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes . A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro . It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner . Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor . The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter . These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes. Biochim Biophys Acta, 1994 Jun 1, 1192(1), 79 - 87 Voltage- and Ca(2+)-dependence of the K+ channel in the vacuolar membrane of Chenopodium rubrum L . suspension cells; Reifarth FW et al.; Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration . Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration . Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions . The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl . the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M . Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+ . Mean open times were approx . 7 ms, and almost independent of both, voltage and Ca2+ . Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M . Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms. Plant Mol Biol, 1994 Jun, 25(3), 401 - 12 Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible; Nelson AJ et al.; The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway . An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) . A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases . The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence . The protein is truncated at its 5' end, and shows reduced sequence conservation in this region as compared to other plant sequences . The rice genome contains a small family of HMGR genes . The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants . It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogen Magnaporthe grisea, causal agent of rice blast disease . This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice. Vaccine, 1994 Feb, 12(2), 159 - 66 Immunogenicity of foot-and-mouth disease virus grown in BHK-21 suspension cells . Correlation with cell ploidy alterations and abnormal expression of the alpha 5 beta 1 integrin; Amadori M et al.; BHK-21 suspension cells were characterized with regard to genetic and phenotypic features which might adversely affect the immunogenic properties of foot-and-mouth disease virus (FMDV) grown therein . A positive correlation was found between number of passages in suspension culture and both prevalence of polyploid cells and reduced cell growth on surfaces . Suspension cells also revealed differences in the expression of RGD-specific integrins and, in particular, of alpha 5 beta 1, which was shown to work as an FMDV receptor structure . These features, along with the notable instability of a few non-structural FMDV A5 proteins in infected cells, outline a new scenario, in which the reduced immunogenicity of FMDV might be accounted for by defined negative influences of the cell environment on viral replication. Plant J, 1993 Nov, 4(5), 855 - 62 The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to Brassica S-locus glycoproteins; van Engelen FA et al.; Non-embryogenic carrot suspension cells secrete the EP1 glycoprotein . A cDNA clone encoding EP1 was isolated and sequenced . The EP1 sequence revealed a region of homology with Brassica S-locus glycoprotein genes, an Arabidopsis S-like gene and putative S-like receptor protein kinases from maize and Arabidopsis . EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot . In developing seeds, expression was most pronounced in both the inner and outer integument epidermis. Int J Hyperthermia, 1993 Nov-Dec, 9(6), 799 - 802 Thermal response of synchronous CHO cells with different shapes; Smith NN et al.; This study examines the differential heat sensitivity of rounded suspension cells versus flattened monolayer cells . G1 populations of two different Chinese hamster ovary lines were used to eliminate possible cell cycle artifacts . The cell populations were heated at 43 and 45 degrees C . In all cases, cells treated in suspension were less sensitive to heat killing than cells treated as monolayers. J Membr Biol, 1993 Oct, 136(1), 43 - 54 Pharmacology of the SV channel in the vacuolar membrane of Chenopodium rubrum suspension cells; Weiser T et al.; Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L . At a holding potential of -100 mV, the SV channel showed half-maximal inhibition with 20 mM tetraethylammonium (TEA), 7 microM 9-amino-acridine, 6 microM (+)-tubocurarine, 300 nM quinacrine, and 35 microM quinine, respectively . The SV channel is also blocked by charybdotoxin (20 nM at -80 mV) but not by apamine . 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J . Gen . Physiol . 61:687-708, 1973) . No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine . Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast . A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred. Plant J, 1993 Sep, 4(3), 545 - 54 Molecular characterization of the gene for carrot cell wall beta-fructosidase; Ramloch-Lorenz K et al.; Carrot cell wall beta-fructosidase, previously purified and cloned, is encoded by a single, wound- and pathogen-inducible gene . The developmental regulation of the gene was studied by determining the steady-state mRNA levels in different organs during carrot development: cell wall beta-fructosidase mRNA was detected in roots and leaves of young plants but not during tap root development . A genomic clone was isolated and characterized . The transcription start site was determined by primer extension analysis . Inspection of the promoter sequence (1488 bp) revealed the presence of sequences with high homology to cis-acting elements for the regulation of plant genes by wounding and infection . The 5'-regulatory sequence was fused to the reporter gene beta-glucuronidase (GUS) and tested in a transient expression assay with carrot suspension cells and wounded carrot root tissue (aged disks of carrot roots) . The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional . In transgenic tobacco plants containing the cell wall beta-fructosidase promoter fused to GUS, the reporter gene was predominantly expressed in the shoot and root meristems of young seedlings . No GUS expression was detected in mature tobacco plants, showing that the development-specific regulation of the cell wall beta-fructosidase promoter seen in carrot was maintained in tobacco plants . In contrast, expression of the GUS reporter gene in transgenic tobacco was not wound inducible . To analyze the functional organization of the cell wall beta-fructosidase promoter, a 5'-deletion series was generated and tested in a transient expression assay in protoplasts of Nicotiana plumbaginifolia . Two regions containing putative silencer elements were identified . A comparison of these regions with known silencer elements identified in both regions one copy of the negative dominant cis-acting element found in a chalcone synthase promoter of petunia. Plant J, 1993 Aug, 4(2), 265 - 78 Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts; Marty I et al.; Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated . The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds . Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks . These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h) . Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes) . Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H4 gene . They seem to be induced in protoplasts by a synergistic effect of wounding and maceration . Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium. Mol Gen Genet, 1993 Jul, 240(1), 1 - 8 Low-temperature-dependent expression of a rice gene encoding a protein with a leucine-zipper motif; Aguan K et al.; We have isolated from rice suspension cells three non-sequence-related cDNAs the expression of which is markedly induced by low, non-freezing temperature . Here we further characterize one of the cDNA clones, lip19 . Expression of lip19 is positively regulated by low temperature, but not affected by high (40 degrees C) temperature . Sequencing and primer extension analyses showed that lip19 has a long (552 bp) 5' non-coding sequence followed by a single open reading frame specifying a protein of 148 amino acids . The deduced amino acid sequence of the protein, Lip19, shows at its amino-terminus a conserved basic region followed by a "leucine-zipper" domain . The reported sequence most similar to Lip19 is maize OCSBF-1, which is a bZip-type DNA binding protein . The possibility is suggested that Lip19 is a transcriptional factor that is positively controlled by low temperature. Plant Cell, 1993 Jun, 5(6), 603 - 13 Scaffold attachment regions increase reporter gene expression in stably transformed plant cells; Allen GC et al.; The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro . To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment . In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs . Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation . Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies . The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays. Plant J, 1993 Apr, 3(4), 619 - 26 Identification of cis elements involved in Commelina yellow mottle virus promoter activity; Medberry SL et al.; Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects a monocot host . A promoter fragment isolated from CoYMV is a strong promoter when assayed after transient introduction into monocot and dicot suspension cells and is highly active in vascular cells of flowers, leaves, stems and roots of stably transformed tobacco plants . Here it is reported that in stably transformed maize calli and transgenic tobacco leaves the CoYMV and CaMV 35S promoters exhibit similar amounts of activity . Deletion of the sequences located distal to nucleotide -230 relative to the start of transcription has no significant effect on promoter strength or tissue specificity . The region between -230 and -200 shares sequence similarity with the as-1 promoter element of the CaMV 35S promoter . Deletion of this as-1-like motif decreases promoter activity in maize suspension cells by 85% . Analysis of deletions affecting the -200 to -52 region ind